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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
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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
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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.
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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
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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
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(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).
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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
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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
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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
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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
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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
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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
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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
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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.
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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
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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
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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
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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
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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
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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 37C 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
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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.
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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
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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
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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
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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
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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.
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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.
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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
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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
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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
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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
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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
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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
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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.
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(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
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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
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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
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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
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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.
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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
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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
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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
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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
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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
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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
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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.
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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
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