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Chapter1: Introduction and Literature Review CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW 1.0 Introduction Ocular infections are one of the major causes of blindness in the developing world. Bacteria, viruses, fungi and parasites cause ocular infections. The important bacteria affecting the eye can be classified as gram positive cocci, gram positive bacilli, gram negative cocci, gram negative bacilli, Mycobacteria and Actinomycetes. These bacteria may cause ocular infections like keratitis, endophthalmitis, conjunctivitis and blepharitis. A few of the infection causing bacteria belong to the normal flora of the eye. The conjunctival sac and the lid margins of the eye harbour a variety of organisms (Moeller et al 2005). Normal flora present in the eye can be arranged in two groups, resident flora and transient flora. The predominant resident flora of the eye are Staphylococcus epidermidis and Corynebacterium xerosis. The important transient flora are Diphtheriods, CoNS, Streptococci, Haemophilus, Moraxella and Neisseria. Members of transient flora are generally of little significance so long as the normal resident flora and host resistance remain intact. Under conditions like trauma, or failure of a surgery or a systemic infection these normal flora are the contributing factors for ocular infections (Sharma et al 1988). Among the group of bacteria isolated from ocular infections the prevalence rate of CoNS is very high (Kunimoto et al 1999, Sharma et al 2002, Pinna et al 2000). Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 1 Chapter1: Introduction and Literature Review However, till date the difference between the coagulase negative staphylococci (CoNS) as a normal flora and as a frequent pathogen are not clearly understood. 1.1 Phylogenetic classification of coagulase negative staphylococci Bergeys manual (Bergys et al 1985) has classified prokaryotes into four divisions and these have been subdivided into classes. Gram positive cocci are included in the section 12 of Bergeys manual. This section consists of fifteen genera, which are phylogenetically, and phenotypically quite diverse. The hierarchical arrangement of taxonomy for the genus Staphylococus is as follows . Domain : Eubacteria Kingdom : Proteobacteria Class : Bacilli Order : Bacillales Family : Micrococcaceae Genus : Staphylococcus The 15 genera are Micrococcus, Planococcus, Deinococcus, Staphylococcus, Stomatococcus, Streptococcus, Leucnostoc, Pediococcus, Aerococcus, Gemella, Peptococcus, Peptostreptococcus, Ruminococcus, Coprococcus, Sarcina. The presence or absence of catalase and cytochrome separates the gram positive into two groups, Micrococcaceae (Staphylococcus, Micrococcus, Planococcus) and Deinococcaceae. Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 2 Chapter1: Introduction and Literature Review Micrococcaceae consists of four genera, Micrococcus, Staphylococcus, Stomatococcus and Planococcus. Members of the genera Staphylococcus and Micrococcus are catalase positive, gram positive cocci and are placed with Stomatococcus and Planococcus in the family Micrococcaceae. Table 1.1 represents the differential characteristics of the genus Micrococcus, Stomatococcus, Planococcus and Staphylococcus. The differential properties of genus Staphylococcus are arrangement of cells which are in clusters, They are facultatively anaerobic genera, positive for catalase reaction, cytochrome is present and are lactate fermentors. The G+C content of DNA is 30%39%. Staphylococci are phylogenetically a cohrent group of organisms. The genus Staphylococcus can be subdivided into at least four species groups on the basis of DNA / DNA relationships and phenotypic characterization. The S epidermidis species group is composed of the species S. epidermidis, S. capitis, S.warneri, S.haemolyticus, S. hominis and S.sacchrolyticus. The S.saprophyticus species group is composed of the species S.saprophyticus, S.cohinni and S. xylosus. The simulans species group is composed of S.simulans and S.carnosus. The S.sciuri species group is composed of S.sciuri and S.lentus. Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 3 Chapter1: Introduction and Literature Review Table 1.1: Differential characteristics of the genus Micrococcus, Stomatococcus, Planococcus and Staphylococcus Characteristics Micrococcus Stomatococcus Planococcus Staphylococcus Irregular clusters + + + + Tetrads + - - - Capsule - + - - Motility - - + - Growth on furazolidine agar + - - - Anaerobic fermentation of glucose- + - + Oxidase and benzidine test + - ND - FDP-aldolase(class) II ND ND I Resistance to lysostaphin R R R S Menaquinones Hydrogenated ND Normal Normal Glycine present in peptidoglycan - - - + Teichoic acid present in cell wall - - - + Mol% G+C of DNA 56-60 39-52 30-39 65-75 Symbols: (+) 90% or more of strains are positive; (-)90% or more are negative; (d) 11-89% of strains are positive; (ND) not determined; (R) resistant; (S) Susceptible. 1.2 Infections caused by coagulase negative staphylococci 1.2.1 Systemic The role of CoNS species in causing nososcomial infections has been recognized and well documented. The infection rate has been correlated with the increase in the use of prosthetic and indwelling medical devices and the growing number of immunocompromised patients in hospitals(Christensen et al 1982). All the species of CoNS cause infections. S.epidermidis has been documented as a pathogen in numerous cases of bacteremia, native and prosthetic valve endocarditis, surgical wounds and urinary tract, cerebrospinal fluid, prosthetic joint, peritoneal dialysis Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 4 Chapter1: Introduction and Literature Review related, ophthalmologic and intravascular catheter related infections. S.saprophyticus is an important opportunistic pathogen in human urinary tract infections, especially in young females (Raz et al 2005). It has been proposed as an agent of non gonnococal urethritis in males or a cause of other sexually transmitted diseases, prostatitis, wound infection and septicemia. S. haemolyticus, the most frequently encountered CoNS species associated with human infections, has been implicated in native valve endocarditis, septicemia, peritonitis, urinary tract infections, wound infections bone and joint infections (Ing et al 1997). S.lugdunensis had been implicated in arthritis, bacteremia, catheter infections, prosthetic joint infections and urinary tract infections. Other CoNS species have been implicated in a variety of infections. For example S.capitis, S.caprae, S. saccharolyticus, S.simulans have been implicated in osteomyelitis, native valve endocarditis, pneumonia and urinary tract infections. In many cases patients with infections caused by these CoNS have predisposing or underlying disease affecting the immune system. 1.2.2 Ocular infections 1.2.2.1 Endophthalmitis Endophthalmitis is an ocular inflammation resulting from the introduction of an infectious agent into the posterior segment of the eye. During infection, irreversible damage to delicate photoreceptor cells of the retina frequently occurs. Infectious agents generally gain access upto the posterior segment of the eye after an intraocular surgery (postoperative endophthalmitis) or following penetrating injury of the globe Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 5 Chapter1: Introduction and Literature Review (posttraumatic endophthalmitis) or haematogenous spread of the bacteria to the eye (endogenous endophthalmitis). Epidemiology Recent endophthalmitis series have recorded that the CoNS are the most frequent etiological agents. The absence of these bacteria from older series suggest that these organisms may have been overlooked as “contaminants”, as was common practice. An increased awareness of delayed onset endophthalmitis, and improved microbiologic investigation also may be factors in the apparent increased prevalence. The source of coagulase negative staphylococcal endophthalmitis is characteristically the endogenous flora of the ocular surface (Callengan et al 2002). The etiologic agents in the acute postoperative enophthalmitis are generally microorganisms of the eyelid margin and periocular tear film. In series from the United States, CoNS are responsible for about 70% of post cataract surgery endophthalmitis, followed by Staphylococcus aureus, viridans group of streptococci, other gram positive microorganisms (Laderbag et al 1998, Ban et al 1996, Speaker et al 1991]. The common bacterial isolates recovered by the endophthalmitis vitrectomy study (EVS) are CoNS (Andrew et al group 1996). A six year review of culture proven endophthalmitis isolates shows CoNS are most common (Matthew et al 2004). The Microbial spectrum isolated from postoperative endophthalmitis at a tertiary eye care centre in Hyderabad shows that 42.9% bacterial isolates are CoNS (Kunimoto et al 1999). Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 6 Chapter1: Introduction and Literature Review Penetrating ocular injuries are accompanied by infection at a much higher rate than occurs with surgery. In most series of the penetrating injuries, 3-17% eyes develop microbial endophthalmitis. Posttraumatic endophthalmitis-associated isolates include a greater variety of organisms than those following ocular surgery and include bacteria derived from the environment. Staphylococci are ranked first in prevalence followed by Bacillus. The Microbial spectrum isolated from posttraumatic endophthalmitis at a tertiary eye care centre in Hyderabad shows that 45.3% isolated cultures are gram positive cocci, out of which 17.3% are CoNS (Kunimoto et al 1999). Endogenous endophthalmitis is relatively rare, accounting for only 2-8% of all endophthalmitis cases (Okada et al 1994). CoNS are not a common cause of endogenous endophthalmitis. Common organisms include S.aureus, B.cereus and gram negative organisms. The most common etiological agent of all cases of endogenous endophthalmitis is the opportunistic fungus Candida albicans. Table 1.2 represents the prevalence of Staphylococcus in various reports of endophthalmitis. Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 7 Chapter1: Introduction and Literature Review Table 1.2: Prevalance of Staphylococcus spp in endophthalmitis in various studies Most No: of prevalant CoNS in organism in Type of Geographic endophthalmitis Area Period patients isolates % of CoNS the study the study Reference Posttraumatic India 7 182 139 17.3 1 CoNS Kunimoto et al 1999 Postoperative India 7 206 176 46% 1 CoNS Kunimoto et al 1999 - 10 223 69.00% 1 CoNS Callengan et al 2002 Postoperative India - 37 62.60% 1 CoNS Renuka et al 2002 Post operative Florida 5 year 278 313 49.98% 2 CoNS Mathew et al 2002 80 No:of Rank of CoNS: coaguase negative staphylococci Clinical features Postoperative endophthalmitis (POE) is defined as severe inflammation involving both the anterior and posterior segments of the eye secondary to an infectious agent. POE occurs after a surgical procedure (cataract, keratoplasty, glaucoma, and trabeculectomy) that breaches the corneo scleral wall of the eye. POE due to CoNS may occur several weeks to years after surgery. This delayed infection is likely due to the sequestration of low virulence organisms introduced at the time of surgery or delayed inoculation of organisms. The three forms of clinical presentation (POE) can de distinguished as acute form, delayed form and moderately severe. Acute form usually occurs 2-4 days post operation, due to S.aureus or Streptococci. Delayed or moderately severe form usually occurs 5-7 days postoperation due to CoNS. The chronic from occurs as early as one month postoperation, due to S.epidermidis or fungus. Examination of the eye Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 8 Chapter1: Introduction and Literature Review demonstrates conjunctival chemosis often with significant amount of yellowish exudates in the conjunctival sac. The upper lid becomes edematous. The cornea demonstrates variable degree of edema, and pigmented cells may accumulate on its posterior surface. The anterior chamber (AC) shows flare and cells, hypopyon is often present in the inferior angle. In extreme cases the AC is filled with exudates and the cornea is white. In vitreous heavy cellular debris will be present. The characteristics of endophthalmitis caused by CoNS include delayed onset, subacute, chronic, and painless infective endophthalmitis. Infection with CoNS is often associated with a good visual outcome (Chen et al 1999). Posttraumatic endophthalmitis (PTE) may be revealed clinically with a wide variety of symptom complexes. PTE is an important complication of open globe injury. The onset of disease after injury accompanies increased pain, intra ocular inflammation, hypopyon and vitreous opacities. It is more often associated with a poor visual outcome. In some instances a seemingly mild injury may not lead the patient to seek care until the signs and symptoms of infection have developed. In other cases, notably with Bacillus infections, the onset of pain and profound visual loss may be explosively rapid. Still other infections are revealed weeks to months after repair of the initial penetrating injury, particularly when the infecting organism is a fungus. Endogenous endophthalmitis comprises approximately 5% to 7% of cases in large series of endophthalmitis. Previously published literature on pathology of endogenous endophthalmitis implicated Staphylococcus aureus as the most common bacterial organism and the most common fungal organism as Candida species (Vivian et al 2004). Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 9 Chapter1: Introduction and Literature Review Most patients with endogenous endophthalmitis have one or more predisposing systemic risk factors, although cases among otherwise healthy, immunocompetent persons have been reported. Endogenous endophthalmitis has been associated with many systemic risk factors, including chronic immune-compromising illnesses (diabetes mellitus, renal failure), indwelling or long-term intravenous catheters, immunosuppressive diseases and therapy (malignancies, human immunodeficiency virus infection or HIV, chemotherapeutic agents), recent invasive surgery, endocarditis, gastrointestinal procedures, hepatobiliary tract infections, and intravenous drug abuse. Ocular symptoms included decreased vision, redness, pain, floaters, and photophobia (Vivian Schiedler et al 2004). Although the symptoms and predisposing factors of endophthalmitis are known, the exact clinical features, predisposing factors and visual outcome of CoNS endophthalmitis are not clearly understood. 1.2.2.2 Keratitis Bacterial keratitis is defined as the inflammatory infiltration of corneal stroma associated with an epithelial defect, from which one or more bacterial species are cultured (Olafur et al 1989). The bacteria that are normally present can be classified in two groups; the resident bacteria that are constantly present in the eye and the transient bacteria, which consist of nonpathogenic or potentially pathogenic bacteria that inhabit the eye for short periods. Almost any species of bacteria can infect the cornea if the integrity of the anatomic barriers or defense mechanisms are compromised (Sharma et al 1998, Brud et al 1994). Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 10 Chapter1: Introduction and Literature Review Epidemiology The spectrum of microorganisms responsible for corneal blindness varies in different geographical locations (Olafur et al 1989). Climatic and other demographic factors interrelate with varying bacterial and host determinants. Epidemiology of bacterial keratitis studied by different groups reveals that gram positive organisms, among which CoNS predominate, are most commonly isolated from the corneal scrapings (Olafur et al 1989). Table 1.3 represents the Staphylococcus prevalence in various reports. Literature survey showed that the age range of bacterial keratitis could be from 7 years – 94 years (Cameron et al 2006). Bacterial keratitis occurs in eyes having a predisposing factor (Bonston et al 1998). Although the eye is constantly exposed to a large number of bacteria, only a small proportion of this results in corneal infection (Bharati et al 2003). Corneal trauma is the leading cause of corneal blindness, others being alteration of any of the local or systemic defense mechanisms (Bourcier et al 2003), eyelid abnormalities and abnormalities of the tear film. The inappropriate use of topical antibiotics could eliminate the natural protection provided by the normal flora and predispose the infections of the cornea. The use of topical corticosteroids can create localized immunosuppression and may be a major risk factor for bacterial keratitis. Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 11 Chapter1: Introduction and Literature Review Clinical features The hallmark of clinical signs distinctive for suspected infectious keratitis includes an ulceration of the epithelium with supuprative stromal inflammation that is either focal or diffuse. The presence of diffuse cellular infiltration in the adjacent stroma is highly suggestive for infectious keratitis. The anterior chamber reaction may range from mild flare cells to hypopyon formation. Symptoms of pain and redness as well as increased epithelial defect and/or stromal ulceration are indications of an infection. Table 1.3: Prevalence of Staphylococcus spp. in bacterial keratitis reported by various investigators Geographic Area Period No: of patients No:of isolates % of CoNS Rank of CoNS in the study prevalant organism in the study Reference New york 30 year 677 494 16 Coagulase positive 2 staphylococci Penny et al 1982 Boston 4 year 175 176 14 Staphylococcus 2 aureus Olafur et al 1989 India - 100 52 42.3 1 CoNS vajpayee et al 2000 India 4 year 102 99 31.1 1 CoNS Kunimoto et al 2000 India 3 year 3183 1043 18.45 Streptococcus 2 pneumoniae Bharti et al 2000 Gram positive cocci typically cause localized round or oval ulceration with greyish white stromal infiltrate that have distinct borders and surrounding epithelial edema. Staphylococcal keratitis is more frequently encountered in immunocompromised corneas, such as those with bullous keratopathy, chronic herpetic keratitis, and keratoconjuctivitis. With delay in presentation and long-standing infection, both Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 12 Chapter1: Introduction and Literature Review coagulase positive and coagulase negative staphylococci cause severe intrastromal abscess and corneal perforation (Jones et al 1981, Jones et al 1973). Both S. aureus and S. epidermidis cause corneal ulceration that have similar have similar appearance with a yellow-white round/oval shaped infiltrate having distinct borders, but the tissue surrounding the ulcer margin is often blurred by a stromal infiltrate and edema. S. aureus causes a more severe microbial ulcer with more complications (Stern et al 1995). 1.2.2.3 Conjunctivitis Epidemiology Bacterial conjunctivitis can be caused by virulent and less virulent bacteria. They disrupt the intact conjunctival surface. It is frequently caused by inoculation from an external source and is often bilateral. The important bacteria causing conjunctivitis are Staphylococcus aureus, Streptococcus, Haemophilus, E. coli, Pseudomonas aeruginosa, Acinetobacter, Corynebacterium, CoNS and Nesseria (Brooks et al 1980). Bacterial conjunctivitis is classified into hyperacute, acute and chronic, based on the duration of illness and severity of clinical findings. CoNS causes chronic conjunctivitis and it is not frequent among acute and hyperacute conjunctivitis. Pathogenic strains of CoNS cause chronic blepharoconjunctivitis. Clinical features Lid margin involvement with loss of lashes, trichiasis, space erythema and telangiectasis are suggestive of staphylococcal infection. Conjunctival infection may Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 13 Chapter1: Introduction and Literature Review be the result of direct infection or release of toxin. Usually low number of organisms are demonstrated. Exotoxins of the CoNS produce a nonspecific conjunctivitis or a superficial punctate keratitis. The patient complains of intense, gritty sensation on eyelid opening in the morning, and the mucopurulent discharge may produce lid stickiness on awakening and crusting on their margins. The symptoms decrease during the day as the ocular surface heals. 1.2.2.4 Blepharitis Epidemiology The most commonly isolated organisms from lids with blepharitis are Staphylococcus epidermidis, Propronibacterium acnes, Corynebacterium spp., Acinetobacter spp., and Staphylococcus aureus. It has been demonstrated that patients with blepharitis are more likely to have normal skin bacteria on their lids and in greater quantities than nonblepharitis patients (Groden et al 1991). Clinical features The squamous type of staphylococcal blepharitis has hard, brittle, fibrinous scales on lid margin. The less common, ulcerative type is characterized by matted hard crusts surrounding the individual cilia. When the crusts are removed, small ulcers of hair can be seen and bleeding can be seen. Characteristics of both types of staphylococcal blepharitis are dilated blood vessels on the lid margins, white lashes, lash loss, trichiasis and scales around the cilia. Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 14 Chapter1: Introduction and Literature Review 1.3 Laboratory diagnosis of keratitis and endophthalmitis 1.3.1 Collection and transport of corneal scrapings and vitreous Corneal samples can be collected using the slit lamp or operating microscope. A heat sterilized Kimura spatula or bent needle or rounded surgical blade (no 15) is used to perform corneal scraping using slit lamp biomicroscopic visualization (Figure 1.1). The ulcerated or infiltrated area is scraped without touching the eyelid margins. Samples collected from the site of lesion, i. e., the infected corneal tissue are the most valuble for microbiological diagnosis. Viable organisms may be present in an inflamed area or localized to one zone such as advancing margin or deep in the ulcer crater (Smith et al 1984). After each set of scraping, material is smeared onto a clean glass slide or is inoculated to a culture medium. A tray (Figure 1.2) containing all requirements for sample collection is kept ready in the laboratory and taken to the clinic on request. Patients suspected of having microbial endophthalmitis are generally taken to the operating room to obtain samples for culture and to initiate antimicrobial therapy. Aqueous and vitreous samples are collected in tuberculin syringe under local anesthesia. Aqueous and vitreous should separately be inoculated into fresh culture plates. The specimens should be inoculated as soon as they are obtained. Drops placed on slides should subsequently be processed for Gram and Giemsa stains (Froster et al 1980). Drops of the fluids are directly inoculated on to culture plates and liquid broths. If therapeutic vitrectomy is done the specimen obtained will be Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 15 Chapter1: Introduction and Literature Review vitreous diluted with infusion fluid. The culture yield of this material can be increased by passing the entire specimen through a sterile membrane filter which is cut into segments, and directly placed onto culture plates. Figure 1.1: Collection of specimen from a corneal ulcer Figure 1.2: corneal scraping collection tray containing culture media, blades, glass slides marker pen and reagents. 1.3.2 Processing of clinical samples 1.3.2.1 Direct Microscopic observation The recommended smears for examination of corneal scrapings are the Gram and Giemsa stains and others such as 10-20% potassium hydroxide (KOH), Grocott’s methenamine silver (GMS) stain, acridine orange stain, calcofluor white stain (CFW), Uvitex 2B, Blankophor and Lectins (Forster et al. 1976, Rao et al 1989). Multiple smears should be obtained by directly transferring corneal/vitreous specimens on to clean glass slides. Differential staining techniques are useful in interpreting smears of corneal material and vitreous. Acridine orange is a suitable Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 16 Chapter1: Introduction and Literature Review initial screening stain to detect the presence of most microorganisms. Gram and Giemsa staining are done on parallel smear. Interpretation of the stained smear begins with recognizing the nature of the inflammatory reaction. The presence of many polymorphonuclear leukocytes suggests infection. Clumps of neutrophils and degenerating epithelial cell space can give an indication as to where to search for microorganisms in the smear. An occasional epithelial cell space can be found with many adherent bacteria. The presence of multiple similar bacteria with acute inflammatory cells is considered as indicative of the responsible microorganisms. 1.3.2.2 Culture methods While smear examination provides preliminary results, culture isolation gives confirmation. Corneal scrapings should ideally be inoculated directly onto culture media. No transport media is recommended. Agar plates and broth tubes recommended for routine processing of corneal scrapings are as given below. Since bacterial, fungal and Acanthamoeba keratitis may overlap clinically, a combination of media is used to allow growth of organism (Sharma et al 2000) 1. Blood agar plate (5 % Sheep blood) 2. Chocolate agar plate (5 % Sheep blood) 3. Thioglycollate broth 4. Anaerobic blood agar plate (5 % sheep blood) 5. Sabouraud dextrose agar plate/tube 6. Brain heart infusion broth 7. Non-nutrient agar with Escherichia coli Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 17 Chapter1: Introduction and Literature Review Solid media are directly inoculated by making a single row of inoculation marks with each collected corneal scraping. Small C-streak marks are made by lightly sweeping both sides of the spatula/blade on the surface of the agar plate without gouging. Each row of inoculation of marks is from one set of scrapings; 3-4 rows of streaks are applied on the plate. The thioglycollate broth tube is inoculated by transferring the specimen from the spatula onto a swab that is dropped into the depth of the culture tube. Other liquid media are directly inoculated by gentle agitation. AC/ vitreous samples are inoculated by placing the drops on solid media (blood agar plate, chocolate agar plate, Sabouraud dextrose gar plate) surface and by directly injecting into liquid media (thioglycollate broth and brain heart infusion broth). Spreading the inoculum with bacteriological loop is not recommended, as it is important to observe growth at the site of inoculum. For seven days all the media are incubated at 37°C . Most bacterial colonies may appear within 24-48 hours. Valid growth appears on the inoculum (C streaks/ drops). Growth occurring away from site of inoculation are considered contaminant. Liquid tubes are observed daily for turbidity. Colonies growing at the site of inoculation are considered for further processing and identification. Colony number, color and size are noted. Staphylococci form raised, white, opaque colonies. A preliminary identification based on colony morphology, Gram reaction and cell morphology is made. Cocci in pairs, tetrads, chains and clusters are subjected to catalase test. Catalase positive organisms belong to the Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 18 Chapter1: Introduction and Literature Review Staphylococcus genus. Genus Staphylococcus is further identified as Staphylococcus aureus if coagulase test is positive and CoNS if coagulase test is negative. 1.3.3 Identification of coagulase negative staphylococci 1.3.3.1 Conventional Methods The isolates are identified by conventional biochemical tests based on the Manual for Clinical Microbiology (Bannerman et al 2003). The various tests are catalase test, coagulase test, urease activity, ornithine decarboxylation, PYRase activity, phosphatase activity, and fermentation of carbohydrates. a. Catalase test The breakdown of hydrogen peroxide into oxygen and water is mediated by the enzyme catalase. When a small amount of an organism, that produces catalase, is emulsified in hydrogen peroxide, rapid effervescence of bubbles of oxygen, the gaseous product of the enzyme activity, is produced indicating catalase positivity. b. Coagulase test The presence of a cell surface associated substance that binds fibrinogen and thus allows aggregation of organisms in plasma containing fibrinogen is detected by observation of clumping of cells. For tube coagulase test a 1 in 10 dilution of the plasma (rabbit) in saline solution is used. A coagulum is formed when a small volume of culture suspension is added to the diluted plasma and incubated at 37°C. Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 19 Chapter1: Introduction and Literature Review c. Urease test Conventional urea broth or agar has been used to detect urease activity in staphylococcal species . The test detects the release of ammonia from urea, resulting in increase in pH that is shown by phenol red indicator changing from yellow to red. S. epidermidis, S. intermidius and most strains of S. saprophyticus are usually urease positive. d. Ornithine decarboxylation: A positive ornithine decarboxylase identifies S. lugdunensis. Ornithine can be identified in a liquid medium containing 1% L ornithine dichloride. A change in a liquid medium from grey to yellow (caused by initial fermentation of glucose) to violet (caused by decarboxylation of ornithine) indicates a positive reaction. A yellow color at 24 hours indicates a negative result. e. PYRase activity: Pyrrolidonyl arylamidase activity is determined by the hydrolysis of pyroglutamyl- naphthylamide into L – pyrrolidone and naphthylamide, which combines with a PYR (p- dimethylaminocinnamaldehyde) reagent to produce a red color. A loopful of overnight culture is added to the tube and the tube is overlaid with mineral oil. The tube is incubated at 37C for 2hours. After incubation, 2 drops of PYR reagent are added to each tube without mixing. The development of yellow, orange or pink color is considered as a negative result. S. haemolyticus, S. schleiferi and S. intermidius are usually PYR positive. Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 20 Chapter1: Introduction and Literature Review f. Phosphatase activity: Phosphatase activity is based on the hydrolysis of pnitrophenyl phosphate into inorganic phosphate (Pi) and P nitro phenyl by the enzyme alkaline phosphatase. Phosphatase activity is indicated by the presence of yellow pnitophenol from colorless substrate. Strains of S. schleiferi , S. intermidus, S. lycus and most strains of S. epidermdis are positive for this test. g. Acid production from carbohydrates: Acid production from carbohydrates can be easily detected in an agar plate containing nutrients and bromocresol purple. Production of acid from maltose and sucrose and absence of acid production for trehalose and mannitol can distinguish S. epidermdis from other novobiocin susceptible species. Production of acid from trehalose, mannose, maltose and sucrose and absence of acid production from mannitol can identify S. lugdunensis, 1.3.3.2 Microbial identification systems Commercially available systems reduce the need for preparing a variety of test media and reagents and the time required for interpretation of results, thereby making the identification of various Staphylococcus species more plausible in the routine laboratory. API Staph, VITEK 2 (bioMérieux, France) and Phoenix system (BD Diagnostic Systems, Sparks, USA) (Eigner et al 2005) are some of the methods used world over. Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 21 Chapter1: Introduction and Literature Review 1.3.3.2.1 Analytical Profile Index(API) api® Staph (bioMérieux, France) consists of a strip containing dehydrated test substrates in individual microtubes. The tests are reconstituted by adding to each tube an aliquot of api® Staph Medium that has been inoculated with the strain to be studied. The strip is then incubated for 18-24 hours at 35-37°C after which the results are read and interpreted with reference to the information contained in this package insert. The identification is facilitated by the use of the api® Staph Analytical Profile Index or the identification software. Positive color reactions are converted to a four-digit profile for species identification. The numerical identification of the observed profile is based on the calculation of how closely the profile corresponds to the taxon relative to all the other taxa in the database (% ID). The identification is rated as good, excellent, acceptable or unacceptable based on the % ID. The advantages of Staph Ident system are, it is easy to inoculate, it is rapid, it requires no special equipment and the database includes 12 species of CoNS. The disadvantages of the system are that the color reactions are difficult to interpret, it requires heavy inoculum, and supplemental testing is required (Olarae et al 1984). Mini API The principle of mini API is similar to API. Mini API is a set of standardized, miniaturized biochemical tests which can be read automatically. The strip is made of thermoformed rigid plastic containing 32 cupules. Each strip carries a screen printed code which is automatically recognized by the reader. The cupules contain a Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 22 Chapter1: Introduction and Literature Review dehydrated substrate where each cupule corresponds to a test. The cupules are inoculated with a bacterial suspension, which reconstitutes the medium. The reactions during the incubation period result in color changes, or an increase in turbidity. The strip is placed on the reader and read automatically. The reader recognizes the strip code, makes the necessary measurements and transfers them to the computer where software establishes the corresponding biochemical profile. The mini API interprets the profiles after automatic reading. For each profile the software calculates the percentage of identification which estimates the relative proximity of the observed profile to the various taxa in the data base. The T index expresses the proximity to the most typical profile in each of the taxa. Its value ranges between 0-1. The software classifies the taxa according to the values of these parameters and provides an identification result. 1.3.3.2.2 Phoenix The BD Phoenix Automated Microbiology System (BD Diagnostic Systems, Sparks, Md. USA) is a newly developed instrument for the reliable and accurate identification and susceptibility testing for the majority of clinically encountered strains. The system is comprised of disposable panels, which combine both identification testing (ID) and antimicrobial susceptibility testing (AST), and performs automatic reading at 20minute intervals during incubation. Phoenix system consists of ID and AST broths PMIC/ID 13 panel for gram-positive cocci. The Phoenix ID broth is inoculated with bacterial colonies from Columbia blood agar and adjusted to a 0.5 to 0.6 McFarland standard using the Crystal Spec Nephelometer (BD Diagnostic Systems). After Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 23 Chapter1: Introduction and Literature Review supplementing the broth with one drop of indicator dye, 25 μl of the ID suspension is transferred to the AST broth to achieve a final inoculum density of 1.5 × 108 CFU/ml. The ID and the AST broths are poured into the respective side of the panel placed on the Phoenix inoculation station. The inoculated panels are closed and placed into the transport caddy, and, after entering the accession number, the panels are placed into the Phoenix instrument (Horstkotte et al 2004). The advantages of PHOENIX System are it gives the capacity to simultaneously perform 1 to 100 ID/AST determinations with the flexibility of random entry on-demand loading custom antibiotic panels, single or batch inoculation, rapid results and minimal waste disposal. When comparing the performance of the Phoenix instrument with the VITEK 2 system Gross et al investigated 400 staphylococcal strains and 121 Enterococcus spp (Gross et al 2002). However, the accuracy rate of this system is not 100% for CoNS isolates. 1.3.3.2.3 VITEK The VITEK 2 system (bioMérieux, France) is a new automated system designed to provide rapid and accurate identification and susceptibility testing results for most clinical isolates. Identification is made on the basis of biochemical reactions, and MIC determinations are made by applying an algorithm to the growth kinetics monitored by the VITEK 2 system. ID-GPC card is used for gram-positive cocci and P 523 panel is used for gram-positive cocci. A sufficient number of colonies is suspended in sterile saline (0.45%) and adjusted to a 0.5 McFarland turbidity. The inoculated tube is placed in a cassette on the VITEK 2 Smart carrier Station. The sample number is entered and associated with an ID and AST card. The sample accession numbers and Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 24 Chapter1: Introduction and Literature Review card identification bar code numbers are scanned, and the information is stored on the cassette memory chip. The SCS cassette with the cards and the test tubes are placed on the VITEK 2 instrument where the inoculation of the AST cards are automatically performed by the instrument (Bannerman et al 1993) The new VITEK 2 system shows high sensitivity, and concordance between mecA PCR and VITEK 2 oxacillin. MICs were observed for almost all S. epidermidis, S. haemolyticus, and S. hominis strains. However, the specificities vary considerably, between 97 and 80%, due to false-positive results, especially for mecA-negative S. saprophyticus, S. cohnii, and S. lugdunensis strains. 1.3.3.2.4 Molecular methods With the advent of molecular biology-based techniques, investigations based on comparative DNA sequence analysis of the genes of conserved macromolecules have become commonplace in microbiology as a tool for classification of microbial organisms. The most useful and extensively investigated taxonomic marker molecules are the larger rRNAs and their corresponding genes, respectively, especially 16S rRNAs and to a lesser extent 23S rRNAs (Hykin, et al 1994). The 5′ end of the gene encoding 16S rRNA (16S rDNA) contains enough information for the identification of almost all staphylococcal species. Molecular identification based on sequence analysis of universal targets offers several advantages, such as improved accuracy and short turnaround time. However, the partial 16S rDNA sequences used are not discriminative enough to differentiate all staphylococcal subspecies. Alexander et al evaluated RNA Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 25 Chapter1: Introduction and Literature Review polymerase B (rpoB) gene. Partial rpoB sequences were determined for 82 culture collection strains and 55 clinical isolates. All staphylococcal type strains were distinguishable by rpoB (Alexendar et al 2006). PCR based assay have been used for the discrimination between invasive and contaminating Staphylococcus epidermidis strains (Frebourg et al 2000). A PCR based assay was developed for the detection of Staphylococi at the genus level. The tuf gene were used to amplify a target region of 884 bp from 11 representative staphylococcal species. The entire amplicon was sequenced for the identification of species (Kontos et al 2003). 1.4 Antibiotic susceptibility profile of coagulase negative staphylococci 1.4.1 Isolates from Systemic infections Antibiotics used for the treatment of systemic infections caused by CoNS are penicillin, oxacillin, tobramycin, gentamicin, ciprofloxacin, vancomycin and rifampacin. Due to frequent use of these antibiotics for therapy or prophylaxis there is a selection of drug resistance in CoNS (Johannes et al 1999). Therefore, periodical check on the antibiotic susceptibility patterns of CoNS causing ocular infections is important. Mulder et al studied the antibiotic susceptibility patterns of 892 CoNS isolated from bacteremia and septicaemia (Mulder et al 1997). The antibiotic susceptibility patterns from their study are shown in the table 1.4. Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 26 Chapter1: Introduction and Literature Review Table 1.4: Percentages of susceptible isolates of CoNS associated with bacteraemia and septicaemia (Mulder et al 1997) Antibiotics Penicillin Oxacillin Tobramycin Gentamicin Trimethoprim Erythromycin Ciprofloxacin Vancomycin Rifampicin Bacteraemia (n=730) % 12 34 37 49 51 55 79 100 95 Septicaemia (n =162) % 7 15 20 28 32 44 64 100 95 1.4.2 Isolates from Ocular infections As described in the section 1.2.2 CoNS, can be isolated in diverse ophthalmic conditions. CoNS forms the part of normal flora, effective management of bacterial keratitis may not be aimed at curtailing CoNS. Antibiotic susceptibility patterns may help in judging the virulence of CoNS. Multiple drug resistant CoNS could be considered as the cause for the infection. Antibiotic resistance of CoNS isolated from keratitis, endophthalmitis and conjunctivitis are shown in table 1.5. Although antibiotic susceptibility patterns to various antibiotics are available in the literature, it is important to check susceptibility patterns periodically. Alternative antibiotics against drug resistant CoNS are not well documented in the literature. Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 27 Chapter1: Introduction and Literature Review Table 1.5: Percentage of susceptible isolates of CoNS associated with ocular infections Antibiotic Gentamicin Tobramycin Neomycin Ciprofloxacin cefazolin Vancomycin Levofloxacin Ceftazidime Chloramphenicol Cephalothin Pencillin Teicoplanin Erythromycin 1 Matthew et al 2004 2 Cameron et al 2006 3 Pinna et al 1999 Endophthalmitis1 Keratitis2 82.7 85 ND 70 ND 85 65.5 90 55.1 ND 100 ND 63.2 ND 65.5 ND ND 85 ND 100 ND ND ND ND ND ND Conjunctivitis/Blepharitis3 78.5 ND ND 92.8 ND ND ND ND ND ND 28.5 97.6 66.6 1.5 Medical management of ocular infections caused by coagulase negative staphylococci The initial therapy for suspected bacterial keratitis should include broad-spectrum antibiotics that are effective against the major pathogens in the community and that this therapy should be altered if the corneal ulcer worsens and microbiological investigations prove that the pathogen is resistant to the initial therapy. The initial therapy for suspected bacterial keratitis should include broad-spectrum antibiotics that are effective against the major pathogens in the community (Baun et al 1979). Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 28 Chapter1: Introduction and Literature Review 1.5.1 Therapeutic agents Aminoglycosides: Gentamicin, tobramycin and amikacin are the group of aminoglycosides used against bacteria causing ocular infections. They have a selective affinity to bacterial 30S and 50S ribosomal subunits to produce a non functional 70S initiation complex that, in turn facilitates the inhibition of bacterial protein synthesis. Gentamicin sulfate and tobramycin are frequently included in the initial treatment of suspected bacterial keratitis. Amikacin is included in the treatment of endophthalmitis. Glycopeptide: Vancomycin is a glycopeptide antibiotic with activity against pencillin resistant staphylococci. Its bactericidal effect is related to the inhibition of biosynthesis of peptidoglycan polymers during bacterial cell wall formation. It is primarily active against gram positive bacteria and is one of the most potent against methicillin resistant CoNS. In ophthalmic use, vancomycin is usually reserved for drug resistant staphylococci (Goodman et al 1988). However, in ocular infections the incidence of vancomycin resistant staphylococci is very rare. Cephalosporins: Cephalosporins contain a - lactam ring that is necessary for bactericidal activity. The important cephalosporins are cefazolin and ceftazidime. Cefazolins are known to have excellent activity against gram positive pathogens causing keratitis and endophthalmitis. It has minimal toxicity after topical Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 29 Chapter1: Introduction and Literature Review administration. It is frequently used with other antibiotics to provide broad spectrum coverage. Fluoroquinolones: These antibiotics act against majority of the ocular pathogens. The bactericidal action is due to inhibition of bacterial DNA gyrase and topoisomerase IV which are enzymes necessary for bacterial DNA synthesis. The second generation fluoroquinolones are frequently used as broad spectrum antibiotics. In case of resistance to second generation, fourth generation fluoroquinolones (gatifloxacin and moxifloxacin) are used. 1.5.2 Treatment of keratitis caused by coagulase negative staphylococci Treatment of bacterial keratitis begins with either combination or single agent therapy which would be effective against the major pathogens in the community. In such cases combined therapy with beta lactam antibiotics such as cefazolin and an aminoglycoside antibiotic such as tobramycin or gentamicin can effectively treat nearly all common causes of bacterial keratitis. After culture results are available, the less effective agent can be stopped. Fixed combination preparations, such as neomycin, polymyxin B and gentamicin, are effective against all gram-positive bacteria (Jones et al 1979). Specific bactericidal therapy can be initiated based on the results of the smears of corneal scrapings (Pepose et al 1995). CoNS are included under gram positive cocci therefore the treatment modalities are similar to gram positive cocci. Initial antibacterial therapy with cephalosporin such Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 30 Chapter1: Introduction and Literature Review as cefazolin or fluoroquinolones such as ciprofloxacin are generally adequate and even optimal for most gram positive cocci. A cephalosporin is an appropriate initial choice when gram positive cocci are identified in smears of corneal scrapings. First generation cephalosporins such as cefazolin or cephapirin are clinically effective. Certain second generation cephalosporins can be effective for many gram-positive cocci. Bacitracin and vancomycin are useful alternatives but are limited by solubility and toxicity problems. Currently, the cornerstone for the successful treatment of infective keratitis is effective topical medication with ciprofloxacin, although for several years fortified antibiotics have been in use. Parks et al compared the efficacy of ciprofloxacin (3mg/ml) for 15 days with combination therapy [cefazolin (50 mg/ml) and gentamicin (9.1 mg/ml)] for 50 days. They did not find any difference in the visual outcome. Similar studies by Bower et al showed that ciprofloxacin therapy was superior to combination therapy. Studies by Parks et al and Bower et al showed ciprofloxacin (0.3%) as a potent drug and it can virtually replace the use of combination therapy (Parks et al 1993, Bower et al 1996). Anti inflammatory therapy: As antimicrobial agents control the infectious process, topical corticosteroids may be used to suppress the local inflammatory response ( Leibowitz et al 1980). The potential advantages of corticosteroids include reducing postinflammatory scarring, limiting the damage produced by neutrophils, minimizing Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 31 Chapter1: Introduction and Literature Review corneal neovascularization and permitting re-epithelialization. Corticosteroids should not be used if the efficacy of antibacterial therapy is uncertain. Adjunctive treatment: Application of tissue adhesive, therapeutic contact lens, lamellar keratectomy and penetrating keratoplasty are among the adjunctive treatment options. These modalities are necessary when the integrity of the eye is compromised, such as extremely thin corneal surface, perforation, and unresponsive disease or endophthalmitis. 1.5.3 Treatment of endophthalmitis caused by coagulase negative staphylococci Management of endophthalmitis is to establish a microbiologic diagnosis, surgically remove by vitrecotmy as much of infection as safely can be achieved and, maintain intravitreal bactericidal concentrations by a combination of intravitreal and intravenous antibiotics. Although intravitreal antibiotic therapy can provide effective bacterial killing during endophthalmitis, vitrectomy is an appealing adjunct to management. Vitrectomy debrides the vitreous cavity of bacteria, inflammatory cells, and other toxic debris; promotes better diffusion of antibiotics; removes inflammatory membranes; permits earlier visualization of the retina; and speedy recovery of vision (Han et al 1999). When endophthalmitis is initially suspected, the pathogen is not typically known, so the choice of antimicrobial agent must be made empirically. Intravitreal administration of antibiotics is a key component of the clinical management of Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 32 Chapter1: Introduction and Literature Review exogenous bacterial endophthalmitis caused by CoNS (Brod et al 1993). Fluoroquinolones penetrate into the inflamed and non inflamed vitreous better than other classes of antibiotics, however, they have not been subjected to rigorous, blinded clinical trials for intravitreal use (Ferencz et al 1999). The three most commonly utilized antibiotics for intravitreal administration include vancomycin, amikacin and ceftazidime. Against CoNS, it is recommended that initial endophthalmitis management incorporates an intravitreal injection of vancomycin together with amikacin and gentamicin. Good results in CoNS endophthalmitis has been reported with use of intravitreal and subconjuctival antibiotics but with out systemic antibiotics (David et al 1999). Anti inflammatory agents: In experimental models of bacterial endophthalmitis, concomitant administration of dexamethasone was reported to be beneficial, had no effect, or was detrimental to infection outcome (Meredith et al 1996). Dexamethasone is frequently used as an adjunct to antibiotic therapy in bacterial endophthalmitis. 1.6 Virulence factors associated with coagulase negative staphylococci 1.6.1 Toxins and enzymes Toxins and enzymes play a major role in the virulence of CoNS infections. Toxins produced by CoNS during growth may alter the normal metabolism of human cells, or sometimes produce deleterious effects on the host. Exotoxins and endotoxins are the two types of toxins produced by the bacteria. CoNS produce Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 33 Chapter1: Introduction and Literature Review exotoxins. They also produce exoenzymes which are capable of disrupting the host cell structure. These pathogenicity factors in the genome of ATCC 12228 (S.epidermidis), RP62A (S.epidermidis) have been categorized into four groups, namely, the exotoxins, exoenzymes, adhesins and others(Kuroda et al 2001). ATCC 12228 is non biofilm forming, non infection associated S.epidermidis strain whereas RP62A is an methicillin resistant S. epidermidis biofilm forming infectious clinical isolate. Table 1.6 summarizes the virulence factors present in the sequenced Staphylococcal genomes. Factors like enterotoxin, exotoxin, toxin shock syndrome, serine protease, staphylokinase, leukotoxin D and alpha haemolysin are present in S.aureus and absent in S.epidermidis. (1) Exotoxins: Aside from haemolysin and haemolysin genes, other potent exotoxin genes, such as the -haemolysin, leucocidin, enterotoxins, toxic-shock syndrome toxins present in S. aureus have not been found in ATCC 12228 and RP62A(Zhang et al 2003, Gill et al 2005). The -haemolysin gene (hld) encodes a short and heat-labile delta toxin which can be secreted from the bacteria and show lytic activity against many types of membranes (Kevitt et al 1990). In the ATCC 12228 genome, hld is located near the 5¢-end of RNAIII in the agr(accessory gene regulator) locus and RNAIII from S. epidermidis can regulate agr-dependent virulence genes in S. aureus strains. Furthermore, S. epidermidis can generate auto-inducing peptides (also called pheromones) which inhibit the agr response of other Staphylococcus members. Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 34 Chapter1: Introduction and Literature Review (2) Exoenzymes. The secreting exoenzymes that degrade or digest lipids are present in ATCC 12228 and RP62A. The geh1 gene which encodes a lipase plays important role in S. epidermidis skin colonization. Other genes coding for proteolytic enzymes, such as the extracellular metalloprotease (SE2219) with elastase activity(Otto et al 1999), serine protease (V8 protease) (SE1543), which catalyses the processing of the epidermin precursor peptide and genes coding for nucleases (Geissler et al 1996) e.g. thermonuclease (SE1004), are found in both the ATCC strains of S. epidermidis. However, genes coding for more virulent exoenzymes, such as staphylocoagulase, staphylokinase and hyaluronidase, which can interact with host tissues or inter tissue components for invasion into deeper tissues, are absent in S. epidermidis. These findings can explain why S. epidermidis strains are common inhabitants of skin or mucous membrane, but usually do not invade deeper tissues. (3) Adhesins Adhesins are the most important pathogenic factors for S. epidermidis. Polysaccharide adhesin (PS/A) and one or more proteins, including the autolysin E (Mack et al 1994) are involved in the initial adherence, whereas the accumulation of cells is due to the production of polysaccharide intercellular adhesin (PIA), the synthesizing enzyme of which is encoded by the ica operon. The ica locus is composed of the genes icaA, icaD, icaB, and icaC. icaA is an Nacetylglucosaminyltransferase, which only reaches low activity without the presence of icaD. icaA and icaD only produce N-acetyl oligomers of up to 20 residues of Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 35 Chapter1: Introduction and Literature Review length. IcaC is responsible for the production of PIA of full length, able to react with anti-PIA antisera. The function of IcaB remains unclear. Table 1.6: Comparison of virulence factors in sequenced Staphylococcus spp. Staphylococcus Staphylococcus Staphylococcus aureus epidermidis epidermidis Virulence factor (N315) (RP62A) (ATCC12228) Enterotoxin P A A Exotoxin P A A Toxin shock syndrome P A A Esterase P P P Serine protease P A A Staphylokinase P A A Serine V 8 Protease P P P Lipase P P P Leukotoxin D P A A Alpha hemolysin P A A Beta hemolysin P P P Delta hemolysin P P P Gamma hemolysin P A A Hemolysin III P P P Thermonuclease P P P Zinc metallo protense P P P Phenol soluble modulin P P P Fironectin binding proteins P P P Inter cellular adhesin proteins P P A P: Present, A: Absent In ATCC 12228, the entire ica operon is absent. When compared to RP62A, the genes adjacent to the putative ica operon region in ATCC 12228 showed inversions, insertions and significant rearrangements. The non-biofilm forming phenotype of ATCC 12228 is due to a genetic defect. Another important gene cluster missing in ATCC 12228 is capA-P. The capsular polysaccharides encoded by cap found in S. Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 36 Chapter1: Introduction and Literature Review aureus are important virulence factors. Other adhesin genes described in S. epidermidis clinical isolates, such as the atIE gene and the fbe gene (Nilsson et al 1998) are present in ATCC 12228. Zhang et al suggest that there could be two groups of commensal S. epidermidis strains. The members of one group of S. epidermidis strains do not contain the ica operon and are ‘innate’ commensals which are unlikely to become invasive. The other group of S. epidermidis strains do have ica operon and can become invasive. (4) Other virulence factors: In S. aureus the enzyme sortase cleaves PXTG motif for anchoring surface proteins to the cell wall (Mazmanian et al 1999). A predicted protein homologue of this enzyme has been detected in ATCC 12228 (SE2076), which indicates that this enzyme is conserved in S. epidermidis strains. The finding of sortase in other species of Staphylococcus supports the suggestion of using this enzyme as a target for development of new antimicrobial drug Mazmanian et al 1999). Another possible pathogenic factor is a 67 kDa myosin-cross reactive protein (SE0776) which may initiate immunopathological responses in host tissues. Coupled with this, there is a CDS (SE0997) which codes for a cardiolipin synthetase with unknown function. (5) Regulatory genes ATCC 12228 does not have more divergent types of regulatory genes than those present in S. aureus. Regulatory genes identified in ATCC 12228 are similar to those reported from clinical isolates of S. epidermidis and S.aureus strains. Regulators Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 37 Chapter1: Introduction and Literature Review involved in pathogenesis including the sigma B operon (SE1668-SE1671), autolysin (atl) transcriptional regulator (SE0749), the staphylococcal accessory regulators (sarA, SE0390, sarR, SE1868), the agr locus (SE1635-SE1638) and the RNAIII regulator are all detected. This indicates that these conserved regulators are important in controlling the expression of S. epidermidis proteins. Iron is an essential nutrient for the survival and pathogenesis of bacteria, but relatively little is known regarding its transport and regulation in S.epidermidis. It is known that ferric-uptake regulatory (fur) genes regulate iron-transport processes in S. aureus. Genome analyis showed that conserved regulatory proteins for iron uptake and a transcriptional regulator of the Fur family are also present in ATCC 12228 (Xiong et al 2000). The most likely candidate for a bona fide virulence factor in S. epidermidis apart from adhesins is the family of small cytokine-stimulating peptides (22 to 44 amino acids in length) previously identified as phenol soluble modulin ( PSM)(otto et al 2004). Members of the PSM family are present in other staphylococci, including S. aureus, but they are more numerous in S. epidermidis, where they appear to have expanded as a result of gene duplication within the genome island. Comparison of the S. epidermidis genomes revealed that a key difference between the ATCC12228 type strain and RP62a is the presence of the intercellular adhesion locus (icaABCD) and the cell wall associated biofilm protein (Bap) or Bap homologous protein (Bhp) (Toledo et al 2001). Table 1.7 shows the important genes which distinguish virulent S. epidermidis (RP62A) and commensal S.epidermidis (ATCC12228). Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 38 Chapter1: Introduction and Literature Review Table 1.7: Virulence genes distinguishing RP62A (pathogenic S. epidermidis) and ATCC12228 (non pathogenic S. epidermidis) Genes ATCC12228 RP62A ica AB A P cap operon P P Bhp A P psm P P mecA A P ppb4 P A P: Present, A: Absent 1.6.2 Biofilm Bacteria interact with the host and cause disease by a variety of complex mechanisms. In order to colonize the host, the bacteria must first adhere to its surface. Biofilm formation is one kind of colonization strategy observed in CoNS (CoNS). The surfaces that allow biofilm formation can range from abiotic surfaces such as intraocular lenses (Dilly et al 1989) and contact lenses to biotic surfaces such as the eukaryotic cells (Merkel et al 2001). Biofilm formation requires the adhesion of bacteria to a solid structure, followed by the bacterial production of a polysaccharide glycocalyx (slime) that prevents antibiotics from gaining access to the microorganisms and reduces the efficacy of host defenses. Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 39 Chapter1: Introduction and Literature Review CoNS are the most frequent microorganisms in bacterial keratitis (Pinna et al 1999). and post-surgical cataract endophthalmitis (Asaria et al 1999). It is a member of the normal ocular and periocular surface micro flora thereby gaining entry into the eye through the incision sites at the time of surgery or wound. It can adhere on scleral buckles after retinal detachment surgery and on soft contact lenses resulting in an increased potential for a hypersensitivity reaction or even infection. There are certain conditions to be fulfilled for such indigenous bacteria to become disease causative bacteria(Miyanaga et al 1997). These conditions could be the following: 1. In vivo, a small breach in epithelium, to which the bacteria may adhere, must be present in the cornea and conjunctiva. 2. Contaminated contact lens or intraocular lens in the eye. 3. Underlying disease or low resistance in host (immunocompromised). 4. Slime production by CoNS. 5. Heavy inoculum (105CFU/ml). The adherence of CoNS to IOLs during implantation is known to be a prominent factor in the pathogenesis of endophthalmitis and pseudophakic chronic intraocular inflammations. These biofilms may prevent antibiotics from gaining access to the microorganisms and reduce the efficacy of host defenses (Kodjikan et al 2003, Nucci et al 2005). In a study by Das et al, Staphylococcus epidermidis adhered to IOL with 108 cfu/mL and 103 cfu/mL bacterial loads and treatment of the IOL with vancomycin reduced the adherence of bacteria to the IOL (Das et al 2002). Georgakopoulos showed the production of slime by CoNS in an Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 40 Chapter1: Introduction and Literature Review experimental animal keratitis model (Georgakopoulos et al 2002). In keratitis, 68.9% of the isolates have been shown to be slime producing (Nayak et al 2000). Seventy nine percent of these slime positive isolates are resistant to three or more drugs (Nayak et al 2000). Several studies proved that the adherence of CoNS to contact lens resulted in inflammation of the cornea (Slusher et al 1987). Slime negative strains have been shown to have less adherence or no adherence to contact lens whereas slime positive strains had good adherence to contact lens. Electron microscopy revealed the presence of staphylococcal biofilms in infectious crystalline keratopathy (Lubniewski et al 1990) and punctal plugs (Sugita et al 2001). 1.6.3 Biofilm inhibitors Once a biofilm has been established in the host, the bacteria inside are less exposed to the host’s immune response and are protected against antibiotic action. Slow penetration of the antibiotic into the biofilm, reduction in the nutrient supply and change in the phenotype of the bacteria are some of the reasons for the failure of antibiotic action against biofilm forming bacteria (Davies et al 2003). Antibiotic resistance is particularly problematic in biofilm-associated infections in staphylococci. In such cases inhibitors that inhibit biofilm formation can act as antimicrobial agents. Biofilm inhibitors can be classified as those that inhibit the quorum sensing mechanism [RNA III induced peptide (Balaban et al 2001,), Acyl homoserine lactone Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 41 Chapter1: Introduction and Literature Review (Saara et al 2006)], those that inhibit slime associated proteins [Aspirin (Muller et al 1998)]and those that inhibit the exoploysaccharide expression. Examples of the compounds that inhibit the exopolysaccharide expression are N acetyl cysteine (Perez et al 1997), Epigallocatechin gallate (Wolinsky et al 2000), 3, 4 dioxopyrazolidines (Yang et al 2006) and Bismuth thiols (Domenico et al 2001). 1.7 Genetic typing Understanding pathogen distribution and relatedness is essential for determining the epidemiology of nosocomial infections and aiding in the design of rational pathogen control methods. The role of pathogen typing is to determine if epidemiologically related isolates are also genetically related. Historically, this analysis of nosocomial pathogens has relied on a comparison of phenotypic characteristics such as biotypes, serotypes, bacteriophage or bacteriocin types, and antimicrobial susceptibility profiles. This approach has begun to change over the past 2 decades, with the development and implementation of new technologies based on DNA, or molecular, analysis. The incorporation of molecular methods for typing of nosocomial pathogens has assisted in efforts to obtain a more fundamental assessment of strain interrelationship. Establishing clonality of pathogens can aid in the identification of the source (environmental or personnel) of organisms, distinguish infectious from noninfectious strains, and distinguish relapse from reinfection. Many of the species that are key hospital-acquired causes of infection are also common commensal organisms, and therefore it is important to be able to determine whether the isolate recovered from the patient is a Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 42 Chapter1: Introduction and Literature Review pathogenic strain that caused the infection or a commensal contaminant that likely is not the likely source of infection. Likewise, it is important to know whether a second infection in a patient is due to reinfection by a strain distinct from that causing the initial infection or whether the infection is likely to be a relapse of the original infection. If the infection is due to relapse, this may be an indication that the initial treatment regimen was not effective, and alternative therapy may be required. Ubiquitous prevalence of CoNS as a commensal makes it difficult for a clinician to decide whether an isolate represents the causative agent of an infection or culture contamination. Research progress into genome research and molecular epidemiology and physiology have given novel and important insights into genome structure and biology as well as the spread of nosocomial staphylococci. The important genetic profile techniques are pulse field gel elctrophoresis (PFGE), Fluorescent amplified fragment length polymorphism (FAFLP), multi locus sequence typing (MLST) and Microarray. Pulse field gel electrophoresis (PFGE) The principle behind this procedure is the ability of the DNA to travel through a gel sieve and get differentiated based on its length. The agarose gel is basically chains of sugar molecules cross linked to each other. Led by an electric current, the negatively charged DNA is forced to travel through this filter in the direction of the positive pole. Smaller pieces of DNA will move more freely than larger pieces, therefore will travel further in the direction of the current. In the case of Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 43 Chapter1: Introduction and Literature Review PFGE, the segments of DNA are very large (the entire bacterial genome is cut with one or two restriction enzymes) and would have a very hard time traveling through the gel. To aid the passage of large DNA fragments, the current is periodically reversed in polarity forcing the pieces of DNA to move in different directions, although the predominant direction is toward the bottom of the gel. This back and forth motion allows the large fragments to ‘snake’ their way through the gel. Based on this principle, PFGE is able to determine the lengths of the DNA fragments in relation to the other samples used. For molecular investigations of CoNS, PFGE was used by Schegal (Schegal et al 2002), pulsed field electrophoresis (PFGE) with Sma I endonuclease demonstrated that CoNS isolates obtained from different patients were unrelated. The CoNS in this study were isolated from intensive care unit. In CoNS isolates from endophthalmitis Bannerman et al (Bannerman et al 1997) used PFGE to show that CoNS present as commensal and CoNS present causing ocular infections are similar. Multilocus sequence typing (MLST) MLST is based on the comparison of the nucleotide sequences of seven housekeeping genes of microorganisms. It can be used to elucidate relationships between strains and to identify ancestral genotypes, as well as to predict patterns of evolutionary divergence within groups of related genotypes. Epidemiological analyses using multilocus sequence typing (MLST) and genetic studies suggest that S. epidermidis isolates in the hospital environment differ from those obtained outside of medical facilities with respect to biofilm formation, antibiotic resistance, and the presence of mobile DNA elements. DNA sequences for Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 44 Chapter1: Introduction and Literature Review internal fragments of seven housekeeping genes were compared in 47 geographically and temporally diverse S. epidermidis isolates that were obtained from clinical infections. In this study twenty three different allelic profiles were detected; 17 of these were represented by single strains and the largest profile group contained 17 isolates (Wang et al 2003). MLST was employed for the clonal analysis of 118 S. epidermidis ica-positive and -negative strains. MLST revealed that the majority of ica-positive and -negative strains were closely related and formed a single clonal complex. Within this complex one sequence type (ST27) was identified which contained exclusively ica-positive isolates and represented the majority of clinical strains tested. ST27 and related ica-positive clones carried different SCCmec cassettes (conferring methicillin resistance) and the insertion sequence IS256 (Kozitskayan et al 2005). However, MLST has not been used in CoNS isolated from ocular infections. Amplified fragment-length polymorphism (AFLP) AFLP or its fluorescent version (FAFLP) is a polymerase chain reaction (PCR)based fingerprinting technology. AFLP involves the restriction of genomic DNA, followed by ligation of adaptors complimentary to the restriction sites and selective PCR amplification of a subset of the adapted restriction fragments. These fragments are visualized on denaturing polyacrylamide gels either through autoradiographic or fluorescence methodologies. Compared to other marker technologies, e.g. randomly amplified polymorphic DNA (RAPD), restriction fragment-length polymorphism (RFLP) or microsatellites, AFLP Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 45 Chapter1: Introduction and Literature Review provides equal or greatly enhanced performance in terms of reproducibility, resolution, and time efficiency. The single greatest advantage of the AFLP technology is its sensitivity to polymorphism detection at the total-genome level. With all of these assets, AFLP markers can be molecular standard for investigations of genetic profiles. Sools et al used FAFLP for typing CoNS from blood infections In this study the applicability of AFLP in epidemiological studies of S. epidermidis was tested on 11 sets of four blood isolates each, from 11 patients with suspected septicaemia. Nine sets had indistinguishable or highly similar AFLP patterns for each isolate per set, while two sets had heterogeneous patterns. These results show that AFLP has high discriminatory power for strain identification in S. epidermidis (Sools et al 1997). So far FAFLP was not used to differentiate ocular CoNS isolates from diseased and normal individuals. Microarray A microarray is a series of nucleic acid targets immobilized on a solid substrate. Hybridization of fluorescently labeled probes made from nucleic acids in the test sample to these targets allows analysis of the relative concentrations of mRNA or DNA in the sample. Microarrays have opened the way for the parallel detection and analysis of the patterns of expression of thousands of genes (currently about 20, 000–40, 000) in a single experiment. The high capacity for data generation with microarray-based approaches has time and resource advantages. Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 46 Chapter1: Introduction and Literature Review Microarray based approaches allow researchers to interrogate host and pathogen genomes without prior bias as to which genes or pathways might be involved in a disease process. Global gene expression enables the investigation of entire biological pathways, many of which might be of previously unknown function. The context in which such genes are upregulated and downregulated provides insights into functional responses of both host and pathogen. Yao et al used microarray based genome-wide comparison of clinical and commensal S. epidermidis strains to identify putative virulence determinants. Their study revealed high genetic variability of S. epidermidis as a species. This study also identified genes with unknown function for use as potential novel drug targets (Yao et al 2005). Keratitis and endophthalmitis caused by coagulase negative staphylococci: An investigation into clinico-microbiologic features, virulence factors and genome profile 47