Accepted Manuscript

Pathogenesis of microbial keratitis

Sahreena Lakhundi, Ruqaiyyah Siddiqui, Naveed Ahmed Khan

PII: S0882-4010(16)30758-6
DOI: 10.1016/j.micpath.2016.12.013
Reference: YMPAT 2034

To appear in: Microbial Pathogenesis

Received Date: 8 November 2016

Revised Date: 30 November 2016
Accepted Date: 5 December 2016

Please cite this article as: Lakhundi S, Siddiqui R, Khan NA, Pathogenesis of microbial keratitis,
Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2016.12.013.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.
 ACCEPTED MANUSCRIPT

3 Pathogenesis of microbial keratitis

PT
1
5 Sahreena Lakhundi, 2Ruqaiyyah Siddiqui, 2Naveed Ahmed Khan*

RI
6

SC
1
7 Department of Biological and Biomedical Sciences, Aga Khan University, Karachi, Pakistan;
2
8 Department of Biological Sciences, Faculty of Science and Technology, Sunway University,

U
9 Selangor, Malaysia.
AN
10
M

11 Short title: Microbial keratitis

12
D
TE

13

14 *Corresponding address: Department of Biological Sciences, Faculty of Science and

EP

15 Technology, Sunway University, Selangor, 47500, Malaysia. Tel: 60-(0)3-7491-8622. Ext: 7176.

16 Fax: 60-(0)3-5635-8630. E-mail: naveed5438@gmail.com

C
AC

17

18

19

1
 ACCEPTED MANUSCRIPT

1 Abstract

2 Microbial keratitis is a sight-threatening ocular infection caused by bacteria, fungi, and

3 protist pathogens. Epithelial defects and injuries are key predisposing factors making the eye

PT
4 susceptible to corneal pathogens. Among bacterial pathogens, the most common agents

5 responsible for keratitis include Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus

RI
6 pneumonia and Serratia species. Fungal agents of corneal infections include both filamentous as

SC
7 well as yeast, including Fusarium, Aspergillus, Phaeohyphomycetes, Curvularia, Paecilomyces,

8 Scedosporium and Candida species, while in protists, Acanthamoeba spp. are responsible for

U
9 causing ocular disease. Clinical features include redness, pain, tearing, blur vision and
AN
10 inflammation but symptoms vary depending on the causative agent. The underlying molecular

11 mechanisms associated with microbial pathogenesis include virulence factors as well as the host
M

12 factors that aid in the progression of keratitis, resulting in damage to the ocular tissue. The

13 treatment therefore should focus not only on the elimination of the culprit but also on the
D

14 neutralization of virulence factors to minimize the damage, in addition to repairing the damaged
TE

15 tissue. A complete understanding of the pathogenesis of microbial keratitis will lead to the

16 rational development of therapeutic interventions. This is a timely review of our current

EP

17 understanding of the advances made in this field in a comprehensible manner. Coupled with the
C

18 recently available genome sequence information and high throughput genomics technology, and
AC

19 the availability of innovative approaches, this will stimulate interest in this field.

20 Key words: Cornea; Keratitis; Protist; Bacteria; Yeast

21

2
 ACCEPTED MANUSCRIPT

1 1. Introduction

2 The unique structure of the human eye along with its exposure to environment, renders it

3 susceptible to a number of agents responsible for causing infection. Injuries and epithelial defects

PT
4 impair defense mechanisms and exposure to pathogenic microbes can lead to corneal

5 inflammation or keratitis. The intact ocular surface thwarts most microorganisms but once

RI
6 anatomical barriers are breached, host defenses against pathogens are less than sufficient to

SC
7 prevent infection that can lead to eventual loss of vision. Microbial or infectious keratitis is a

8 potentially sight-threatening ocular condition caused by bacteria, fungi, protists etc. It is the

U
9 inflammation of the cornea caused by pathogenic microbes that eventually invades the corneal
AN
10 stroma causing inflammation, and ultimately destruction of these structures (Ansari et al., 2013;

11 Wong et al., 2012). The most common pre-disposing factors to develop infectious keratitis
M

12 include the use of contact lenses, especially overnight or extended wear lenses, inadequate

13 disinfecting solutions, trauma, ocular surgery especially corneal surgery, chronic ocular surface
D

14 disease, systemic disease like diabetes mellitus and/or extended use of topical corticosteroids
TE

15 (Stapleton et al., 2012; Wong et al., 2012). Patients usually present with redness, tearing, rapid

16 onset of pain and blur vision. Clinical presentation may vary depending on the causative agent
EP

17 responsible for causing keratitis. The condition should be treated as a medical emergency and
C

18 adequate treatment should commence promptly. If appropriate antimicrobial treatment is

AC

19 delayed, only 50% of the eye gains good visual acuity (Jones, 1981). Appropriate management

20 and timely onset of treatment can reduce the incidence of severe visual loss restricting corneal

21 damage. Here, we present a concise review of the bacterial, fungal and protist keratitis.

22 Numerous microorganisms can infect the eye either by direct or indirect introduction into the

23 eye. The most common clinically important microorganisms involved in eye infections are
3
 ACCEPTED MANUSCRIPT

1 reviewed in this article with relation to the anatomical part of the eye involved in the disease,

2 along with a discussion of the pathogenic mechanisms and management of the disease.

3 2. Acanthamoeba keratitis

PT
4 Acanthamoeba keratitis is a rare but sight threatening corneal infection, caused by an

5 opportunistic protist pathogen belonging to the genus Acanthamoeba. They are ubiquitous, free-

RI
6 living protists dispersed in a variety of environments including air, soil, freshwater, tap water,

SC
7 hospital equipments, surgical instruments, showers, ventilation ducts, air-conditioning units,

8 chlorinated swimming pools, sewage etc. (De Jonckheere, 1991; Kilvington & White, 1994).

U
9 Phenotypic switching into a cyst form enables Acanthamoeba to withstand adverse
AN
10 environmental conditions. The Acanthamoeba trophozoite has an amoeboid shape that contains

11 spike-like structures known as acanthopodia and during this stage, amoebae feed and reproduce
M

12 under favourable environmental conditions (Marciano-Cabral & Cabral, 2003; Visvesvara et al.,
D

13 2007). However, under extreme situations such as lack of nutrients, hyperosmolarity,

TE

14 desiccation, extreme pH, temperatures, and the presence of antimicrobials; the trophozoite

15 rounds up and confines itself within a double-walled resistant cyst form that has minimal
EP

16 metabolic activity. The cyst stage presents a major problem in the successful treatment of

17 Acanthamoeba infections as they are resistant to various antimicrobial agents, often leading to
C

18 recurrence of the disease upon discontinuation of therapy (Niederkorn et al., 1999). Although
AC

19 exposure to Acanthamoeba spp. appears to be common due to its ubiquitous nature, the incidence

20 of Acanthamoeba keratitis is less common. The main risk factors for Acanthamoeba keratitis are

21 contact lens wear for extended periods, corneal trauma, non-sterile contact lens rinsing,

22 swimming while wearing contact lenses and biofilm formation on contact lens (Marciano-Cabral

4
 ACCEPTED MANUSCRIPT

1 & Cabral, 2003; Trabelsi et al., 2012). While contact lens wear is the leading risk factor,

2 Acanthamoeba can cause keratitis in non-contact lens wearers as well (Dart et al., 2009).

3 However, it is often overlooked in non-CLs users as a causative agent of keratitis, where it is

4 usually associated with trauma and/or exposure to contaminated water, soil and organic matter.

PT
5 In addition, Acanthamoeba keratitis has been reported after invasive or radial keratoplasty and/or

RI
6 laser-assisted in situ keratomileusis (LASIK) where the lesions generally worsen due to a delay

7 in the diagnosis and treatment (Garg et al., 2010; Sharma et al., 2011).

SC
8 Clinical manifestations of Acanthamoeba keratitis include excruciating pain characterized

U
9 by redness, epiphora, lacrimation, eyelid diptosis, conjonctival hyperhemia, foreign body
AN
10 sensation and photophobia (Niederkorn et al., 1999; Mahgoub, 2010; Alizadeh et al., 1994). As

11 the disease progresses, stromal involvement results in infiltration of inflammatory cells

M

12 displaying a characteristic ring infiltrate. If not promptly diagnosed and treated aggressively,

13 cornea becomes ulcerated leading to perforation, ring infiltrate, stromal abscess formation, loss
D

14 of visual acuity and eventually blindness and enucleation.

TE

15 Acanthamoeba was first recognized as an ocular pathogen in 1973 in the U.S.A where the
EP

16 first case was reported by an ocular microbiology group in Dallas (Clarke et al., 2012). However,

17 the first published report of Acanthamoeba keratitis emerged in the UK in 1974 and was
C

18 associated with minor eye injury (Naginton et al., 1974). The first case in a contact lens wearer
AC

19 was reported 10 years later in 1984 from a patient wearing soft contact lenses (Samples et al.,

20 1984). Since then the number of cases has been on the rise especially in recent years, mainly due

21 to an increase in the number of contact lens users, better diagnostics and increased awareness.

22

5
 ACCEPTED MANUSCRIPT

1 2.1. Pathogenesis

2 2.1.1. Adhesion

3 The first step in the pathogenesis of Acanthamoeba keratitis is the ability of amoebae to

PT
4 bind to the corneal epithelium, a factor that determines the degree of pathogenicity of different

5 isolates. The pathogenic cascade begins via amoebal binding to mannose glycoproteins on the

RI
6 corneal surface through adhesin expressed on the trophozoite membrane called the mannose-

SC
7 binding protein (MBP) (Panjwani, 2010; Clarke & Niederkorn, 2006; Garate et al., 2004). This

8 adherence is a crucial prerequisite for producing infection. In addition. mild trauma or corneal

U
9 abrasion is required for the establishment of corneal infection. Abrasion or trauma results in
AN
10 increased expression of mannose glycoproteins on the corneal epithelium and hence there is

11 increased adhesion of amoebae to the damaged cornea, compared with the healthy cornea
M

12 (Clarke & Niederkorn, 2006). The contact lenses, in addition to serving as a vector for the
D

13 introduction of trophozoite onto the corneal surface, also up regulate mannose glycoproteins on
TE

14 the corneal epithelium. This results in an increased number of trophozoites binding to the contact

15 lens-conditioned cornea compared to the normal cornea thus leading to findings that more than
EP

16 80% of Acanthamoeba keratitis cases are associated with the use of CLs (Alizadeh et al., 2005).

17 The second important element involved in adhesion is the number of acanthopodia on the
C

18 amoebic surface. Pathogenic amoebae have more than 100 acanthopodia/cell compared to non-
AC

19 pathogenic amoebae. Therefore, non-pathogenic amoeba present very low binding levels to host

20 cells compared to pathogenic amoebae (Khan, 2001; Panjwani, 2010). Hence the number of

21 acanthopodia is also believed to be closely related to the rate of adhesion to the corneal surface.

6
 ACCEPTED MANUSCRIPT

1 Once attached, intracellular signaling processes trigger the pathogenic cascade involved in the

2 pathogenesis of Acanthamoeba keratitis.

3 2.1.2. Cytopathic effect

PT
4 Trophozoite binding to corneal surface is tailed by extensive desquamation of the corneal

5 epithelium, leading to penetration of the underlying Bowman’s membrane. Trophozoite-

RI
6 mediated cytopathic effect proceed via several mechanisms such as direct cytolysis, phagocytosis

SC
7 and apoptosis (Alizadeh et al., 1999). It has been observed that Acanthamoeba-mediated direct

8 cytolysis is dependent on calcium channel activity and on cytoskeletal elements, as the inhibition

U
9 of both via the calcium channel blocker, Bepridil and actin polymerization inhibitor, cytochalasin
AN
10 D overcomes Acanthamoeba-mediated cytolysis by over 98% (Taylor et al., 1995). In addition

11 the presence of structures like food cups or amoebastomes on the surface of Acanthamoeba
M

12 suggests that the amoeba binding to host cells leads to secondary events like phagocytosis by
D

13 which the amoeba bites or engulfs host cells. It has been observed that Acanthamoeba
TE

14 phagocytose and/or engulfs corneal epithelial cells and that this activity is mediated via

15 amoebastomes present on the surface of amoebae (Khan, 2001).

EP

16 Apart from inducing direct cell death, several studies indicate that Acanthamoeba induces

17 apoptosis of keratocytes, iris ciliary body cells, retinal pigment epithelial cells, corneal epithelial
C

18 cells, neuroblastoma cells etc. (Marciano-Cabral & Cabral, 2003; Niederkorn et al., 1999; Clarke
AC

19 et al., 2005; Stopak et al., 1991). Acanthamoeba induces membrane blebbing, formation of

20 apoptotic bodies, DNA laddering, nuclear chromatin condensation of host cell, all known

21 markers for apoptosis (Alizadeh et al., 1994; 1994a; Pettit et al., 1996). It has been observed that

22 exposure to mannose stimulates the production of a 133-kDa protease termed mannose-induced

7
 ACCEPTED MANUSCRIPT

1 protein (MIP133) by Acanthamoeba trophozoites. This production of MIP133 appears to be

2 another vital element of the pathogenic cascade of Acanthamoeba keratitis. It initiates apoptosis

3 of corneal epithelial cells in a caspase-3-dependent pathway in vitro and it is possible that the

4 trophozoite uses this pathway in the desquamation of the corneal epithelial cells in vivo (Hurt et

PT
5 al., 2003a; Hurt et al., 2003a). Furthermore, clinical isolates of Acanthamoeba but not soil

RI
6 isolates produce MIP133 and can cause disease in animals, signifying an association between

7 MIP133 production and pathogenic potential. Hence the trophozoite-mediated cytopathic effect

SC
8 to destroy epithelial cells occurs by three independent mechanisms i.e., direct cytolysis,

9 phagocytosis and apoptosis.

U
AN
10 2.1.3. Stromal invasion

11 Following binding and desquamation of the corneal epithelium, the next step in
M

12 Acanthamoeba keratitis involves the penetration and dissolution of the underlying collagenous
D

13 stroma. The Acanthamoeba trophozoites secrete a variety of proteases with nonspecific

TE

14 collagenolytic activity to facilitate the invasion and degradation of stroma. These include serine

15 proteases, cysteine proteases, metalloproteinases, elastase, collagenolytic enzyme, phospholipase

EP

16 A and a novel plasminogen activator (Cao et al., 1998; Cho et al., 2000; Hadas & Mazur, 1993).

17 Studies have shown the direct role of extracellular proteases in Acanthamoeba keratitis by co-
C

18 incubating corneal epithelial cells with Acanthamoeba conditioned medium, which resulted in
AC

19 host cell cytotoxicity (He et al., 1990). This cytotoxic effect was abolished in the presence of a

20 serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), suggesting a crucial role played

21 by serine proteases in the pathogenicity of Acanthamoeba keratitis (Dudley et al., 2008).

22 Furthermore, pathogenic Acanthamoeba exhibit higher protease activity compared to non-

8
 ACCEPTED MANUSCRIPT

1 pathogenic amoebae (Khan et al., 2000). Moreover, serine proteases are also able to degrade

2 collagen, fibronectin, laminin, secretory IgA, IgG, plasminogen, BSA, fibrin, fibrinogen, and

3 rabbit corneal proteins (Siddiqui & Khan, 2012). These properties of proteases are important

4 because collagen type I is the main structural protein in the corneal stroma which is important for

PT
5 the maintenance of its integrity. In addition sIgA, the main immunoglobulin in tears and a

RI
6 primary barrier against pathogenic microbes are also degraded by these proteases. Other

7 extracellular enzymes like elastase and phospholipase A are capable of degrading connective

SC
8 tissues. These findings suggest that collagenolytic enzymes have a role in corneal lesions and the

9 generation of ring-like stromal infiltrates which are characteristic features of Acanthamoeba

10 keratitis (He et al., 1990).

U
AN
11 Pathogenic Acanthamoeba can also use plasminogen activator that is constitutively present
M

12 in ocular isolates, to catalyze the cleavage of host plasminogen to plasmin, which in turn

13 activates matrix metalloproteinases (MMPs) to degrade components of extracellular matrix

D

14 (Mitra et al., 1995; Berman, 1993). They result in ulceration of normally quiescent cornea by
TE

15 degrading the components of basement membrane and extracellular matrix, including type I and

16 II collagens, laminin and fibronectin (Parks, 1999). Corneal stroma, unlike other extracellular
EP

17 matrices is vital for the normal functioning of the eye. Its degradation affects normal vision and
C

18 can lead to blindness. Studies have also shown that pathogenic isolates of Acanthamoeba are
AC

19 able to secrete ecto-ATPases which play a role in protecting the cells from effector cells of the

20 immune system. Secondly, they play a role in repolarization of the membrane after

21 depolarization by an active compound by acting as a proton pump. Lastly, these enzymes are

22 thought to be related to adherence to the host cell (Siddiqui & Khan 2012).

9
 ACCEPTED MANUSCRIPT

1 2.1.4. Neuritis

2 Acanthamoeba spp. are highly motile and secrete a plethora of proteases that augment their

3 pathogenesis by desquamating corneal epithelium, penetrating Bowman’s membrane and

PT
4 invading the corneal stroma. This cascade of events comes to a halt and trophozoites almost are

5 never able to penetrate the corneal endothelium and the anterior chamber of the eye (Clarke &

RI
6 Niederkorn, 2006). However, they seem to cluster around the corneal nerves producing radial

SC
7 keratoneuritis (Alizadeh et al., 2005). Studies have demonstrated the chemotactic response of

8 Acanthamoeba trophozoites towards extracts of neuronal cells and neural-crest-derived cells but

U
9 not towards the corneal epithelial or stromal cells (Pidherney et al., 1993). Moreover, in vitro
AN
10 studies have also demonstrated the killing of nerve cells by trophozoites via direct cytolysis

11 and/or apoptosis (Pettit et al., 1996). These cytopathic effects contribute to nerve damage and
M

12 may account for the excruciating pain experienced by the patient, coupled with this infection in

13 vivo.
D
TE

14 2.2. Treatment

15 Currently the treatment for Acanthamoeba keratitis involves topical application of cocktail
EP

16 of drugs, as there is no single effective drug treatment against Acanthamoeba keratitis. This is

17 mainly because of the degree of virulence of the infecting isolate which is different for each
C

18 individual case and therefore makes it impossible to establish a correlation between in vitro and
AC

19 in vivo efficacies. The best therapeutic outcomes are expected only when, it is diagnosed early,

20 treated adequately and aggressively with a high level of patient compliance. Acanthamoeba

21 trophozoites are sensitive to most available drugs; however the cysts present a major issue in the

22 course of treatment and are the main reason for the recurrence of infection upon discontinuation
10
 ACCEPTED MANUSCRIPT

1 of therapy. Thus, successful management most often necessitates extended therapy with a

2 combination of well tolerated drugs with minimal toxicity to hosts cells that results in a

3 favourable visual consequences with reduced need for surgical interventions.

PT
4 The most effective topical agents currently used against Acanthamoeba trophozoites and

5 cysts are the biguanides (polyhexamethylene biguanide (PHMB) 0.02 – 0.06% or chlorhexidine

RI
6 0.02 – 0.2%) in combination with diamidine (propamidine isethionate 0.1% or hexamidine 0.1%)

SC
7 (Wright et al., 1985; Khan, 2003; Dart et al., 2009; Lorenzo-Morales et al., 2013). The former

8 drugs are membrane-acting cationic biocides that interact with negatively charged surface

U
9 proteins of Acanthamoeba resulting in leakage of the cellular components (Perrine et al., 1995).
AN
10 The latter however exert their amoebicidal effects via cationic surface-active properties inducing

11 structural changes resulting in cell permeability and are effective DNA synthesis inhibitors
M

12 (Duguid et al., 1997). In vitro studies revealed that cationic agents have the best and most

13 constant amoebicidal and cysticidal activity (Elder et al., 1994; Hay et al., 1994). If these drugs
D

14 are applied early on in the development of infection, at high frequency along with neomycin and
TE

15 0.15% dibromopropamidine, they show successful prognosis (Lorenzo-Morales et al., 2013).

16 Hourly drops of PHMB and hexamidine day and night may be tapered off after 48 hours to
EP

17 alleviate epithelial toxicity, to hourly drops during the day for the next 72 hours. This therapy
C

18 reduces viable trophozoites when they are more susceptible and prevent them from turning into
AC

19 fully mature cysts. This treatment is then reduced to 2-hourly application during the day for the

20 next 3 – 4 weeks and then gradually tailored depending on the individual case. Treatment

21 regimes on average last over a period of 6 months, ranging from 0.5 to 29 months (Radford et

22 al., 1998).

11
 ACCEPTED MANUSCRIPT

1 In the case of inflammation along with persistent infection, corticosteroids have been

2 implicated however, there use is controversial as they cause suppression of host immune

3 response (Lorenzo-Morales et al., 2013). Recent reports also point towards the use of the azoles

4 as an adjuvant to biguanide and diamidine therapy in resistant Acanthamoeba keratitis cases

PT
5 (Bang, et al., 2010). Therefore topical and intrastromal voriconalzole drops (1%) have been used

RI
6 successfully in 3 cases with resistant Acanthamoeba keratitis. Surgical management via epithelial

7 debridement, corneal graft surgery (keratoplasty) and/or DALK (deep lamellar keratoplasty) may

SC
8 be required in some cases where the corneas are permanently scarred or where the topical and

9 oral treatments have failed (Illingworth & Cook, 1998; Chen et al., 2004; Sony et al., 2002;

10
U
Jiang et al., 2006; Parthasanrathy & Tan, 2007). Recently, photorefractive surgery seems to be a
AN
11 very promising modality and have been used in four cases of early stage Acanthamoeba keratitis,

where the patients developed large corneal abscesses in the upper third thickness of stroma
M

12

13 (Kandori et al., 2010). Another relatively new approach is collagen cross-linking which has
D

14 shown promising results in clinically (Garduno-Vieyra et al., 2011; Khan et al., 2011; Muller et
TE

15 al., 2012). It is presumed that collagen probably prevents further tissue damage and prevents

16 reproduction of amoebae and hence the patients showed rapid reduction in ocular symptoms and
EP

17 ulcer size.
C

18 Moon et al., (2015) recently reported that autophagy inhibitors have anti-amoebic effects
AC

19 and if used with low concentrations of PHMB (0.00125%) has very low cytopathic effect on

20 human corneal cells and a high cytopathic effect on Acanthamoeba cells. In addition, Aqeel et

21 al., (2015) suggested photochemotherapeutic strategy against Acanthamoeba infections and

22 showed that mannose-conjugated porphyrin has the potential for targeted photodynamic therapy

23 of Acanthamoeba infections. Regardless of significant advances in Acanthamoeba keratitis

12
 ACCEPTED MANUSCRIPT

1 treatment over the last decade or so, prevention still seems to be the best option. In addition,

2 contact lens wearers must be thoroughly educated on the proper hygiene to handle contact lenses

3 to avoid this serious sight-threatening infection.

PT
4 3. Mycotic keratitis

5 Mycotic keratitis is the fungal infection of the cornea caused by either filamentous and/or

RI
6 yeast-like fungi. They may account for more than 50% of all culture-positive microbial keratitis

SC
7 especially in tropical and sub-tropical countries (Xie et al., 2006; Nath et al., 2011; Thomas &

8 Kaliamurthy, 2013). A strong geographical correlation has been reported to exist between the

U
9 occurrences of different types of keratomycosis. For example, the proportion of keratitis due to
AN
10 yeast-like fungi show a tendency to increase towards temperate climates whereas corneal ulcers

11 caused by filamentous fungi appear to be more common towards tropical latitudes (Leck et al.,
M

12 2002).
D

13 Filamentous fungi such as Fusarium, Aspergillus, Phaeohyphomycetes, Curvularia,

TE

14 Paecilomyces and Scedosporium apiospermum are most commonly associated with keratitis

15 caused by filamentous fungi (Thomas & Kaliamurthy, 2013). However Candida albicans and
EP

16 other Candida species are most common keratitis-causing yeast-like fungi. Former is reported to

17 be due to ocular trauma which is usually the most important predisposing factor in healthy young
C

18 males engaged in agricultural or other outdoor activities (Nath et al., 2011; Gopinathan et al.,
AC

19 2009; Thomas & Kaliamurthy, 2013). These fungi do not invade the intact cornea and

20 penetration only occurs once the epithelium is abraded. Traumatizing agents of animal origin or

21 vegetative matter, soil or dust particle either directly implant fungal conidia on abraded corneal

22 epithelium for fungal invasion (Nath et al., 2011; Gopinathan et al., 2009; Thomas &
13
 ACCEPTED MANUSCRIPT

1 Kaliamurthy, 2013). The use of corticosteroids, ocular surgery, ocular surface disease and

2 contact lens wear has now also been increasingly identified as a significant risk factor.

3 Conversely, C. albicans and related fungi are only linked to keratitis when there is a pre-existing

4 ocular condition like insufficient tear secretion, defective eye closure, or some systemic illness

PT
5 such as diabetes mellitus or immunosuppression (Thomas, 2003). This form of keratomycosis

RI
6 may also supervene on a pre-existing epithelial defect caused by herpes keratitis or abrasion due

7 to contaminated contact lenses.

SC
8 Fungal infections of the cornea need to be promptly addressed to facilitate full recovery. In

U
9 case of filamentous fungal keratitis, clinical manifestations include sudden onset of pain along
AN
10 with photophobia, discharge with reduced vision and opacity on the surface of the cornea

11 suggestive of an ulcer (Ansari et al., 2013). It may involve any part of the cornea and show firm,
M

12 sometimes dry elevated slough, hyphate lines extending into the normal cornea beyond the edge

13 of the ulcers, multifocal granular or feathery grey-white satellite stromal infiltrates, immune ring,
D

14 Descemet’s fold and mild iritis (Thomas, 1994; Rosa et al., 1994; Srinivasan, 2004; Thomas &
TE

15 Kaliamurthy, 2013). Although each fungal keratitis exhibits these basic features, they may vary

16 depending on the etiological agent. Severe, chronic filamentous keratitis somewhat resemble
EP

17 bacterial suppuration and may involve the entire cornea. However those due to yeast-like and
C

18 related fungi may resemble bacterial keratitis with an overlying epithelial defect, discrete
AC

19 infiltrate and slow progression (Sun et al., 2007).

20 3.1 Mechanism

21 3.1.1 Adhesion

14
 ACCEPTED MANUSCRIPT

1 Interaction of pathogenic fungi with host cells is the key factor in the pathogenesis of

2 mycotic keratitis. Adherence of microorganisms to host cells is a pre-requisite for the initiation

3 of the infection. Hence fungal infections of the cornea begin via adhesion of fungi to the

4 damaged cornea. Fungal pathogens display a variety of adhesins that are capable of adhering to

PT
5 various cell types and interact with a variety of host proteins and glycoproteins present in host

RI
6 cells (Hostetter, 1994). The outer fibrillar layer of yeast and filamentous fungal cell wall is

7 composed of mannan or mannoprotein (Imwidthaya, 1995; Thomas, 2003). The adhesive

SC
8 mannoproteins play an essential role in fungal binding to corneal tissues. These lectin-like

9 proteins recognize D-mannose or mannose glycoproteins on the surface of corneal epithelial

10
U
cells. Damage to the cornea results in the upregulation of mannose glycoproteins on the corneal
AN
11 surface (Clarke & Niederkorn, 2006); it may therefore play an important role in the adhesion and

pathogenesis of fungal keratitis. Ocular trauma is the most important pre-disposing factor in
M

12

13 mycotic keratitis (Nath et al., 2011; Gopinathan et al., 2009; Thomas & Kaliamurthy, 2013); the
D

14 absence of which prevents fungal penetration into the cornea. Thus, the correlation of ocular
TE

15 trauma with an increased expression of mannose glycoproteins on the corneal surface and

16 presence of adhesive mannoproteins on the surface of fungal cell wall is likely involved in the
EP

17 pathophysiology of fungal keratitis.

C

18 The corneal epithelium also possess other potential fungal binding sites like laminin,
AC

19 fibronectin, collagen etc. which also play a role in keratitis (Thomas, 2003). Tronchin et al.,

20 (1988) demonstrated the importance of an outer fibrillar layer of the germ tube in the process of

21 adhesion to plastics. Bouchara et al., (1990) reported that these major components of the fibrillar

22 cell wall layer of the germ tube act as a receptor for laminin, fibrinogen and mediates its

23 attachment to membranes. The involvement of specific interaction of Aspergillus fumigatus with

15
 ACCEPTED MANUSCRIPT

1 finrinogen and its role in cell adhesion has shown that fungal conidia adhere avidly to wells

2 coated with laminin, fibrinogen, collagen and fibronectin substrates (Coulot et al., 1994). This

3 was further supported by Bromley & Donaldson (1996), who showed the ability of fibrinogen

4 and laminin to inhibit the adherence of conidia to pulmonary epithelial cell lines. In addition

PT
5 sialic-acid dependent lectins have also been reported to play an important role in recognition of

RI
6 laminin and fibrinogen by Aspergillus and Penicillium conidia.

SC
7 Furthermore, the outer layer of conidia also plays a crucial role in the early stages of

8 infectious process. The surface of resting conidia has been reported to have proteins belonging to

U
9 the hydrophobin family (Parta et al., 1994; Thau et al., 1994) which are detected in all
AN
10 filamentous fungi and thought to play a role in adherence (Scholtmeijer et al., 2001).

11 3.1.2 Invasiveness and Morphogenesis

M

12 The etiological agent in Fusarium keratitis is able to invade the cornea gaining access to
D

13 the anterior chamber of the eye. There, at the pupillary area, it forms a lens-iris-fungal mass
TE

14 affecting the normal drainage of aqueous humor leading to an increase in the intraocular pressure

15 causing fungal malignant glaucoma (Kuriakose & Thomas, 1991). Initially, malignant glaucoma
EP

16 was thought to occur only in Fusarium keratitis, however recently Jain et al., (2007), reported a

17 case of Aspergillus-induced malignant glaucoma where the uniform shallowing of the anterior
C

18 chamber was present with raised IOP unresponsive to antiglaucoma measures. Eventually A.
AC

19 flavus was isolated from the anterior chamber of the eye.

20 Fungal invasiveness is directly related to the fungal load and inversely proportional to the

21 intensity of inflammatory response (Vemuganti et al., 2002). Therefore, the heavier the load of

16
 ACCEPTED MANUSCRIPT

1 the fungus, the greater the extent of invasion and the smaller the number of inflammatory cells.

2 In early mycotic keratitis, the heavy fungal load with deep tissue penetration may overcome the

3 inflammatory response in corneal tissue, encouraging the progression of the disease. Conversely

4 the inflammatory response in early keratitis may be mild and therefore the fungi multiply

PT
5 extensively and penetrate deep into the tissues. The putative sequence of events in which it

RI
6 occurs still needs to be confirmed.

SC
7 Phenotypic switching or morphogenesis is an important method of adaptation used by

8 some microbes in different microenvironments to survive inside infected hosts. This permits

U
9 fungi to survive in the presence of anti-fungal drugs and resist anti-microbial therapy. The same
AN
10 intrahyphal hyphae or hypha-in-hypha and thickened fungal cell walls are seen in corneal tissues

11 infected with L. theobromae and in corneas infected with F. solani keratitis treated earlier with
M

12 corticosteroids, as seen in the presence of antifungal drugs (Thomas et al., 1991; Kiryu et al.,

13 1991). This suggests that these morphological alterations may allow fungi to evade host defenses
D

14 and present a barrier against antifungal drugs. Jackson et al., (2007) demonstrated the role of
TE

15 EFG1-regulated SAP6 gene of C. albicans which encodes for a unique secreted aspartyl

16 proteinase. They concluded that proteases contribute to corneal pathogenicity in C. albicans

EP

17 keratitis and are also associated with morphogenic transformation from yeast to invasive
C

18 filamentous forms.
AC

19 3.1.3 Toxigenicity

20 Various keratitis causing fungi are known to produce mycotoxins; however their exact role

21 in the pathogenesis of keratitis is still unknown. Several toxins produced by Fusarium spp.

22 include nivalenol, T-2 toxin, deoxynivalenol, diacetoxyscirpenol and fusaric acid. Raza et al.,
17
 ACCEPTED MANUSCRIPT

1 (1994) studied the relationship of toxin production with the severity of disease and concluded

2 that toxin production in vitro did not relate to the clinical presentation of severity of keratitis and

3 the outcome of the treatment. The toxic effects of aflatoxins on the cornea of chicks has been

4 established (Kant et al., 1984). It was demonstrated that when aflatoxin was administered to

PT
5 chicks, haziness of the cornea and separation of corneal lamellae was observed in addition to

RI
6 infiltration by polymorphonuclear leukocytes. Leema et al., (2010) demonstrated that A. flavus

7 isolated from keratitis patients produce significantly more aflatoxin compared to those of the

SC
8 environmental isolates. However the reason behind this phenomenon is unclear and requires

9 further study.

U
AN
10 The ability of fungi to produce various enzymes could also damage tissues, facilitate

11 invasion and eventually influence the severity and outcome of the disease. Zhu et al., (1990)
M

12 examined the role of fungal proteases in the pathogenesis of mycotic keratitis. It was observed

13 that the clinical isolate of A. flavus, isolated from a patient with severe keratitis, secrete variety
D

14 of proteases including serine proteases, cysteine proteases, metalloproteases and concluded that
TE

15 fungal collagenases are the mediator of the severe corneal destruction observed in keratitis.

16 When attempted to compare the presence of fungal proteases in vitro and in vivo, the corneal
EP

17 isolates of A. flavus and F. solani predominantly secreted serine proteases and little
C

18 metalloproteases in vitro. In vivo, however the protease profile shifted to metalloproteases and no
AC

19 serine protease activity was detected (Gopinathan et al., 2001). Matrix metalloproteinases are

20 thought to play a pathological role in the degradation of extracellular matrix components such as

21 basement membrane collagen; same as those found in corneal basement membrane and stroma

22 (Boveland et al., 2010). They have also been shown to be upregulated in ulcerative fungal

23 keratitis in rabbit as well as in the tear film of horses with ulcerative keratitis and hence are
18
 ACCEPTED MANUSCRIPT

1 thought to play a probable role in the pathogenesis of fungal keratitis (Gopinathan et al., 2001;

2 Ollivier et al., 2004). However, the exact role of fungal proteases in mycotic keratitis needs

3 further investigations.

PT
4 3.2 Treatment

5 Fungal keratitis is a complex entity with many considerations when it comes to treatment.

RI
6 On the whole, treatment generally entails of chemotherapy consisting of topical and/or systemic

SC
7 drugs, alone or in combination with surgical treatment. However, in developing countries with

8 limited care and economical barriers, mycotic keratitis is of major concern as it can cause visual

U
9 loss in a demographic population that has limited access to care. Each antifungal has its own
AN
10 benefits and limitations and therefore must be selected carefully based on etiological agent and

11 susceptibility testing. There are several classes of antifungals available like polyenes, azoles and
M

12 fluorinated pyrimidines, each with their own advantages and disadvantages. Polyenes, including
D

13 natamycin, nystatin and amphotericin B, disrupt fungal cell by binding to the cell wall ergosterol
TE

14 of both filamentous as well as yeast-like fungi. However their tissue penetrating ability is poor

15 and therefore is mostly recommended in cases of superficial corneal infections (Thomas, 2003;
EP

16 Ansari et al., 2013). Administration is every 30 min during the first 24 hours and then every hour

17 for the next 24 hours and then gradually tapered off according to the response. While
C

18 amphotericin B is commonly administered as a topical solution, intracameral administration is an

AC

19 effective alternative in reducing time to disappearance of hypopyon and final improvement

20 (Yoon et al., 2007). Although active against both forms of fungi, it has shown variable activity

21 against Fusarium species. Owing to its side effect profile and lack of coverage against Fusarium

22 keratitis, it is not considered as a first line agent (Ansari et al., 2013). Natamycin on the other

19
 ACCEPTED MANUSCRIPT

1 hand has a broad-spectrum activity against filamentous fungi and is the first line treatment

2 against fungal keratitis and the drug of choice for Fusarium keratitis. The drug can only be given

3 topically and hence can only treat superficial fungal keratitis as opposed to deep stromal fungal

4 invasion. The presence of deep lesions may therefore require other antifungals like azoles

PT
5 administered via subconjunctival or intravenous routes (Tanure et al., 2000).

RI
6 The azoles including imidazoles and triazoles, at low concentrations inhibit ergosterol

SC
7 biosynthesis while, cause direct damage to cell walls at higher concentrations (Tatsumi et al.,

8 2013). Fluconazole and ketoconazole show good intraocular penetration and are therefore good

U
9 agents against keratitis with deep lesions (Panda et al., 1996). They are the preferred treatment
AN
10 against both candida and filamentous fungi however, fluconazole has narrow coverage against

11 filamentous organisms but ketoconazole is active against Aspergillus, Candida as well as

M

12 Curvularia species (Rao et al., 1997; Fitzsimons & Peters, 1986; Ansari et al., 2013).

13 Voriconazole is a good alternative with minimal toxicity and is not only active against Candida
D

14 but also against filamentous fungi such as Fusarium spp. (Marangon et al., 2004; Lalitha et al.,
TE

15 2007). In refractory FK cases, topical voriconalzole has been used as an adjunct to natamycin

16 along with intrastromal injections of voriconalzole with success (Hariprasad et al., 2008).
EP

17 Hariprasad et al., (2008) reviewed 40 case-reports and concluded that voriconalzole is a safe
C

18 alternative against major ocular fungal infections but shouldn’t be used as a single agent for
AC

19 initial treatment.

20 The fluorinated pyrimidines such as flucytosine etc. are converted into thymidine analog

21 blocking fungal thymidine synthesis. They are usually administered with an azole or

22 amphotericin B for synergistic effects and to avoid development of resistance. Subconjunctival

20
 ACCEPTED MANUSCRIPT

1 injections have also been used in patients with severe keratitis. Regardless of the drug choice,

2 successful therapy requires prolonged frequent drug administration and therefore adherence of

3 patients to treatment is also an important factor affecting the outcome of treatment. In addition,

4 liposomal preparation of anti-fungal drugs are also under trail and has demonstrated some

PT
5 efficacy in rabbit models (Habib et al., 2010; Abdel-Rhaman et al., 2012). Furthermore Rasool et

RI
6 al., (2012) compared the efficacy of topical clotrimazole-β-cyclodextrin (CBC), a comparatively

7 new anti-fungal compared to amphotericin B and concluded that CBC reduces the period of

SC
8 treatment on average by a duration of 1 week. The authors also reported greater efficacy of CBC

9 in cases of Candida keratitis.

U
AN
10 Surgical interventions are required in case of complications of acute infectious processes as

11 well as if the disease is refractory to medical management. Penetrating keratoplasty (PK) is the
M

12 most common surgical treatment used to excise lesions of the cornea and replaced with a donor

13 corneal graft in case of refractory or severe cases of fungal keratitis (Prakash et al., 2008). In
D

14 addition, it is an effective way to treat corneal perforations as a result of fungal infections (Xie et
TE

15 al., 2007). As an alternative to PK, debridment is also used where causative agent and

16 necrotizing material is removed thereby enhancing the penetration of anti-fungal medications by

EP

17 the removal of epithelium. It is recommended where the necrotizing tissues are hindrance to the
C

18 healing of corneal ulcers.

AC

19 Alternate surgical procedure includes lamellar keratoplasty (LK) in which only diseased

20 layers of the corneal surface are excised thereby leaving the underlying structures of the cornea

21 intact. It is recommended when the fungal invasion is only focal (Price et al., 2005; Reddy et al.,

21
 ACCEPTED MANUSCRIPT

1 2013). In thisway, the risk of endothelial rejection, the most severe type of corneal graft

2 rejection, is minimized.

3 The challenging nature of fungal keratitis along with less than ideal outcome of current

PT
4 treatment regimes, various experimental advances have also been made. Corneal collagen cross-

5 linking (CXL) is a relatively new technique currently been investigated in conjunction with

RI
6 photo-activated riboflavin (PAR) in refractory keratitis cases (Li et al., 2013; Panda et al., 2012).

SC
7 Investigations into the use of nanoparticle technology with terbinafine and silver have also

8 shown promising results (Tayel et al., 2013; Xu et al., 2013). In addition, topical aqueous garlic

U
9 extracts have been tested on rabbit keratitis models infected with A. flavus (Ismaiel et al., 2012).
AN
10 Nonetheless, fungal keratitis is a complex entity with many considerations when it comes to

11 treatment. However, prompt identification of the etiological agent along with targeted therapy
M

12 may result in favourable outcome.

D

13 4 Bacterial keratitis
TE

14 The major bacterial agents of infectious keratitis include Staphylococcus aureus,

15 Pseudomonas aeruginosa, Streptococcus pneumonia and Serratia species (Wong et al., 2012).
EP

16 The community acquired cases of bacterial keratitis are usually resolved with an empirical

17 treatment, however if left un-attempted, may result in perforation, endophthalmitis and loss of
C

18 vision (Callegan et al., 1994; Wong et al., 2012). Clinical presentation of bacterial keratitis
AC

19 include acute pain, redness, photophobia and corneal ulceration (Stapleton et al., 2007).

20 Pseudomonas ulcers are more severe at presentation than other bacterial ulcers and are often

21 difficult to treat, resulting in worse visual outcome than other bacterial ulcers (Sy et al., 2012;

22 Green et al., 2008).

22
 ACCEPTED MANUSCRIPT

1 Bacterial keratitis accounts for approximately 90% of all microbial keratitis cases (Musa et

2 al., 2010) with P. aeruginosa as the most common culprit, worldwide (Dutta et al., 2012).

3 Corneal infection of Pseudomonas is most commonly associated with the use of contact lenses

4 and was rarely reported as a problem prior to the emergence of contact lens (Marquart &

PT
5 Callaghan, 2013; Ramirez et al., 2012). The resistance of Pseudomonas to disinfectants, coupled

RI
6 with its adherence capability to plastics, facilitates its introduction into the eye where it can react

7 with defective corneal epithelium, gaining further entrance into the corneal stroma. S. pneumonia

SC
8 on the other hand is the major cause of corneal ulcers in developing countries however, some

9 reports emphasize on the fact that Streptococcus are most commonly encountered after P.

10
U
aeruginosa and/or S. aureus eye infections (Bharathi et al., 2010; Yilmaz et al., 2007; Marquart
AN
11 & Callaghan, 2013). Unlike P. aeruginosa, pneumococcal keratitis is not commonly associated

with the use of contact lenses and the predisposing factors often include ocular trauma or surgery
M

12

13 (Marquart and Callaghan, 2013; Wagoner et al., 2007; Cosar et al., 2001; Lifshitz et al., 2005;
D

14 Mulet et al., 2010; Rehany et al., 2004).

TE

15 Along with P. aeruginosa, S. aureus is also a common etiological agent (Marquart &

16 Callaghan, 2013; Liesegang, 1998; Wong et al., 2011; Jeng et al., 2010; Palmer & Hyndiuk,
EP

17 1993). It is a commensal organism that can readily gain access into the eye, given the
C

18 opportunity (Mohammadinia et al., 2012; Tacconelli & Johnson, 2011; Speaker et al., 1991).
AC

19 Individuals whose eyes are compromised due to various reasons including ocular surgery or

20 trauma, contact lens use, viral infection or other eye illnesses are at high risk of developing this

21 infection (Cheung & Slomovic, 1995; Ormerod et al., 1987; Coster & Badenoch, 1987). In

22 particular, the ability of S. aureus to develop antibiotic resistance makes this infection among the

23 most difficult one to treat (Asbell et al., 2008). This, along with Serratia marcescens, which once
23
 ACCEPTED MANUSCRIPT

1 was considered a harmless saprophyte, has now been increasingly isolated from the corneal

2 surfaces of keratitis patients (Parment, 1997). The isolation rate of S. marcescens from contact

3 lens-coupled corneal infections ranges from 5 – 28%; almost comparable to the isolation rate of

4 P. aeruginosa in contact lens-related keratitis (Schein et al., 1989; Hume et al., 2007). S.

PT
5 marcescens is a motile gram negative rod abundantly dispersed in nature and contributes to

RI
6 contact lens-related keratitis (Parment et al., 1996; Parment, 1997).

SC
7 4.1 Mechanism

8 4.1.1 Adhesion

9
U
Bacteria initiate infection by engaging with the host cell-surface receptors via various
AN
10 adhesins, which mediate bacterial binding to corneal epithelial cells. Microbial adhesins not only
M

11 play a role in bacterial attachment to the surface of epithelial cells but may also play an active

12 role in subsequent interactions and infective process. They may act as toxins initiating microbial
D

13 invasion and contribute to subsequent pathogenic cascade. Bacteria display several adhesins on
TE

14 their surface such as pili or fimbriae, which recognize specific carbohydrates or proteins on the

15 surface of host cell. The adherence of P. aeruginosa, S. pneumonia and S. aureus is significantly
EP

16 higher to damaged corneal epithelium compared to other bacteria accounting for their frequent

17 isolation from keratitis cases.

C
AC

18 Studies have shown that purified pili successfully compete with cold bacteria for binding to

19 ocular surface and have been used to protect against P. aeruginosa keratitis. Corneal epithelial

20 glycoproteins act as surface receptor mediating pilus binding activity and amino sugar sialic acid

21 was able to completely inhibit pilus binding to mouse corneal epithelial cells (Hazlett et al.,

24
 ACCEPTED MANUSCRIPT

1 1992; Rudner et al., 1992). Bacterial flagella on the other hand are filamentous organelles,

2 responsible for bacterial motility and therefore are also responsible for dissemination of

3 infection. More than 95% of P. aeruginosa clinical isolates are flagellated and flagella-deficient

4 mutants have been observed to be non-virulent (Ansorg et al., 1984). In addition the anti-

PT
5 flagellar antibody homologous to the infecting strain protects mice from Pseudomonas corneal

RI
6 infection (Rudner et al., 1992). Furthermore, the glycocalyx may also play an important role in

7 bacterial adhesion by producing slime aggregates resistant to phagocytosis thereby enabling

SC
8 them to adhere to cells (Hyndiuk, 1981).

U
9 S. aureus surface adhesins, collectively known as MSCRAMMS (microbial surface
AN
10 components recognizing adhesive matrix molecules), have also been recognized to mediate

11 bacterial adherence to host extracellular matrix components, collagen, fibronectin, fibrinogen,

M

12 laminin and elastin (Jett & Gilmore, 2002). Rhem et al., (2000), studied the role of MSCRAMM

13 Cna (collagen-binding adhesin) in S. aureus keratitis and concluded that collagen-binding

D

14 adhesin is a virulence factor for S. aureus keratitis and is involved in the early events of
TE

15 pathogenesis of S. aureus infection of the cornea. Similarly, Jett & Gilmore (2002) studied the

16 role of S. aureus fibronectin-binding protein as an epithelial and/or endothelial cell

EP

17 adhesin/invasion protein. Their data suggested that FnBPs is a surface ligand for human corneal
C

18 cells and play a key role in host-parasite interactions. It serves as an important adhesin and
AC

19 triggers invasion in ulcerative keratitis caused by S. aureus.

20 Likewise, Streptococci colonize different sites on human body by expressing multiple

21 adhesins and hence their attachment to human tissues is mediated by a diverse group of bacterial

22 surface proteins, MSCRAMMS (Hammerschmidt, 2006). Plasmin and fibronectin binding

25
 ACCEPTED MANUSCRIPT

1 protein A (PfbA), a MSCRAMMS, is known to promote adherence and invasion of bacteria to

2 human epithelial cells by recognizing molecules like fibronectin. In addition, pneumococcal

3 surface adhesin A (PsaA), pneumococcal surface protein A (PspA), pneumolysin (ply),

4 pneumococcal adherence and virulence factor A (PavA), choline-binding protein A

PT
5 (CbpA/PcpA), putative protease maturation protein A (PpmA), IgAI protease (IgAIp), and the

RI
6 streptococcal lipoprotein rotamase A (SIsA) have all been shown to be associated with

7 pneumococcal adherence and virulence (Rajam et al., 2008). Streptococcal cell surface may

SC
8 contain fibrillar structure like pili and fibrils which may also mediate attachment to cell surface

9 to initiate infection. The pneumococcal surface protein C (PspC) promotes adherence and uptake

10
U
of pneumococci into nasopharyngeal epithelial cells (Hammerschmidt et al., 2000). Also,
AN
11 antibodies against PsaA which code for pneumococcal surface adhesin A (PsaA) resulted in

reduced adherence to nasopharyngeal epithelial cells (Romero-Steiner et al., 2003). Therefore,

M

12

13 these adhesins are likely involved in the initial stages of Streptococcal ocular infection.
D

14 Serratia marcescens on the other hand possess mannose-specific adhesins, associated with
TE

15 flagella. These adhesins probably interact with mannose glycoproteins expressed on the surface

16 of corneal epithelial cells as a result of abrasion or trauma due to the use of contact lenses.
EP

17 Labbate et al., (2007), identified type I fimbriae as the critical adhesin that mediate attachment of
C

18 S. marcescens to human corneal epithelial cell surface. They also identified two AHL (N-
AC

19 acylhomoserine lactone)-regulated genes, bsmA and bsmB, to be involved in adhesion to biotic

20 surface and the expression of these genes leads to the attachment of S. marcescens to HCE cells.

21 In 2007, Shanks et al., demonstrated the role of oxyR and type I fimbrial genes and concluded

22 that they are involved in cell-cell and cell-biotic surface interactions. In addition, two classes of

26
 ACCEPTED MANUSCRIPT

1 pili, mannose-resistant and mannose-sensitive pili may also play a role in the attachment of

2 bacteria to epithelial cells (Hejazi & Falkiner, 1997).

3 4.1.2 Bacterial invasion and cytotoxic effects

PT
4 Once adhered to the epithelial surface, the pathogen invades into the corneal stroma. This

5 invasion is facilitated via proteases, exotoxins resulting in degradation of basement membrane

RI
6 and extracellular matrix, causing cells to lyse. A number of exotoxins including heat-stable

SC
7 haemolysin, phospholipases, exotoxins play a role in invasion of bacteria into the cornea with

8 eventual stromal necrosis (O’Brien, 2003). Once bacterial invasion into the cornea has ensued,

U
9 the infection progresses rapidly towards melting of cornea facilitated by bacterial proteases,
AN
10 activation of metalloproteases and stimulation of immune response resulting in further damage

11 via release of reactive oxygen intermediates and host proteases (Thidodeaux et al., 2007;
M

12 Marquart & Callaghan, 2013). Proteases contribute to the pathogenesis of keratitis by degrading
D

13 basement membrane, laminin, proteoglycans, collagen, and extracellular matrix (Heck et al.,
TE

14 1986; Twining et al., 1986).

15 P. aeruginosa is capable of secreting at least 7 different proteases including elastase A and

EP

16 B, modified elastase, alkaline protease, protease IV, P. aeruginosa small protease and large

17 exoprotease, some of which play a potential role in the pathogenesis of keratitis (Twining et al.,
C

18 1993; Marquart et al., 2005). Metalloproteases especially elastase B (Las B) and alkaline
AC

19 protease (AP), play a considerable role in keratitis as Las B injection into the corneal stroma

20 result in significant corneal damage (Ijiri et al., 1993; Kessler et al., 1977). Protease IV (PIV) on

21 the other hand cleaves variety of host defense proteins including immunoglobulins, complement

22 components, antimicrobial peptides and surfactants and hence contributes to the virulence of the
27
 ACCEPTED MANUSCRIPT

1 organism (Engel et al., 1998; Alionte et al., 2001). Pseudomonas aeruginosa small protease

2 (PASP) is able to cleave collagen, the chief structural component of the corneal stroma and

3 hence it could play an important role in the obliteration of cornea (Tang et al., 2009). Purified

4 PASP when injected into the rabbit cornea resulted in the destruction of the epithelium and the

PT
5 formation of erosions that can reach into the stroma. These findings suggest that PIV provides

RI
6 bacteria with a defense arm against multiple host defense molecules, while PASP degrads

7 collagen-based structural component of the eye, thereby mediating the pathophysiology of

SC
8 keratitis.

U
9 Mochizuki et al., (2014) determined the role of MucD protease of P. aeruginosa in
AN
10 keratitis in the cornea of mice. The number of bacteria and clinical score in eyes infected with

11 mucD deficient strain was significantly less compared to the parent or rescued strain. In addition
M

12 large number of infiltrating PMN cells were observed in mucD deficient eyes along with higher

13 MIP2 levels. It was concluded that MucD protease of P. aeruginosa suppressed IL-1β, KC and
D

14 MIP2 as well as inhibited neutrophil recruitment in the cornea and hence plays a critical role in
TE

15 P. aeruginosa keratitis by facilitating the evasion of immune response.

EP

16 Karthikeyan et al., (2013), examined the expression of three main exotoxins, ExoS, ExoT

17 and ExoU, in clinical isolates of P. aeruginosa and showed that majority of strains (84%) were
C

18 ExoS+ and ExoT+ but lacked ExoU. However, 5 out of 7 strains that were expressing ExoU
AC

19 were also positive for ExoS which is usually a rare phenotype. These exotoxins are directly

20 injected into the host cell via type III secretion system of P. aeruginosa where they exert their

21 cytotoxic effects onto the host cell (Hauser, 2009). ExoU expressing strains cause rapid cell lysis

28
 ACCEPTED MANUSCRIPT

1 whereas, ExoS strains are invasive in nature causing membrane blebbing within epithelial cells

2 which are utilized as a site for bacterial replication and motility (Fleiszig et al., 1996).

3 The capsular polysaccharide of S. pneumoniae which once was considered the central

PT
4 dogma in pneumococcal virulence is proven to be untrue in the case of keratitis; as noncapsular

5 strains of S. pneumoniae are also able to cause severe keratitis in rabbit keratitis infection models

RI
6 (Norcross et al., 2010; Reed et al., 2005). Other than capsule, the most studied virulence factor

SC
7 of pneumococcus is pneumolysin, a toxin belonging to the family of bacterial cholesterol-

8 dependent cytolysin. It causes direct cell damage by binding to the cholesterol in the host cell

U
9 membrane followed by polymerization into 30-50 mers, thereby forming pores in the host cell
AN
10 membranes (Morgan et al., 1995; Tilley et al., 2005). In addition to forming pores, it initiates

11 immune-derived damage by activating complement system and induces inflammation (Paton et

M

12 al., 1984). Futhermore, reduced corneal virulence was demonstrated by of pneumolysin-deficient

13 strain of S. pneumoniae in a rabbit model of intrastromal infection. It was shown that mutation in
D

14 the complement activation domain of pneumolysin resulted in reduced polymorphonuclear

TE

15 leukocyte (PMN) recruitment and therefore less corneal damage and virulence compared to the

16 wild-type strain (Johnson et al., 1995).

EP

17 Proteins other than pneumolysin, includes choline-binding proteins like pneumococcal

C

18 surface proteins A and C (PspA and PspC), neuraminidase A (NanA) and a variety of other
AC

19 surface-associated proteins such as those involved in adherence, immune evasion, activation and

20 enzymatic reactions have been identified to be involved in non-ocular models of infection

21 Recently a metalloprotease, ZmpC, has been shown to induce ectodomain shedding of the

22 membrane-associated mucin (MAM) from conjunctival and corneal epithelial cells leading to the

29
 ACCEPTED MANUSCRIPT

1 loss of glycocalyx barrier function and enhanced internalization of bacterium. It was concluded

2 that the removal of MAMs may serve to be an important virulence mechanism employed by S.

3 pneumoniae (Govindarajan et al., 2012).

PT
4 The alpha-toxin is a pore-forming lytic cytotoxin produced by nearly all strains of S.

5 aureus. Once it gains entry into the cytoplasmic membrane, it moves laterally until seven

RI
6 subunits combine in a circular arrangement (Menestrina et al., 2001; Pany et al., 2004;

SC
7 Vijayvargia et al., 2004). The individual toxin molecules interact with cavoline-1, thereby

8 forming pores in the membrane. Callegan et al., (1994) studied the role of alpha-toxin in the

U
9 corneal virulence of S. aureus and concluded that it plays a major role in staphylococcal keratitis,
AN
10 causing destruction of corneal tissues in the infected eye. Later, Moreau et al., (1997) showed

11 that even nanogram quantity of purified alpha-toxin causes extensive sloughing of the rabbit
M

12 corneal epithelium, corneal edema and severe iritis. Gamma-toxin, a two component toxin from

13 S. aureus is composed of an F component and an S component that are non-toxic on their own.
D

14 The S component binds to the target cell followed by binding of the F component both of which
TE

15 then move laterally in the cell membrane joining the other F-S pairs to form a ring that penetrates

16 the membrane resulting in cell lysis (Gravet et al., 1998; Szmigieski et al., 1999). The
EP

17 pathogenic role of gamma- and alpha-toxin were determined by Dajcs et al., (2002) who
C

18 illustrated that the virulence of strain, Newman is mediated by both gamma- and alpha-toxin,
AC

19 with alpha-toxin mediating corneal epithelial erosions. The strain deficient in either toxins were

20 considerably less virulent compared to parent or rescued strains. S. aureus secretes several two

21 component toxins each of which has its own S and F components (Gravet et al., 1998). This two

22 component toxin system of S. aureus is complicated by the fact that S component of one toxin

23 can also bind to the F component of the other toxin thereby creating several unique combinations
30
 ACCEPTED MANUSCRIPT

1 with their own specific toxicity. The role of these toxins in corneal virulence is yet to be

2 determined. The protein produced by setnm-1 gene has been shown to cause extensive corneal

3 damage; an attribute mediated by its protease activity and is now considered to be important in

4 the virulence of S. aureus keratitis (Caballero et al., 2008). In addition, mutant deficient in this

PT
5 gene is considerably less virulent compared to its parent or rescued strain (Caballero et al.,

RI
6 2010).

SC
7 Similarly, intra-corneal injections of sub-microgram amounts of Serratia metalloprotease

8 were able to elicit a rapid and extensive corneal tissue damage of rabbit corneas by causing

U
9 liquefactive necrosis and descemetocele formation (Kreger & Griffin, 1975; Lyerly & Kreger,
AN
10 1979). Lyerly et al., (1981) showed that acute inflammation, liquefactive necrosis of rabbit

11 cornea and descemetocele formation occurred after intra-corneal injection of Serratia protease.
M

12 In addition, they also showed the in vitro solubilization of the stromal proteoglycan ground

13 substance. Ultramicroscopic examination of damaged cornea revealed loss of ruthenium red

D

14 staining of the proteoglycan ground substance and dispersal of ultra-structurally normal collagen
TE

15 fibrils. Therefore, the major corneal damage after the injection of Serratia protease is due to the

16 solubilization and loss of ground substance of the tissue and the proteases are at least in part
EP

17 involved in the production of severe corneal damage caused by this bacterium. Kamata et al.,
C

18 (1985) identified a 56-kDa protease as one of the major factors contributing to the pathogenesis
AC

19 and tissue destruction caused by this bacterium. Vaccination of rabbits with purified protease

20 preparations from S. marcescens exhibited significantly less corneal destruction upon corneal

21 encounter with live bacteria, indicating that proteases are important virulence factors during the

22 development of serratial keratitis (Kreger et al., 1986). Marty et al., (2002) showed that mutant

23 strains of S. marcescens that are deficient in the production of 56 kDa metalloprotease are
31
 ACCEPTED MANUSCRIPT

1 considerably less cytotoxic to mammalian cells. It was further revealed that the culture filtrates

2 of wild-type bacteria pre-treated with EDTA or 1,10-phenanthroline (inhibitor of

3 metalloproteases) exhibited significantly reduced cytotoxicity.

PT
4 Gram negative lipopolysaccharide is also an important virulence factor in infectious

5 keratitis. It contributes to the pathogenesis of keratitis by mediating the attachment to the cornea

RI
6 and contact lenses, bacterial internalization and survival inside corneal epithelial cells. In

SC
7 addition, it confers resistance to the complement-mediated killing and stimulates neutrophil

8 migration and infiltration into the cornea with subsequent corneal scarring and opacification.

U
9 Also, exopolysaccharide formation by both gram positive and gram negative bacteria results in
AN
10 local immunosuppressive effects as well as interference with phagocytosis.

11 4.1.3 Stromal necrosis and production of ring infiltrate

M

12 Bacterial exotoxins and proteases are constitutively released during multiplication of

D

13 bacteria. These toxins and enzymes persist in the cornea for a prolonged period of time causing
TE

14 continual stromal destruction and can deprive eye of its vision. Most exotoxins are heat-labile

15 and have antigenic properties. The LPS, an endotoxin within the cell wall of gram negative
EP

16 bacteria is released resulting in the production of stromal rings. These rings consist of

17 polymorphonuclear leucocytes within the corneal stroma, which are chemo-attracted by the
C

18 alternate complement pathway.

AC

19 4.2 Treatment

20 Due to the rapid progression of bacterial keratitis, it should be treated as a medical

21 emergency and empirical antibiotic treatment should be promptly commenced. The objective for

32
 ACCEPTED MANUSCRIPT

1 the initial therapy is the rapid eradication of the corneal pathogen. Basically, no single antibiotic

2 is effective against all bacterial keratitis-causing organism and therefore an agent having broad

3 spectrum activity covering both, gram negative and gram positive organisms is desirable.

4 Broadly speaking, two treatment options are available; fluoroquinolones monotherapy and/or

PT
5 combination therapy of fortified antibiotics including cefazolin and tobramycin or gentamicin

RI
6 (Gokhale, 2008). The aminoglycosides in fortified drops provides excellent coverage against

7 gram-negative organisms and are also active against Staphylococci and some Streptococci.

SC
8 Cephalosporin on the other hand gives good coverage for non-penicillinase producing gram-

9 positive bacteria. Fluoroquinolone antibiotics have a broad spectrum activity against both gram-

10
U
negative and gram-positive organisms including penicillinase-producing and methicillin resistant
AN
11 Staphylococci. They inhibit DNA gyrase and topoisomerase IV; the key enzymes involved in

DNA replication and transcription leading to bactericidal effect of these antibiotics (Wong et al.,
M

12

13 2012; Pestova et al., 2000; Blondeau, 2004).

D

14 The frequency of application of antibiotic drops depends on the severity of infection but
TE

15 usually they are administered every half an hour for the first 24 – 36 hours (O’Brien, 2003;

16 Gokhale, 2008). In severe cases, an initial loading dose is achieved via a drop every 5 min for the
EP

17 first 30 min. The concept behind the loading dose is the vascularity of the cornea and poor
C

18 penetration of the drug into the corneal stroma which requires frequent topical applications to
AC

19 achieve minimal inhibitory concentration. The therapy is then tapered off gradually based on the

20 clinical response of the patient. Other antibiotics used include amikacin, vancomycin,

21 methicillin, clotrimoxazole, and clarithromycin depending on the causative agent responsible for

22 keratitis (Gokhale, 2008).

33
 ACCEPTED MANUSCRIPT

1 Owing to the rich innervation of the cornea, the disease is frequently associated with

2 considerable pain and therefore analgesics or pain control medications are recommended, which

3 result in improved patient comfort and effective delivery of the treatment regimen. The use of

4 corticosteroids as anti-inflammatory agents are controversial and are best avoided until the

PT
5 infection is completely eradicated or at least under control (O’Brien, 2003; Gokhale, 2008). In

RI
6 patients with corneal ulceration the use of non-steroidal anti-inflammatory drugs should also be

7 avoided due to increased risk of corneal melting and perforation. Topical cycloplegics are used

SC
8 to relieve ciliary spasm, pain and formation of synechiae. Secondary glaucoma may result in

9 keratitis due to increased intraocular inflammation and hence must be treated with topical

10 antiglaucoma medications.
U
AN
11 In the case of marked corneal ulceration, the temporary use of therapeutic soft contact
M

12 lenses may facilitate stromal repair and promote re-epithelialization by protecting corneal surface

13 from mechanical trauma. They may act as a tear film antibiotic retention device thereby
D

14 facilitating penetration by prolonging contact time (Matoba & McCulley, 1985; Busin &
TE

15 Spitznas, 1988). In addition, collagen corneal shields soaked in antibiotic solution have also been

16 used as an effective adjunct and have shown to increase antibiotic penetration (O’Brien et al.,
EP

17 1988; Unterman et al., 1988). Furthermore, temporary intracanalicular collagen implants and
C

18 liposomal systems have also been designed to prolong drug retention and interaction (Gilbert et
AC

19 al., 1986; Schaeffer & Krohn, 1982).

20 Therapy for bacterial keratitis is not only to eradicate the causative agent but also to

21 prevent tissue destruction and irreversible structural alterations caused by enzymes and toxins

22 released by the bacteria. Enzyme inhibitors like disodium edetate, acetylcysteine, Heparin have

34
 ACCEPTED MANUSCRIPT

1 been shown to be effective experimentally (Slansky et al., 1971; Ellison & Poirier, 1976; Mehra

2 et al., 1975). Synthetic inhibitors of matrix metalloproteases have been shown to inhibit P.

3 aeruginosa proteases in experimental Pseudomonas keratitis (Barletta et al., 1996). The corneal

4 stroma which constitutes about 90% of corneal thickness is made up of regularly arranged

PT
5 collagen fibrils. Thus the collagenases from bacteria, digest the human collagen causing the

RI
6 cornea to melt. Moreover, collagen cross linking is a useful technique which uses riboflavin and

7 UVA irradiation to strengthen corneal tissues thereby enhancing its rigidity (Spoerl et al., 1998;

SC
8 Spoerl & Seiler, 1999; Wollensak et al., 2003). The interaction between riboflavin and UVA

9 strengthens chemical bond formation between collagen fibrils and hence increasing its resistance

10
U
to enzymatic digestion (Spoerl et al., 2004). In addition, the photoactivation of riboflavin has
AN
11 cidal effect on microorganisms by causing damage to microbial DNA and/or RNA (Wong et al.,

2012).
M

12

13 In case of progressive corneal thinning or perforations, less than 2 mm cyanoacrylate tissue

D

14 adhesives may be used, which may also have some inherit antibacterial activity as well
TE

15 (Eiferman & Snyder, 1983; Gokhale, 2008; O’Brien, 2003; Kirkness et al., 1991). Tissue

16 adhesives are only suitable for small perforations, as they are toxic to corneal endothelium. In
EP

17 case of large perforations, therapeutic penetrating keratoplasty may be required. Corneal patch
C

18 grafting may be an alternative to tissue adhesives and conjunctival flaps may help selected cases
AC

19 of refractory ulceration to assist healing. Severe bacterial keratitis may also result in cataract

20 formation due to bacterial toxins, iridocyclitis and treatment toxicity (Lotti & Dart, 1992).

21 Surgical intervention may be required in such cases depending on the degree of corneal scarring

22 and opacification.

35
 ACCEPTED MANUSCRIPT

1 5 Concluding remarks

2 Microbial keratitis, is a complex entity with many considerations when it comes to its

3 treatment. It is a major public health concern particularly in developing countries where access to

PT
4 care is limited and economic barriers are huge where it can become a leading cause of visual loss

5 in a population that is young. As with all corneal infections, proper identification of the causative

RI
6 agent followed by appropriate targeted therapy can abate the risk of complications. A better

SC
7 understanding of the pathogenic cascade would no doubt lead to improved clinical treatment.

8 However the emergence of drug-resistant strains along with the recurrence of infections

U
9 emphasise on the need for more effective therapeutic modalities. Additionally, the need to
AN
10 identify the genetic basis of virulence factors responsible for the disease is also important, as the

11 pathogenicity of keratitis is a sum of multiple events occurring together in time and space for the
M

12 successful transmission of the pathogen to the susceptible host, overcoming its physiological

13 barriers and eventually producing disease. Subsequently, a complete understanding of

D

14 pathogenesis of the particular organism will undoubtly lead to the identification of potential
TE

15 therapeutic interventions.
EP

61

62 Acknowledgments: This work was supported by Aga Khan University, Pakistan and Sunway
C

63 University, Malaysia.
AC

64 Conflict of Interests: The authors declare that there is no conflict of interests regarding the

65 publication of this paper.

66

36
 ACCEPTED MANUSCRIPT

1 References

2 Abdel-Rhaman MS, Soliman W, Habib F, Fathalla D (2012): A new long-acting liposomal

3 topical antifungal formula: human clinical study. Cornea 31: 126-129.

PT
4 Alizadeh H, Apte S, El-Agha MS, Li L, Hurt M, Howard K, Cavanagh HD, McCulley JP, &

5 Niederkorn JY (2001): Tear IgA and serum IgG antibodies against Acanthamoeba in

RI
6 patients with Acanthamoeba keratitis. Cornea 20: 622-627.

SC
7 Alizadeh H, Pidherney MS, McCulley JP, & Niederkorn JY (1994): Acanthamoeba keratitis. In

8 Ocular infection and immunity. eds Pepose J. S., Holland G. N., Wilhelmus K. R. (Mosby,

9 St. Louis, Mo), 1062–1071.

U
AN
10 Alizadeh H, Pidherney MS, McCulley JP, Niederkorn JY (1994): Apoptosis as a mechanism of
M

11 cytolysis of tumor cells by a pathogenic free-living amoeba. Infect Immun 62: 1298-1303.
D

12 Alizadeh H, Neelam S, Hurt M, Niederkorn JY (2005): Role of contact lens wear, bacterial flora,
TE

13 and mannose-induced pathogenic protease in the pathogenesis of amoebic keratitis. Infect

14 Immun 73: 1061-1068.

EP

15 Alionte LG, Cannon BM, White CD, Caballero AR, O'Callaghan RJ, Hobden JA (2001):

16 Pseudomonas aeruginosa LasA protease and corneal infections. Curr Eye Res 22: 266-271.
C
AC

17 Ansari Z, Miller D, Galor A (2013): Current thoughts in fungal keratitis: diagnosis and

18 treatment. Curr Fungal Infect Rep 7: 209-218.

37
 ACCEPTED MANUSCRIPT

1 Ansorg RA, Knoche ME, Spies AF, Kraus CJ (1984): Differentiation of the major flagellar

2 antigens of Pseudomonas aeruginosa by the slide coagglutination technique. J Clin

3 Microbiol 20: 84-88.

PT
4 Aqeel Y, Siddiqui R, Anwar A, Shah MR, Khoja S, Khan NA (2015): Photochemotherapeutic

5 strategy against Acanthamoeba infections. Antimicrob Agents Chemother 59: 3031-3041.

RI
6 Asbell PA, Sahm DF, Shaw M, Draghi DC, Brown NP (2008): Increasing prevalence of

SC
7 methicillin resistance in serious ocular infections caused by Staphylococcus aureus in the

8 United States: 2000 to 2005. J Cataract Refract Surg 34: 814-818.

9
U
Bang S, Edell E, Eghrari AO, Gottsch JD (2010): Treatment with voriconazole in 3 eyes with
AN
10 resistant Acanthamoeba keratitis. Am J Ophthalmol 149: 66-69.
M

11 Barletta JP, Angella G, Balch KC, Dimova HG, Stern GA, Moser MT, van Setten GB, Schultz

12 GS (1996): Inhibition of pseudomonal ulceration in rabbit corneas by a synthetic matrix

D

13 metalloproteinase inhibitor. Invest Ophthalmol Vis Sci 37: 20-28.

TE

14 Berman MB (1993): Regulation of corneal fibroblast MMP-1 collagenase secretion by plasmin.

EP

15 Cornea 12: 420-432.

16 Bharathi MJ, Ramakrishnan R, Shivakumar C, Meenakshi R, Lionalraj D (2010): Etiology and

C

17 antibacterial susceptibility pattern of community-acquired bacterial ocular infections in a

AC

18 tertiary eye care hospital in south India. Indian J Ophthalmol 58: 497-507.

19 Blondeau JM (2004): Fluoroquinolones: mechanism of action, classification, and development of

20 resistance. Surv Ophthalmol 49 Suppl 2: S73-78.

38
 ACCEPTED MANUSCRIPT

1 Bouchara JP, Tronchin G, Annaix V, Robert R, Senet JM (1990): Laminin receptors on Candida

2 albicans germ tubes. Infect Immun 58: 48-54.

3 Bouchara JP, Sanchez M, Chevailler A, Marot-Leblond A, Lissitzky JC, Tronchin G, Chabasse

PT
4 D (1997): Sialic acid-dependent recognition of laminin and fibrinogen by Aspergillus

5 fumigatus conidia. Infect Immun 65: 2717-2724.

RI
6 Boveland SD, Moore PA, Mysore J, Krunkosky TM, Dietrich UM, Jarrett C, Paige Carmichael

SC
7 K (2010): Immunohistochemical study of matrix metalloproteinases-2 and -9, macrophage

8 inflammatory protein-2 and tissue inhibitors of matrix metalloproteinases-1 and -2 in

U
9 normal, purulonecrotic and fungal infected equine corneas. Vet Ophthalmol 13: 81-90.
AN
10 Bromley IM, Donaldson K (1996): Binding of Aspergillus fumigatus spores to lung epithelial
M

11 cells and basement membrane proteins: relevance to the asthmatic lung. Thorax 51: 1203-

12 1209.
D

13 Busin M, Spitznas M (1988): Sustained gentamicin release by presoaked medicated bandage

TE

14 contact lenses. Ophthalmol 95: 796-798.

EP

15 Caballero AR, McCormick CC, Tang A, O'Callaghan RJ (2008): Isolation and characterization

16 of a new Staphylococcus aureus protease. Invest Ophthalmol Vis Sci 49: 5514.
C

17 Caballero AR, Foster TJ, Monk IR, Tang A, Balzli CL, O'Callaghan RJ (2010): Ocular
AC

18 pathology of a Staphylococcus aureus mutant lacking a recently discovered virulence factor.

19 Invest Ophthalmol Vis Sci 51: 3891.

39
 ACCEPTED MANUSCRIPT

1 Callegan MC, Engel LS, Hill JM, O'Callaghan RJ (1994): Corneal virulence of Staphylococcus

2 aureus: roles of alpha-toxin and protein A in pathogenesis. Infect Immun 62: 2478-2482.

3 Cao Z, Jefferson DM, Panjwani N (1998): Role of carbohydrate-mediated adherence in

PT
4 cytopathogenic mechanisms of Acanthamoeba. J Biol Chem 273: 15838-15845.

5 Chen WL, Wu CY, Hu FR, Wang IJ (2004): Therapeutic penetrating keratoplasty for microbial

RI
6 keratitis in Taiwan from 1987 to 2001. Am J Ophthalmol 137: 736-743.

SC
7 Cheung J, Slomovic AR (1995): Microbial etiology and predisposing factors among patients

8 hospitalized for corneal ulceration. Can J Ophthalmol 30: 251-255.

U
AN
9 Cho JH, Na BK, Kim TS, Song CY (2000): Purification and characterization of an extracellular

10 serine proteinase from Acanthamoeba castellanii. IUBMB Life 50: 209-214.

M

11 Clarke B, Sinha A, Parmar DN, Sykakis E (2012): Advances in the diagnosis and treatment of
D

12 Acanthamoeba keratitis. J Ophthalmol Article ID: 484892.

TE

13 Clarke DW, Niederkorn JY (2006): The pathophysiology of Acanthamoeba keratitis. Trends

14 Parasitol 22: 175-180.

EP

15 Clarke DW, Alizadeh H, Niederkorn JY (2005): Failure of Acanthamoeba castellanii to produce

C

16 intraocular infections. Invest Ophthalmol Vis Sci 46: 2472-2478.

AC

17 Cosar CB1, Cohen EJ, Rapuano CJ, Laibson PR (2001): Clear corneal wound infection after

18 phacoemulsification. Arch Ophthalmol 119: 1755-1759.

19 Coster DJ, Badenoch PR (1987): Host, microbial, and pharmacological factors affecting the

20 outcome of suppurative keratitis. Br J Ophthalmol 71: 96-101.

40
 ACCEPTED MANUSCRIPT

1 Costerton JW (1980). Pseudomonas aeruginosa in nature and diseases. In: Sabath LD (ed.).

2 Pseudomonas aeruginosa: The Organisms, The Disease it Causes and their treatment. Hans

3 Huber: Bern, 1980.

PT
4 Coulot P, Bouchara JP, Renier G, Annaix V, Planchenault C, Tronchin G, Chabasse D (1994):

5 Specific interaction of Aspergillus fumigatus with fibrinogen and its role in cell adhesion.

RI
6 Infect Immun 62: 2169-2177.

SC
7 Dajcs JJ, Austin MS, Sloop GD, Moreau JM, Hume EB, Thompson HW, McAleese FM, Foster

8 TJ, O'Callaghan RJ (2002): Corneal pathogenesis of Staphylococcus aureus strain Newman.

U
9 Invest Ophthalmol Vis Sci 43: 1109-1115.
AN
10 Dart JK, Saw VP, Kilvington S (2009): Acanthamoeba Keratitis: Diagnosis and Treatment
M

11 Update 2009. Am J Ophthalmol 148: 487-499.

12 De Jonckheere JF (1991): Ecology of Acanthamoeba. Rev Infect Dis 13 Suppl 5: S385-7.

D
TE

13 Dudley R, Alsam S, Khan NA (2008): The role of proteases in the differentiation of

14 Acanthamoeba castellanii. FEMS Microbiol Lett 286: 9-15.

EP

15 Duguid IG, Dart JK, Morlet N, Allan BD, Matheson M, Ficker L, Tuft S (1997): Outcome of

16 Acanthamoeba keratitis treated with polyhexamethyl biguanide and propamidine.

C

17 Ophthalmol 104: 1587-1592.

AC

18 Dutta D1, Cole N, Willcox M (2012): Factors influencing bacterial adhesion to contact lenses.

19 Mol Vis 18: 14-21.

41
 ACCEPTED MANUSCRIPT

1 Eiferman RA, Snyder JW (1983): Antibacterial effect of cyanoacrylate glue. Arch Ophthalmol

2 101: 958-960.

3 Elder MJ, Kilvington S, Dart JK (1994): A clinicopathologic study of in vitro sensitivity testing

PT
4 and Acanthamoeba keratitis. Invest Ophthalmol Vis Sci 35: 1059-1064.

5 Ellison A, Poirier R (1976): Therapeutic effects of heparin on Pseudomonas-induced corneal

RI
6 ulceration. Am J Ophthalmol 82: 619-627.

SC
7 Engel LS, Hill JM, Moreau JM, Green LC, Hobden JA, O'Callaghan RJ (1998): Pseudomonas

8 aeruginosa protease IV produces corneal damage and contributes to bacterial virulence.

9 Invest Ophthalmol Vis Sci 39: 662-665.

U
AN
10 Fitzsimons R, Peters AL (1986): Miconazole and ketoconazole as a satisfactory first-line
M

11 treatment for keratomycosis. Am J Ophthalmol 101: 605-608.

D

12 Fleiszig SM, Zaidi TS, Preston MJ, Grout M, Evans DJ, Pier GB (1996): Relationship between
TE

13 cytotoxicity and corneal epithelial cell invasion by clinical isolates of Pseudomonas

14 aeruginosa. Infect Immun 64: 2288-2294.

EP

15 Garate M, Cao Z, Bateman E, Panjwani N (2004): Cloning and characterization of a novel

16 mannose-binding protein of Acanthamoeba. J Biol Chem 279: 29849-29856.

C
AC

17 Garduño-Vieyra L, Gonzalez-Sanchez CR, Hernandez-Da Mota SE (2011): Ultraviolet-a light

18 and riboflavin therapy for Acanthamoeba keratitis: a case report. Ophthalmol 118: 291-295.

19 Garg P, Chaurasia S, Vaddavalli PK, Muralidhar R, Mittal V, Gopinathan U (2010): Microbial

20 keratitis after LASIK. J Refract Surg 26: 209-216.

42
 ACCEPTED MANUSCRIPT

1 Gilbert ML, Wilhelmus KR, Osato MS (1986): Intracanalicular collagen implants enhance

2 topical antibiotic bioavailability. Cornea 5: 167-171.

3 Gokhale NS (2008): Medical management approach to infectious keratitis. Indian J Ophthalmol

PT
4 56: 215-220.

5 Gopinathan U, Ramakrishna T, Willcox M, Rao CM, Balasubramanian D, Kulkarni A,

RI
6 Vemuganti GK, Rao GN (2001): Enzymatic, clinical and histologic evaluation of corneal

SC
7 tissues in experimental fungal keratitis in rabbits. Exp Eye Res 72: 433-442.

8 Gopinathan U, Sharma S, Garg P, Rao GN (2009): Review of epidemiological features,

9
U
microbiological diagnosis and treatment outcome of microbial keratitis: experience of over a
AN
10 decade. Indian J Ophthalmol 57: 273-279.
M

11 Govindarajan B, Menon BB, Spurr-Michaud S, Rastogi K, Gilmore MS, Argüeso P, Gipson IK

12 (2012): A metalloproteinase secreted by Streptococcus pneumoniae removes membrane

D

13 mucin MUC16 from the epithelial glycocalyx barrier. PLoS One 7: e32418.
TE

14 Gravet A, Colin DA, Keller D, Girardot R, Monteil H, Prévost G (1998): Characterization of a

EP

15 novel structural member, LukE-LukD, of the bi-component staphylococcal leucotoxins

16 family. FEBS Lett 436: 202-208.

C

17 Green M, Apel A, Stapleton F (2008): Risk factors and causative organisms in microbial
AC

18 keratitis. Cornea 27: 22-27.

19 Habib FS, Fouad EA, Abdel-Rhaman MS, Fathalla D (2010): Liposomes as an ocular delivery

20 system of fluconazole: in-vitro studies. Acta Ophthalmol 88: 901-904.

43
 ACCEPTED MANUSCRIPT

1 Hadas E, Mazur T (1993): Proteolytic enzymes of pathogenic and non-pathogenic strains of

2 Acanthamoeba spp. Trop Med Parasitol 44: 197-200.

3 Hamilton AJ, Jeavons L, Youngchim S, Vanittanakom N, Hay RJ (1998): Sialic acid-dependent

PT
4 recognition of laminin by Penicillium marneffei conidia. Infect Immun 66: 6024-6026.

5 Hammerschmidt S (2006): Adherence molecules of pathogenic pneumococci. Curr Opin

RI
6 Microbiol 9: 12-20.

SC
7 Hariprasad SM, Mieler WF, Lin TK, Sponsel WE, Graybill JR (2008): Voriconazole in the

8 treatment of fungal eye infections: a review of current literature. Br J Ophthalmol 92: 871-

9 878.
U
AN
10 Hay J, Kirkness CM, Seal DV, Wright P (1994): Drug resistance and Acanthamoeba keratitis:
M

11 the quest for alternative antiprotozoal chemotherapy. Eye (Lond). 8: 555-563.

D

12 Hazlett LD, Zucker M, Berk RS (1992): Distribution and kinetics of the inflammatory cell
TE

13 response to ocular challenge with Pseudomonas aeruginosa in susceptible versus resistant

14 mice. Ophthalmic Res 24: 32-39.

EP

15 Hazlett LD (1995): Analysis of ocular microbial adhesion. Methods Enzymol 253: 53-66.
C

16 He YG, Niederkorn JY, McCulley JP, Stewart GL, Meyer DR, Silvany R, Dougherty J (1990):
AC

17 In vivo and in vitro collagenolytic activity of Acanthamoeba castellanii. Invest Ophthalmol

18 Vis Sci 31: 2235-2240.

19 Heck LW, Morihara K, McRae WB, Miller EJ (1986): Specific cleavage of human type III and

20 IV collagens by Pseudomonas aeruginosa elastase. Infect Immun 51: 115-118.

44
 ACCEPTED MANUSCRIPT

1 Hejazi A, Falkiner FR (1997): Serratia marcescens. J Med Microbiol 46: 903-912.

2 Hostetter MK (1994): Adhesins and ligands involved in the interaction of Candida spp. with

3 epithelial and endothelial surfaces. Clin Microbiol Rev 7: 29-42.

PT
4 Hume EB, Zhu H, Cole N, Huynh C, Lam S, Willcox MD (2007): Efficacy of contact lens

5 multipurpose solutions against serratia marcescens. Optom Vis Sci 84: 316-320.

RI
6 Hurt M, Niederkorn J, Alizadeh H (2003): Effects of mannose on Acanthamoeba castellanii

SC
7 proliferation and cytolytic ability to corneal epithelial cells. Invest Ophthalmol Vis Sci 44:

8 3424-3431.

U
AN
9 Hurt M, Neelam S, Niederkorn J, Alizadeh H (2003): Pathogenic Acanthamoeba spp. secrete a

10 mannose-induced cytolytic protein that correlates with the ability to cause disease. Infect
M

11 Immun 71: 6243-6255.

D

12 Hyndiuk RA (1981): Experimental Pseudomonas keratitis: I. Sequential electron microscopy. II.

TE

13 Comparative therapy trials. Trans Am Ophthalmol Soc 79: 541.

14 Ijiri Y1, Yamamoto T, Kamata R, Aoki H, Matsumoto K, Okamura R, Kambara T (1993): The
EP

15 role of Pseudomonas aeruginosa elastase in corneal ring abscess formation in pseudomonal

16 keratitis. Graefes Arch Clin Exp Ophthalmol 231: 521-528.

C
AC

17 Illingworth CD, Cook SD (1998): Acanthamoeba keratitis. Surv Ophthalmol 42: 493-508.

18 Imwidthaya P (1995): Mycotic keratitis in Thailand. J Med Vet Mycol 33: 81-82.

19 Ismaiel AA, Rabie GH, Kenawey SE, Abd El-Aal MA (2012): Efficacy of aqueous garlic extract

20 on growth, aflatoxin B1 production, and cyto-morphological aberrations of Aspergillus

45
 ACCEPTED MANUSCRIPT

1 flavus, causing human ophthalmic infection: topical treatment of A. flavus keratitis. Braz J

2 Microbiol 43: 1355-1364.

3 Jackson BE, Wilhelmus KR, Hube B (2007): The role of secreted aspartyl proteinases in

PT
4 Candida albicans keratitis. Invest Ophthalmol Vis Sci 48: 3559-3565.

5 Jain V, Maiti A, Shome D, Borse N, Natarajan S (2007): Aspergillus-induced malignant

RI
6 glaucoma. Cornea 26: 762-763.

SC
7 Jeng BH, Gritz DC, Kumar AB, Holsclaw DS, Porco TC, Smith SD, Whitcher JP, Margolis TP,

8 Wong IG (2010): Epidemiology of ulcerative keratitis in Northern California. Arch

9 Ophthalmol 128: 1022-1028.

U
AN
10 Jett BD, Gilmore MS (2002): Host-parasite interactions in Staphylococcus aureus keratitis. DNA
M

11 Cell Biol 21: 397-404.

D

12 Jiang C, Sun X, Wang Z, Zhang Y (2006): Acanthamoeba keratitis: clinical characteristics and
TE

13 management. Ophthalmol 113: 412-416.

14 Johnson MK, Callegan MC, Engel LS, O'Callaghan RJ, Hill JM, Hobden JA, Boulnois GJ,
EP

15 Andrew PW, Mitchell TJ (1995): Growth and virulence of a complement-activation-

16 negative mutant of Streptococcus pneumoniae in the rabbit cornea. Curr Eye Res 14: 281-
C

17 284.
AC

18 Jones DB (1981): Decision-making in the management of microbial keratitis. Ophthalmol 88:

19 814-820.

46
 ACCEPTED MANUSCRIPT

1 Kamata R, Matsumoto K, Okamura R, Yamamoto T, Maeda H (1985): The serratial 56K

2 protease as a major pathogenic factor in serratial keratitis. Clinical and experimental study.

3 Ophthalmol 92: 1452-1459.

PT
4 Kandori M, Inoue T, Shimabukuro M, Hayashi H, Hori Y, Maeda N, Tano Y (2010): Four cases

5 of Acanthamoeba keratitis treated with phototherapeutic keratectomy. Cornea 29: 1199-

RI
6 1202.

SC
7 Kant S, Srivastava D, Misra RN (1984): Toxic effects of aflatoxins on eyes--an experimental

8 clinico-histopathological evaluation. Indian J Ophthalmol 32: 424-426.

9
U
Karthikeyan RS, Priya JL, Leal SM Jr, Toska J, Rietsch A, Prajna V, Pearlman E, Lalitha P
AN
10 (2013): Host response and bacterial virulence factor expression in Pseudomonas aeruginosa
M

11 and Streptococcus pneumoniae corneal ulcers. PLoS One 8: e64867.

12 Kessler E, Kennah HE, Brown SI (1977): Pseudomonas protease. Purification, partial

D

13 characterization, and its effect on collagen, proteoglycan, and rabbit corneas. Invest
TE

14 Ophthalmol Vis Sci 16: 488-497.

EP

15 Khan NA (2001): Pathogenicity, morphology, and differentiation of Acanthamoeba. Curr

16 Microbiol 43: 391-395.

C

17 Khan NA (2003): Pathogenesis of Acanthamoeba infections. Microb Pathog 34: 277-285.

AC

18 Khan NA, Jarroll EL, Panjwani N, Cao Z, Paget TA (2000): Proteases as markers for

19 differentiation of pathogenic and nonpathogenic species of Acanthamoeba. J Clin Microbiol

20 38: 2858-2861.

47
 ACCEPTED MANUSCRIPT

1 Khan YA, Kashiwabuchi RT, Martins SA, Castro-Combs JM, Kalyani S, Stanley P, Flikier D,

2 Behrens A (2011): Riboflavin and ultraviolet light a therapy as an adjuvant treatment for

3 medically refractive Acanthamoeba keratitis: report of 3 cases. Opthalmol 118: 324-331.

PT
4 Kirkness CM, Ficker LA, Steele AD, Rice NS (1991): The role of penetrating keratoplasty in the

5 management of microbial keratitis. Eye (Lond) 5 ( Pt 4): 425-431.

RI
6 Kiryu H1, Yoshida S, Suenaga Y, Asahi M (1991): Invasion and survival of Fusarium solani in

SC
7 the dexamethasone-treated cornea of rabbits. J Med Vet Mycol 29: 395-406.

8 Kreger AS, Griffin OK (1975): Cornea-damaging proteases of Serratia marcescens. Invest

9 Ophthalmol 14: 190-198.

U
AN
10 Kreger AS, Lyerly DM, Hazlett LD, Berk RS (1986): Immunization against experimental
M

11 Pseudomonas aeruginosa and Serratia marcescens keratitis. Vaccination with

12 lipopolysaccharide endotoxins and proteases. Invest Ophthalmol Vis Sci 27: 932-939.
D
TE

13 Kuriakose T, Thomas PA (1991): Keratomycotic malignant glaucoma. Indian J Ophthalmol 39:

14 118-121.
EP

15 Labbate M, Zhu H, Thung L, Bandara R, Larsen MR, Willcox MD, Givskov M, Rice SA,

16 Kjelleberg S (2007): Quorum-sensing regulation of adhesion in Serratia marcescens MG1 is

C

17 surface dependent. J Bacteriol 189: 2702-2711.

AC

18 Lalitha P, Shapiro BL, Srinivasan M, Prajna NV, Acharya NR, Fothergill AW, Ruiz J,

19 Chidambaram JD, Maxey KJ, Hong KC, McLeod SD, Lietman TM (2007): Antimicrobial

48
 ACCEPTED MANUSCRIPT

1 susceptibility of Fusarium, Aspergillus, and other filamentous fungi isolated from keratitis.

2 Arch Ophthalmol 125: 789-793.

3 Leck AK, Thomas PA, Hagan M, Kaliamurthy J, Ackuaku E, John M, Newman MJ, Codjoe FS,

PT
4 Opintan JA, Kalavathy CM, Essuman V, Jesudasan CA, Johnson GJ (2002): Aetiology of

5 suppurative corneal ulcers in Ghana and south India, and epidemiology of fungal keratitis.

RI
6 Br J Ophthalmol 86: 1211-1215.

SC
7 Leema G, Kaliamurthy J, Geraldine P, Thomas PA (2010): Keratitis due to Aspergillus flavus:

8 clinical profile, molecular identification of fungal strains and detection of aflatoxin

U
9 production. Mol Vis 16: 843-854.
AN
10 Li Z, Jhanji V, Tao X, Yu H, Chen W, Mu G (2013): Riboflavin/ultravoilet light-mediated
M

11 crosslinking for fungal keratitis. Br J Ophthalmol 97: 669-671.

12 Liesegang TJ (1998): Conjunctival changes associated with glaucoma therapy: implications for
D

13 the external disease consultant and the treatment of glaucoma. Cornea 17: 574-583.
TE

14 Lifshitz T, Levy J, Klemperer I (2005): Bacterial keratitis after laser subepithelial keratectomy. J
EP

15 Refract Surg 21: 94-96.

16 Liu X, Hazlett L, Berk R (1990): Systemic and topical protection studies using Pseudomonas
C

17 flagella or pili. Invest Ophthalmol Vis Sci 31: 449.

AC

18 Lorenzo-Morales J, Martín-Navarro CM, López-Arencibia A, Arnalich-Montiel F, Piñero JE,

19 Valladares B (2013): Acanthamoeba keratitis: an emerging disease gathering importance

20 worldwide? Trends Parasitol 29: 181-187.

49
 ACCEPTED MANUSCRIPT

1 Lotti R, Dart JK (1992): Cataract as a complication of severe microbial keratitis. Eye (Lond) 6 (

2 Pt 4): 400-403.

3 Lyerly D, Kreger A (1979): Purification and characterization of a Serratia marcescens

PT
4 metalloprotease. Infect Immun 24: 411-421.

5 Lyerly D, Gray L, Kreger A (1981): Characterization of rabbit corneal damage produced by

RI
6 Serratia keratitis and by a serratia protease. Infect Immun 33: 927-932.

SC
7 Mahgoub AMA (2010): Acanthamoeba keratitis. Parasitologist Unit J 3: 9-18.

U
8 Marciano-Cabral F, Cabral G (2003): Acanthamoeba spp. as agents of disease in humans. Clin
AN
9 Microbiol Rev 16: 273-307.

10 Marangon FB, Miller D, Giaconi JA, Alfonso EC (2004): In vitro investigation of voriconazole
M

11 susceptibility for keratitis and endophthalmitis fungal pathogens. Am J Ophthalmol 137:

D

12 820-825.
TE

13 Marquart ME, Caballero AR, Chomnawang M, Thibodeaux BA, Twining SS, O'Callaghan RJ

14 (2005): Identification of a novel secreted protease from Pseudomonas aeruginosa that

EP

15 causes corneal erosions. Invest Ophthalmol Vis Sci 46: 3761-3768.

C

16 Marquart ME, O'Callaghan RJ (2013): Infectious keratitis: secreted bacterial proteins that
AC

17 mediate corneal damage. J Ophthalmol ID: 369094.

18 Marty KB, Williams CL, Guynn LJ, Benedik MJ, Blanke SR (2002): Characterization of a

19 cytotoxic factor in culture filtrates of Serratia marcescens. Infect Immun 70: 1121-1128.

50
 ACCEPTED MANUSCRIPT

1 Matoba AY, McCulley JP (1985): The effect of therapeutic soft contact lenses on antibiotic

2 delivery to the cornea. Ophthalmol 92: 97-99.

3 Mehra KS, Singh R, Bhatia RP, Sen PC, Singh H (1975): Lysozyme in corneal ulcer. Ann

PT
4 Ophthalmol 7: 1470-1472.

5 Menestrina G, Serra MD, Prévost G (2001): Mode of action of beta-barrel pore-forming toxins of

RI
6 the staphylococcal alpha-hemolysin family. Toxicon 39: 1661-1672.

SC
7 Mitra MM, Alizadeh H, Gerard RD, Niederkorn JY (1995): Characterization of a plasminogen

8 activator produced by Acanthamoeba castellanii. Mol Biochem Parasitol 73: 157-164.

U
AN
9 Mochizuki Y, Suzuki T, Oka N, Zhang Y, Hayashi Y, Hayashi N, Gotoh N, Ohashi Y (2014):

10 Pseudomonas aeruginosa MucD protease mediates keratitis by inhibiting neutrophil

M

11 recruitment and promoting bacterial survival. Invest Ophthalmol Vis Sci 55: 240-246.
D

12 Mohammadinia M, Rahmani S, Eslami G, Ghassemi-Broumand M, Aghazadh Amiri M, Aghaie

TE

13 G, Tabatabaee SM, Taheri S, Behgozin A (2012): Contact lens disinfecting solutions

14 antibacterial efficacy: comparison between clinical isolates and the standard ISO ATCC
EP

15 strains of Pseudomonas aeruginosa and Staphylococcus aureus. Eye (Lond) 26: 327-330.

16 Moon EK, Kim SH, Hong Y, Chung DI, Goo YK, Kong HH (2015): Autophagy inhibitors as a
C

17 potential anti-amoebic treatment for Acanthamoeba keratitis. Antimicrob Agents Chemother

AC

18 59: 4020-4045.

19 Moreau JM, Sloop GD, Engel LS, Hill JM, O'Callaghan RJ (1997): Histopathological studies of

20 staphylococcal alpha-toxin: effects on rabbit corneas. Curr Eye Res 16: 1221-1228.

51
 ACCEPTED MANUSCRIPT

1 Morgan PJ, Hyman SC, Rowe AJ, Mitchell TJ, Andrew PW, Saibil HR (1995): Subunit

2 organisation and symmetry of pore-forming, oligomeric pneumolysin. FEBS Lett 371: 77-

3 80.

PT
4 Mulet ME, Pérez-Santonja JJ, Ferrer C, Alió JL (2010): Microbial keratitis after intrastromal

5 corneal ring segment implantation. J Refract Surg 26: 364-369.

RI
6 Müller L, Thiel MA, Kipfer-Kauer AI, Kaufmann C (2012): Corneal cross-linking as

SC
7 supplementary treatment option in melting keratitis: a case series. Klin Monbl Augenheilkd

8 229: 411-415.

9
U
Musa F, Tailor R, Gao A, Hutley E, Rauz S, Scott RA (2010): Contact lens-related microbial
AN
10 keratitis in deployed British military personnel. Br J Ophthalmol 94: 988-993.
M

11 Naginton J, Watson PG, Playfair TJ, McGill J, Jones BR, Steele AD (1974): Amoebic infection

12 of the eye. Lancet 2(7896): 1537-1540.

D
TE

13 Nath R1, Baruah S, Saikia L, Devi B, Borthakur AK, Mahanta J (2011): Mycotic corneal ulcers

14 in upper Assam. Indian J Ophthalmol 59: 367-371.

EP

15 Niederkorn JY, Alizadeh H, Leher H, McCulley JP (1999): The pathogenesis of Acanthamoeba

16 keratitis. Microbes Infect 1: 437-443.

C
AC

17 Norcross EW, Tullos NA, Taylor SD, Sanders ME, Marquart ME (2010): Assessment of

18 Streptococcus pneumoniae capsule in conjunctivitis and keratitis in vivo neuraminidase

19 activity increases in nonencapsulated pneumococci following conjunctival infection. Curr

20 Eye Res 35: 787-798.

52
 ACCEPTED MANUSCRIPT

1 O'Brien TP, Sawusch MR, Dick JD, Gottsch JD (1988): Topical ciprofloxacin treatment of

2 Pseudomonas keratitis in rabbits. Arch Ophthalmol 106: 1444-1446.

3 O'Brien TP (2003): Management of bacterial keratitis: beyond exorcism towards consideration of

PT
4 organism and host factors. Eye (Lond) 17: 957-974.

5 Ollivier FJ, Brooks DE, Van Setten GB, Schultz GS, Gelatt KN, Stevens GR, Blalock TD,

RI
6 Andrew SE, Komaromy AM, Lassaline ME, Kallberg ME, Cutler TJ (2004): Profiles of

SC
7 matrix metalloproteinase activity in equine tear fluid during corneal healing in 10 horses

8 with ulcerative keratitis. Vet Ophthalmol 7: 397-405. Ormerod LD, Hertzmark E, Gomez

U
9 DS, Stabiner RG, Schanzlin DJ, Smith RE (1987): Epidemiology of microbial keratitis in
AN
10 southern California. A multivariate analysis. Ophthalmol 94: 1322-1333.
M

11 Palmer ML, Hyndiuk RA (1993): Contact lens-related infectious keratitis. Int Ophthalmol Clin

12 33: 23-49.
D

13 Panda A, Sharma N, Angra SK (1996): Topical fluconazole therapy of Candida keratitis. Cornea
TE

14 15: 373-375.
EP

15 Panjwani N (2010): Pathogenesis of Acanthamoeba keratitis. Ocul Surf 8: 70-79.

16 Pany S, Vijayvargia R, Krishnasastry MV (2004): Caveolin-1 binding motif of alpha-hemolysin:

C

17 its role in stability and pore formation. Biochem Biophys Res Commun 322: 29-36.
AC

18 Parks WC (1999): Matrix metalloproteinases in repair. Wound Repair Regen 7: 423-432.

53
 ACCEPTED MANUSCRIPT

1 Parment PA, Colucci B, Nyström B (1996): The efficacy of soft contact lens disinfection

2 solutions against Serratia marcescens and Pseudomonas aeruginosa. Acta Ophthalmol

3 Scand 74: 235-237.

PT
4 Parment PA (1997): The role of Serratia marcescens in soft contact lens associated ocular

5 infections. A review. Acta Ophthalmol Scand 75: 67-71.

RI
6 Parta M, Chang Y, Rulong S, Pinto-DaSilva P, Kwon-Chung KJ (1994): HYP1, a hydrophobin

SC
7 gene from Aspergillus fumigatus, complements the rodletless phenotype in Aspergillus

8 nidulans. Infect Immun 62: 4389-4395.

9
U
Parthasarathy A, Tan DT (2007): Deep lamellar keratoplasty for Acanthamoeba keratitis. Cornea
AN
10 26: 1021-1023.
M

11 Paton JC, Rowan-Kelly B, Ferrante A (1984): Activation of human complement by the

12 pneumococcal toxin pneumolysin. Infect Immun 43: 1085-1087.

D
TE

13 Perrine D, Chenu JP, Georges P, Lancelot JC, Saturnino C, Robba M (1995): Amoebicidal

14 efficiencies of various diamidines against two strains of Acanthamoeba polyphaga.

EP

15 Antimicrob Agents Chemother 39: 339-342.

16 Pestova E, Beyer R, Cianciotto NP, Noskin GA, Peterson LR (1999): Contribution of

C

17 topoisomerase IV and DNA gyrase mutations in Streptococcus pneumoniae to resistance to

AC

18 novel fluoroquinolones. Antimicrob Agents Chemother 43: 2000-2004.

54
 ACCEPTED MANUSCRIPT

1 Pettit DA, Williamson J, Cabral GA, Marciano-Cabral F (1996): In vitro destruction of nerve cell

2 cultures by Acanthamoeba spp.: a transmission and scanning electron microscopy study. J

3 Parasitol 82: 769-777.

PT
4 Pidherney MS, Alizadeh H, Stewart GL, McCulley JP, Niederkorn JY (1993): In vitro and in

5 vivo tumoricidal properties of a pathogenic/free-living amoeba. Cancer Lett 72: 91-98.

RI
6 Prakash G, Sharma N, Goel M, Titiyal JS, Vajpayee RB (2008): Evaluation of intrastromal

SC
7 injection of voriconazole as a therapeutic adjunctive for the management of deep recalcitrant

8 fungal keratitis. Am J Ophthalmol 146: 56-59.

9
U
Price MO, Price FW Jr, Maclellan D (2005): Effect of gatifloxacin 0.3% and moxifloxacin 0.5%
AN
10 ophthalmic solutions on human corneal epithelium following 2 dosing regimens. J Cataract
M

11 Refract Surg 31: 2137-2141.

12 Radford CF, Lehmann OJ, Dat JK (1998): Acanthamoeba keratitis: multicentre survey in
D

13 England 1992-6. National Acanthamoeba keratitis Study Group. Br J Ophthalmol 82: 1387-
TE

14 1392.
EP

15 Rajam G, Anderton JM, Carlone GM, Sampson JS, Ades EW (2008): Pneumococcal surface

16 adhesin A (PsaA): a review. Crit Rev Microbiol 34: 163-173.

C

17 Rao SK, Madhavan HN, Rao G, Padmanabhan P (1997): Fluconazole in filamentous fungal
AC

18 keratitis. Cornea 16: 700.

55
 ACCEPTED MANUSCRIPT

1 Ramirez JC, Fleiszig SM, Sullivan AB, Tam C, Borazjani R, Evans DJ (2012): Traversal of

2 multilayered corneal epithelia by cytotoxic Pseudomonas aeruginosa requires the

3 phospholipase domain of exoU. Invest Ophthalmol Vis Sci 53: 448-453.

PT
4 Rasool BK, Salmo HM (2012): Development and clinical evaluation of clotrimazole-β-

5 cyclodextrin eyedrops for the treatment of fungal keratitis. AAPS PharmSciTech 13: 883-

RI
6 889.

SC
7 Raza SK1, Mallet AI, Howell SA, Thomas PA (1994): An in-vitro study of the sterol content and

8 toxin production of Fusarium isolates from mycotic keratitis. J Med Microbiol 41: 204-208.

9
U
Reddy JC, Tibbetts MD, Hammersmith KM, Nagra PK, Rapuano CJ (2013): Successful
AN
10 management of Burkholderia cepacia keratitis after LASIK. J Refract Surg 29: 8-9.
M

11 Reed JM, O'Callaghan RJ, Girgis DO, McCormick CC, Caballero AR, Marquart ME (2005):

12 Ocular virulence of capsule-deficient Streptococcus pneumoniae in a rabbit keratitis model.

D

13 Invest Ophthalmol Vis Sci 46: 604-608.

TE

14 Rehany U, Balut G, Lefler E, Rumelt S (2004): The prevalence and risk factors for donor corneal
EP

15 button contamination and its association with ocular infection after transplantation. Cornea

16 23: 649-654.
C

17 Rhem MN, Lech EM, Patti JM, McDevitt D, Höök M, Jones DB, Wilhelmus KR (2000): The
AC

18 collagen-binding adhesin is a virulence factor in Staphylococcus aureus keratitis. Infect

19 Immun 68: 3776-3779.

56
 ACCEPTED MANUSCRIPT

1 Romero-Steiner S, Pilishvili T, Sampson JS, Johnson SE, Stinson A, Carlone GM, Ades EW

2 (2003): Inhibition of pneumococcal adherence to human nasopharyngeal epithelial cells by

3 anti-PsaA antibodies. Clin Diagn Lab Immunol 10: 246-251.

PT
4 Rosa RH Jr, Miller D, Alfonso EC (1994): The changing spectrum of fungal keratitis in south

5 Florida. Ophthalmol 101: 1005-1013.

RI
6 Rudner XL, Zheng Z, Berk RS, Irvin RT, Hazlett LD (1992): Corneal epithelial glycoproteins

SC
7 exhibit Pseudomonas aeruginosa pilus binding activity. Invest Ophthalmol Vis Sci 33: 2185-

8 2193.

9
U
Samples JR, Binder PS, Luibel FJ, Font RL, Visvesvara GS, Peter CR (1984): Acanthamoeba
AN
10 keratitis possibly acquired from a hot tub. Arch Ophthalmol 102: 707-710.
M

11 Schaeffer HE, Krohn DL (1982): Liposomes in topical drug delivery. Invest Ophthalmol Vis Sci

12 22: 220-227.
D
TE

13 Schein OD, Ormerod LD, Barraquer E, Alfonso E, Egan KM, Paton BG, Kenyon KR (1989):

14 Microbiology of contact lens-related keratitis. Cornea 8: 281-285.

EP

15 Scholtmeijer K, Wessels JG, Wösten HA (2001): Fungal hydrophobins in medical and technical

16 applications. Appl Microbiol Biotechnol 56: 1-8.

C
AC

17 Shanks RM, Stella NA, Kalivoda EJ, Doe MR, O'Dee DM, Lathrop KL, Guo FL, Nau GJ

18 (2007): A Serratia marcescens OxyR homolog mediates surface attachment and biofilm

19 formation. J Bacteriol 189: 7262-7272.

57
 ACCEPTED MANUSCRIPT

1 Sharma DP, Sharma S, Wilkins MR (2011): Microbial keratitis after corneal laser refractive

2 surgery. Future Microbiol 6: 819-831.

3 Siddiqui R, Khan NA (2012): Biology and pathogenesis of Acanthamoeba. Parasit Vectors 5: 6.

PT
4 Slansky HH, Dohlman CH, Berman MB (1971): Prevention of corneal ulcers. Trans Am Acad

5 Ophthalmol Otolaryngol 75: 1208-1211.

RI
6 Sony P, Sharma N, Vajpayee RB, Ray M (2002): Therapeutic keratoplasty for infectious

SC
7 keratitis: a review of the literature. CLAO J 28: 111-118.

U
8 Speaker MG, Milch FA, Shah MK, Eisner W, Kreiswirth BN (1991): Role of external bacterial
AN
9 flora in the pathogenesis of acute postoperative endophthalmitis. Ophthalmol 98: 639-649.

10 Spoerl E, Huhle M, Seiler T (1998): Induction of cross-links in corneal tissue. Exp Eye Res 66:
M

11 97-103.
D

12 Spoerl E, Seiler T (1999): Techniques for stiffening the cornea. J Refract Surg 15: 711-713.
TE

13 Spoerl E, Wollensak G, Seiler T (2004): Increased resistance of crosslinked cornea against

EP

14 enzymatic digestion. Curr Eye Res 29: 35-40.

15 Srinivasan M (2004): Fungal keratitis. Curr Opin Ophthalmol 15: 321-327.

C
AC

16 Stapleton F, Keay LJ, Sanfilippo PG, Katiyar S, Edwards KP, Naduvilath T (2007): Relationship

17 between climate, disease severity, and causative organism for contact lens-associated

18 microbial keratitis in Australia. Am J Ophthalmol 144: 690-698.

58
 ACCEPTED MANUSCRIPT

1 Stapleton F, Carnt N (2012): Contact lens-related microbial keratitis: how have epidemiology

2 and genetics helped us with pathogenesis and prophylaxis. Eye (Lond) 26: 185-193.

3 Stopak SS, Roat MI, Nauheim RC, Turgeon PW, Sossi G, Kowalski RP, Thoft RA (1991):

PT
4 Growth of Acanthamoeba on human corneal epithelial cells and keratocytes in vitro. Invest

5 Ophthalmol Vis Sci 32: 354-359.

RI
6 Sun RL, Jones DB, Wilhelmus KR (2007): Clinical characteristics and outcome of Candida

SC
7 keratitis. Am J Ophthalmol 143: 1043-1045.

8 Sy A, Srinivasan M, Mascarenhas J, Lalitha P, Rajaraman R, Ravindran M, Oldenburg CE, Ray

9
U
KJ, Glidden D, Zegans ME, McLeod SD, Lietman TM, Acharya NR (2012): Pseudomonas
AN
10 aeruginosa keratitis: outcomes and response to corticosteroid treatment. Invest Ophthalmol
M

11 Vis Sci 53: 267-272.

12 Szmigielski S, Prévost G, Monteil H, Colin DA, Jeljaszewicz J (1999): Leukocidal toxins of

D

13 staphylococci. Zentralbl Bakteriol 289: 185-201.

TE

14 Tacconelli E, Johnson AP (2011): National guidelines for decolonization of methicillin-resistant

EP

15 Staphylococcus aureus carriers: the implications of recent experience in the Netherlands. J

16 Antimicrob Chemother 66: 2195-2198.

C

17 Tang A, Marquart ME, Fratkin JD, McCormick CC, Caballero AR, Gatlin HP, O'Callaghan RJ
AC

18 (2009): Properties of PASP: a Pseudomonas protease capable of mediating corneal erosions.

19 Invest Ophthalmol Vis Sci 50: 3794-3801.

59
 ACCEPTED MANUSCRIPT

1 Tanure MA, Cohen EJ, Sudesh S, Rapuano CJ, Laibson PR (2000): Spectrum of fungal keratitis

2 at Wills Eye Hospital, Philadelphia, Pennsylvania. Cornea 19: 307-312.

3 Tatsumi Y, Nagashima M, Shibanushi T, Iwata A, Kangawa Y, Inui F, Siu WJ, Pillai R,

PT
4 Nishiyama Y (2013): Mechanism of action of efinaconazole, a novel triazole antifungal

5 agent. Antimicrob Agents Chemother 57: 2405-2409.

RI
6 Tayel SA, El-Nabarawi MA, Tadros MI, Abd-Elsalam WH (2013): Positively charged polymeric

SC
7 nanoparticle reservoirs of terbinafine hydrochloride: preclinical implications for controlled

8 drug delivery in the aqueous humor of rabbits. AAPS PharmSciTech 14: 782-793.

9
U
Taylor WM, Pidherney MS, Alizadeh H, Niederkorn JY (1995): In vitro characterization of
AN
10 Acanthamoeba castellanii cytopathic effect. J Parasitol 81: 603-609.
M

11 Thau N, Monod M, Crestani B, Rolland C, Tronchin G, Latgé JP, Paris S (1994): Rodletless

12 mutants of Aspergillus fumigatus. Infect Immun 62: 4380-4388.

D
TE

13 Thibodeaux BA1, Caballero AR, Marquart ME, Tommassen J, O'Callaghan RJ (2007): Corneal

14 virulence of Pseudomonas aeruginosa elastase B and alkaline protease produced by

EP

15 Pseudomonas putida. Curr Eye Res 32: 373-386.

16 Thomas PA, Garrison RG, Jansen T (1991): Intrahyphal hyphae in corneal tissue from a case of
C

17 keratitis due to Lasiodiplodia theobromae. J Med Vet Mycol 29: 263-267.

AC

18 Thomas PA (1994): Mycotic keratitis--an underestimated mycosis. J Med Vet Mycol 32: 235-

19 256.

60
 ACCEPTED MANUSCRIPT

1 Thomas PA (2003): Current perspectives on ophthalmic mycoses. Clin Microbiol Rev 16: 730-

2 797.

3 Thomas PA, Kaliamurthy J (2013): Mycotic keratitis: epidemiology, diagnosis and management.

PT
4 Clin Microbiol Infect 19: 210-220.

5 Tilley SJ, Orlova EV, Gilbert RJ, Andrew PW, Saibil HR (2005): Structural basis of pore

RI
6 formation by the bacterial toxin pneumolysin. Cell 121: 247-256.

SC
7 Trabelsi H, Dendana F, Sellami A, Sellami H, Cheikhrouhou F, Neji S, Makni F, Ayadi A

8 (2012): Pathogenic free-living amoebae: epidemiology and clinical review. Pathol Biol

9 (Paris) 60: 399-405.

U
AN
10 Tronchin G, Bouchara JP, Robert R, Senet JM (1988): Adherence of Candida albicans germ
M

11 tubes to plastic: ultrastructural and molecular studies of fibrillar adhesins. Infect Immun 56:

12 1987-1993.
D
TE

13 Tronchin G, Pihet M, Lopes-Bezerra LM, Bouchara JP (2008): Adherence mechanisms in human

14 pathogenic fungi. Med Mycol 46: 749-772.

EP

15 Twining SS, Davis SD, Hyndiuk RA (1986): Relationship between proteases and descemetocele

16 formation in experimental Pseudomonas keratitis. Curr Eye Res 5: 503-510.

C
AC

17 Twining SS, Kirschner SE, Mahnke LA, Frank DW (1993): Effect of Pseudomonas aeruginosa

18 elastase, alkaline protease, and exotoxin A on corneal proteinases and proteins. Invest

19 Ophthalmol Vis Sci 34: 2699-2712.

61
 ACCEPTED MANUSCRIPT

1 Unterman SR, Rootman DS, Hill JM, Parelman JJ, Thompson HW, Kaufman HE (1988):

2 Collagen shield drug delivery: therapeutic concentrations of tobramycin in the rabbit cornea

3 and aqueous humor. J Cataract Refract Surg 14: 500-504.

PT
4 Vemuganti GK, Garg P, Gopinathan U, Naduvilath TJ, John RK, Buddi R, Rao GN (2002):

5 Evaluation of agent and host factors in progression of mycotic keratitis: A histologic and

RI
6 microbiologic study of 167 corneal buttons. Ophthalmol 109: 1538-1546.

SC
7 Vijayvargia R, Kaur S, Sangha N, Sahasrabuddhe AA, Surolia I, Shouche Y, Krishnasastry MV

8 (2004): Assembly of alpha-hemolysin on A431 cells leads to clustering of Caveolin-1.

U
9 Biochem Biophys Res Commun 324: 1124-1129.
AN
10 Visvesvara GS, Moura H, Schuster FL (2007): Pathogenic and opportunistic free-living
M

11 amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia

12 diploidea. FEMS Immunol Med Microbiol 50: 1-26

D

13 Wagoner MD, Al-Swailem SA, Sutphin JE, Zimmerman MB (2007): Bacterial keratitis after
TE

14 penetrating keratoplasty: incidence, microbiological profile, graft survival, and visual

outcome. Ophthalmol 114: 1073-1079.

EP

15

16 Wollensak G, Spoerl E, Seiler T (2003): Riboflavin/ultraviolet-a-induced collagen crosslinking

C

17 for the treatment of keratoconus. Am J Ophthalmol 135: 620-627.

AC

18 Wong VW, Lai TY, Chi SC, Lam DS (2011): Pediatric ocular surface infections: a 5-year review

19 of demographics, clinical features, risk factors, microbiological results, and treatment.

20 Cornea 30: 995-1002.

62
 ACCEPTED MANUSCRIPT

1 Wong RL, Gangwani RA, Yu LW, Lai JS (2012): New treatments for bacterial keratitis. J

2 Ophthalmol ID:831502.

3 Wright P, Warhurst D, Jones BR (1985): Acanthamoeba keratitis successfully treated medically.

PT
4 Br J Ophthalmol 69: 778-782.

5 Xie L, Zhai H, Zhao J, Sun S, Shi W, Dong X (2001): Antifungal susceptibility for common

RI
6 pathogens of fungal keratitis in Shandong Province, China. Am J Ophthalmol 146: 260-265.

SC
7 Xie L, Zhong W, Shi W, Sun S (2006): Spectrum of fungal keratitis in north China. Ophthalmol

8 113: 1943-1948.

U
AN
9 Xu Y, Gao C, Li X, He Y, Zhou L, Pang G, Sun S (2013): In vitro antifungal activity of silver

10 nanoparticles against ocular pathogenic filamentous fungi. J Ocul Pharmacol Ther 29: 270-
M

11 274.
D

12 Yilmaz S, Ozturk I, Maden A (2007): Microbial keratitis in West Anatolia, Turkey: a

TE

13 retrospective review. Int Ophthalmol 27: 261-268.

14 Yoon KC, Jeong IY, Im SK, Chae HJ, Yang SY (2007): Therapeutic effect of intracameral
EP

15 amphotericin B injection in the treatment of fungal keratitis. Cornea 26: 814-818.

C

16 Zhu WS, Wojdyla K, Donlon K, Thomas PA, Eberle HI (1990): Extracellular proteases of
AC

17 Aspergillus flavus. Fungal keratitis, proteases, and pathogenesis. Diagn Microbiol Infect Dis

18 13: 491-497.

63
 ACCEPTED MANUSCRIPT
Highlights

• Microbial keratitis is caused by bacteria, fungi, and protist pathogens

• Epithelial defects and injuries are key predisposing factors in microbial keratitis

• Factors from the host and the pathogen contribute to the disease.

• Successful prognosis requires early diagnosis and aggressive treatment.

PT
• This is a timely review of our current understanding of microbial keratitis.

RI
U SC
AN
M
D
TE
C EP
AC