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Indian Journal of Medical Microbiology, (2003) 21 (4):239-245

Original Article

EPIDEMIOLOGY OF BACTERIAL KERATITIS IN A REFERRAL

CENTRE IN SOUTH INDIA
*MJ Bharathi, R Ramakrishnan, S Vasu, R Meenakshi, C Shivkumar, R Palaniappan

Abstract
Purpose: To study the epidemiological characteristics of bacterial keratitis seen at a tertiary eye care
referral centre in south India. Methods: A retrospective review of medical records of all culture-positive
bacterial keratitis which were seen over a 3 years period, from September 1999 through August 2002
was performed. After clinical evaluation corneal scrapings were collected and subjected to culture and
microscopy using standard protocols in all patients. Results: Out of 3183 corneal ulcers evaluated,
1043(32.77%) were found to be of bacterial aetiology. A total of 1109 bacterial pathogens were isolated
from 1046 eyes with keratitis. The predominant bacterial species isolated was Streptococcus pneumoniae
(37.5%). Males were 592(56.76%) and 451(43.24%) were females. There were 564(54.07%) rural residents
and 479(45.93%) urban residents; this difference was statistically significant (p< 0.0001). Patients with
age more than 50 years (60.2%) were affected significantly more than patients aged less than 50 years
(30.8%). While 57.62% of patients were non-agricultural workers, 42.38% were farmers; this difference
was statistically significant (p<0.0001). Co-existing ocular diseases predisposing to corneal ulceration
were identified in 703(67.4%) patients, compared to other predisposing risk factors in 340(32.6%) patients.
One hundred and seventy seven (16.97%) had corneal injury with soil and/or sand, compared to
115(11.03%) patients who had injury due to other materials and the difference was statistically significant.
There was lower incidence of bacterial keratitis from June to September. Conclusions: The epidemiological
characteristics of bacterial keratitis vary geographically. This study describing the features of bacterial
keratitis would greatly help the practising ophthalmologist and other medical practitioners in the
management of their patients.

Key words: Bacterial keratitis, culture, epidemiology, risk factors, seasonal variation

Microbial keratitis is a common, potentially sight-
threatening ocular infection that may be caused by
bacteria, fungi, viruses or parasites.1 Bacterial keratitis
rarely occurs in the normal eye because of the human
cornea’s natural resistance to infection. However,
predisposing factors such as corneal injury, contact lens
wear, ocular adnexal dysfunction (including tear
deficiencies), corneal abnormalities and other exogenous
factors, systemic diseases and immunosuppression may
alter the defense mechanisms of the outer eye and permit
bacteria to invade the cornea.2,3 Bacterial corneal
ulceration is an ocular emergency due to the often rapid
progression of this corneal infection with the threat of
visual loss and potential corneal perforation.4 The
*Corresponding author
Department of Microbiology (MJB) and Cornea Service
(RR, SV, RM, CS), Aravind Eye Care System, Aravind
Eye Hospital and Postgraduate Institute of
Ophthalmology, Tirunelveli - 627 001, Tamil Nadu,
India; Postgraduate Department of Microbiology (RP),
Sri Paramakalyani College, Alwarkuruchi, Tirunelveli -
627 412, Tamil Nadu, India.
Received : 13-03-2003
Accepted : 29-06-2003
majority of bacteria cultured from infections of cornea
are of the same species that normally are present in the
conjunctival sac, on the lids or periocular skin, and in
the adjacent nasal passages.2
Considering the importance of corneal ulceration as
an important cause of visual loss, many studies have
reported the prevalence of microbial pathogens and
identified the risk factors predisposing a population to
corneal infection in south India.5-7 The aetiological and
epidemiological patterns of corneal ulceration have been
found to vary with the patient population,health of the
cornea, geographic location and climate, and also tends
to vary somewhat over time.8 Hence, an understanding
of the epidemiological features, risk factors and
aetiological agents that occur in specific region are
important in rapid recognition, timely institution of
therapy, optimal management and prevention of this
disease entity. In order to start specific therapy, it is
necessary to do meticulous laboratory investigations, and
this includes microscopy and culture of corneal
scrapings.
The purpose of this study was to determine the risk
factors and other epidemiological characteristics, and to

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240 Indian Journal of Medical Microbiology
identify specific bacterial pathogens causing bacterial
keratitis in patients presenting at a tertiary referral eye
care centre in south India.
Materials and Methods
Patients
A retrospective analysis of all patients with culture-
proven bacterial keratitis seen over a period of three
years from September 1999 to August 2002 was
performed. A total of 3183 consecutive patients with
corneal ulceration were analysed. Ulceration was
defined as a loss of the corneal epithelium with
underlying stromal infiltration and suppuration
associated with signs of inflammation with or without
hypopyon. Ulcers with typical features of viral infection
and healing ulcers were excluded as were Mooren’s
ulcers, interstitial keratitis, sterile neurotropic ulcers, and
any ulcer associated with autoimmune conditions. Data
related to sociodemographic features, duration of
symptoms, predisposing factors, history of corneal
trauma, traumatic agents, associated ocular conditions,
other systemic diseases, therapy received prior to
presentation, visual acuity at the time of presentation and
all clinical findings were collected from medical records
of the patients.
All patients had undergone thorough slit-lamp
biomicroscopic examination by an ophthalmologist.
After a detailed ocular examination, using standard
techniques, corneal scrapings were taken under aseptic
conditions from each ulcer by an ophthalmologist using
a sterile Bard-Parker blade (No 15).6,9,10 The procedure
was performed under the magnification of a slit-lamp
or operating microscope after instillation of 4%
lignocaine (lidocaine) without preservative. The material
scraped from the leading edge and the base of each ulcer
was initially directly inoculated onto the surface of solid
media such as 7% sheep blood agar, chocolate agar and
Sabouraud glucose neopeptone agar (pH 5.6 + 0.2,
Carlier modification of Sabouraud formulation) in a row
of C- shaped streaks. Material was also inoculated into
the depth of liquid media such as brain heart infusion
broth and thioglycollate medium. The material obtained
by scraping was also spread onto labeled slides in a thin,
even manner to prepare a 10% potassium hydroxide
(KOH) wet mount and to prepare smear for Gram
staining and Giemsa staining. In cases of suspected
actinomycete keratitis, Kinyoun’s method of acid fast
staining was performed. When KOH smears revealed
cysts with the morphology of Acanthamoeba spp. further
corneal scrapings were performed and the material was
inoculated onto non-nutrient agar. Meticulous care was
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Vol.21, No.4
taken in the collection of material and in transferring it
aseptically to the appropriate culture media.
Laboratory Procedures
All inoculated media were incubated aerobically.
The inoculated Sabouraud dextrose agar were incubated
at 270 C, examined daily, and discarded at three weeks
if no growth was seen. The inoculated plates of blood
agar and chocolate agar, and tubes of thioglycollate
broth and brain heart infusion broth were incubated at
370 C, examined daily, and discarded at 7 days if growth
was not seen. Broth tubes were held upright in racks.
The inoculated non-nutrient agar plates were incubated
at 370 C after overlaying with Escherichia coli broth
culture and were examined daily for the presence of
Acanthamoeba spp. and discarded at three weeks, if
there were no signs of growth. All laboratory methods
followed standard protocols.6,9,10 Microbial cultures
were considered significant if growth of the same
organism was demonstrated on more than one solid
phase medium, and/or if there was confluent growth at
the site of inoculation on one solid medium, and/or if
growth of one medium was consistent with direct
microscopy findings (i.e., appropriate staining and
morphology with Gram stain) and/or if the same
organism was grown from repeated scraping. Pearson’s
chi-square test was used to carry out the statistical
analysis wherever required.
Results
Microbiological profile
Bacteria alone were recovered in culture from the
corneal scrapings of 1043(32.77%) patients, fungi alone
from 1095(34.4%), Acanthamoeba alone from
33(1.04%) and both bacteria and fungi from 76(2.39%).
There was no growth in culture from the corneal
scrapings of 936(29.41%) patients. Of the 1043 culture
positive bacterial keratitis cases, one eye was infected
in 1040(99.71%) patients and both eyes were infected
in 3(0.29%) patients. Of the 1046 culture positive eyes,
983(93.98%) eyes had a single species of bacterial
growth and 63(6.02%) eyes had two species of bacterial
growth. A total of 1109 bacterial pathogens were
isolated from the 1046 eyes which yielded only bacterial
growth in culture. Of the 1109, 722(65.1%) were gram
positive cocci, 274(24.71%) were gram negative bacilli,
55(4.96%) were gram positive bacilli, 46(4.15%) were
aerobic actinomycetes (Nocardia spp.) and 12(1.08%)
were gram negative cocci and coccobacilli. The
predominant bacterial species isolated were
Streptococcus pneumoniae 416(37.5%) followed by
Pseudomonas aeruginosa 200(18.03%) and
Staphylococcus epidermidis 193(17.4%).
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October, 2003 Bharathi et al - Bacterial keratitis 241

Table 1 : Bacterial pathogens isolated from 1046 culture-positive bacterial keratitis eyes
Name of the bacterial Pure Mixed with Total
isolates isolates other bacteria bacterial isolates (%)
Total gram positive cocci 659 63 722 (65.1)
S.pneumoniae 411 5 416
S.epidermidis 150 43 193
S.aureus 33 10 43
Miccrococcus spp. 6 0 6
Alphahaemolytic streptococci 44 5 49
Beta haemolytic streptococci 6 6
Non-haemolytic streptococci 9 9
Total gram positive bacilli 33 22 55 (4.96)
Bacillus spp. 12 15 27
Corynebacterium spp. 21 7 28
Total gram negative cocci and 12 12 (1.08)
cocco bacilli
Moraxella spp. 9 9
Neisseria spp. 3 3
Total aerobic actinomycetes 39 7 46 (4.15)
Nocardia spp. 39 7 46
Total gram negative bacilli 240 34 274 (24.71)
Pseudomonas spp. 173 27 200
Enterobacter spp. 24 5 29
Klebsiella spp. 10 2 12
Proteus spp. 6 6
Alkaligens spp. 6 6
Haemophilus spp. 6 6
Acinetobacter spp. 6 6
E.coli 4 4
Serratia spp. 3 3
Citrobacter spp. 2 2
Total no. of isolates (%) 983 (88.64) 126 (11.36) 1109 (100)

Demographics Co-existing ocular diseases predisposing to corneal

ulceration were identified in 703 (67.4%) patients,
Out of 1043 patients 592(56.76%) were males and
compared to other predisposing risk factors in 340
451 (43.24%) were females. There were 564 (54.07%)
(32.6%) patients (p< 0.0001). The co-existing ocular
rural residents and 479 (45.93%) urban residents and the
diseases are shown in Table 2. A history of corneal
difference was statistically significant (p <0.0001).
injury was recorded in 292(28%) patients, of which 177
Patients above the age of 50 years (628, 60.2%) were
(16.97%) had corneal injury with soil and/or sand and
significantly (P<0.0001) more than patients below 50
115 (11.03%) had injury due to other materials. The
years (415, 39.8%). Non-agricultural workers were
percentage of patients who reported a history of corneal
significantly (P<0.001) more in number than were
injury with soil and/or sand was significantly higher than
farmers (442, 42.38%).
the percentage reporting injury due to other traumatising
agents (p<0.0001).

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242 Indian Journal of Medical Microbiology Vol.21, No.4

Table 2 : Demographic characteristics of 1043 cases of culture-positive bacterial keratitis

Demogrphics Particulars n (%)
Occupation Agricultural worker 442 (42.38)
Labourer 246 (23.58)
Students / Children 217 (20.80)
Household 105 (10.07)
Unemployed 18 (1.73)
Tradesman / Professional 15 (1.44)
Age in years 0 - 10 years 55 (5.27)
11 - 20 years 116 (11.12)
21 - 30 years 82 (7.86)
31 - 40 years 67 (6.42)
41 - 50 years 95 (9.11)
51 - 60 years 231 (22.15)
61 - 70 years 287 (27.52)
71 - 80 years 100 (9.59)
> 80 years 10 (0.96)
Predisposing factors Corneal trauma 292 (28)
Co-existing ocular diseases: 703 (67.41)
chronic dacryocystitis 242 (23.2)
spheroidal degeneration 234 (22.44)
blepharitis 74 (7.09)
suture infiltrates 30 (2.88)
conjunctivitis 26 (2.49)
dry eye syndrome 24 (2.3)
bullous keratoplasty 19 (1.82)
pre-existing viral keratitis 13 (1.25)
lid abnormalities 12 (1.15)
bell’s palsy 10 (0.96)
lagophthalmos 8 (0.77)
trichiasis 6 (0.58)
adherent leucoma 5 (0.48)
Contact lens usage 33 (3.16)
Use of topical steroids 7 (0.67)
Systemic diseases: 8 (0.77)
Stevens-Johnson syndrome 2 (0.19)
Leprosy 6 (0.58)
Traumatic agents Total No. of trauma 292 (28)
Soil/Sand/Stone 177 (16.97)
Vegetative matters 26 (2.49)
Dirt 26 (2.49)
Miscellaneous items 63 (6.04)

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October, 2003
Of 1046 patients 550(52.58%) patients had bacterial
corneal ulcers in the left eye, 493(47.13%) in the right
eye and 3(0.29%) in both eyes. Figure depicts the
seasonal distribution of 1043 culture positive bacterial
keratitis cases analyzed over a period of three years. Our
data revealed that in south India, there was lower
incidence of bacterial keratitis from June to September
than other months.
Figure : Seasonal observation of 1043 culture-positive
cases of bacterial keratitis seen at tertiary eye care
referral centre in south India from September 1999
to August 2002
60
38.82
39.31
26.39
% of Bacterial keratitis
40
20
0 study, but it is not statistically significant (p= 0.075)).
1999-2000
Oct - Jan Feb - May
Discussion
At birth the eyes are sterile but they soon become
invaded by various bacteria and other microorganisms.
The conjunctival sac and lid margins of the eye harbour
a variety of bacteria. The interior structures are sterile. 11
The bacteria that are normally present can be arranged
in two groups; the resident bacteria which are constantly
present in the eye and which if disturbed, promptly re-
establish themselves (Corynebacterium spp.); the
transient bacteria which consist of non-pathogenic or
potentially pathogenic bacteria that inhabit the eye for
short periods.11 Almost any species of bacteria can infect
the cornea if the integrity of the natural anatomic barriers
or defense mechanisms is compromised.11, 8 The present
study describes features of 1043 culture-proven cases of
bacterial keratitis diagnosed at a tertiary eye care centre
in south India over a period of three years.
In this study, bacterial keratitis accounted for
32.77% out of 3183 corneal ulcers evaluated. Bacterial
keratitis has been reported to account for 32.3% of all
cases of corneal ulcer evaluated in Madurai (south
India),6 29.3% in Thiruchirapalli (south India),5 35.6%
in south Florida12 and 25% in southern Ghana.13 In
marked contrast, a study performed in Nepal
documented the occurrence of a bacterial aetiology in
63.2% of all corneal ulcers.14 Bacteria are responsible
Bharathi et al - Bacterial keratitis 243
for a larger proportion of corneal ulceration in temperate
climates such as Britain 15 and northern United States 16
than in tropical regions such a south India, but in the
sub-tropical urban climates of Hong Kong bacteria are
a predominant cause for microbial keratitis.17
Of 1043 patients, 1040(99.71%) had unilateral
bacterial keratitis and 3(0.29%) bilateral bacterial
keratitis and there was a total of 1109 bacterial isolates.
Of the 1046 eyes, 983(93.98%) had single species of
bacterial growth and 63(6.02%) had two species of
bacterial growth. Streptococcus pneumoniae (37.51%)
was the predominant bacterial species in this study and
was similar to the reports from Madurai,6 Trichirapalli5
and Nepal. 14 In contrast, predominance of Pseudomonas
36.62
41.03
23.94
33.03
37.06
24.72
aeruginosa in Ghana 13 and Hong Kong, 17 and
Staphylococcus spp in south Florida, 12 Switzerland18 and
Hyderabad19 have been reported. These reports show
that there is distinct pattern of geographical variation in
the aetiology.
Male preponderance (56.76%) was noted in our
2000-2001 2001-2002 The incidence of bacterial keratitis was 45.93% in urban
Year and 54.07% in rural area and this difference was
Jun - Sep statistically significant. Urban residents have 2.98(95%
Confidence Interval: 2.54 - 3.4) times higher odds of
getting bacterial keratitis. In marked contrast,
significantly higher incidence of Nocardia keratitis has
been reported among rural residents and accounted for
24(77.24%) of 31 culture-positive Nocardia keratitis
evaluated in our previous study.20
There was significantly higher incidence of bacterial
keratitis among patients other than agricultural workers
(p<0.0001). This study shows that there is significant
association between occupation and bacterial keratitis.
Those who are occupied elsewhere are 2.88(95% CI:
2.47 - 3.36) times more prone to bacterial keratitis than
those who are engaged in farm work, while the higher
incidence of Nocardia spp. keratitis has been reported
among farmers (71%) in our previous study.20
Bacterial keratitis is significantly higher (60.21%)
among those aged >50 years in our present study
(p<0.0001). Those aged >50 years are 6.11(95% CI:
5.19% - 7.19%) times more prone to bacterial keratitis
than those less than 50 years. This is in contrast to
fungal keratitis which affects more of younger age group
(21-50 years).6
Corneal trauma is the leading cause of microbial
keratitis.6,14,21 However, in the present study the most
common risk factors identified for the development of
bacterial keratitis was co-existing ocular diseases. There
is significant (p<0.0001) association between associated
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244 Indian Journal of Medical Microbiology
ocular conditions and bacterial keratitis. Eyes that have
co-existing ocular diseases are 33.99(95% CI+: 27.37 -
42.21) times more prone to bacterial keratitis than those
that do not have any. Similarly Schaefer et al reported
that co-existing ocular diseases and contact lens wear
were predominant predisposing factor (80%) for the
development of bacterial keratitis.18 In marked contrast,
corneal injury has been reported as the predominant
predisposing factor for the development of Nocardia
keratitis in our previous study20 and Gopinathan et al
reported higher incidence of fungal keratitis due to
corneal injuries in Hyderabad.22 Chronic dacryocystitis
and spheroidal degeneration were the most common risk
factors and they were encountered in 242(23.2%) and
234(22.44%) patients respectively in our study. Severe
blepharitis was present in 74(7.09%) patients. Loose
suture infiltrates extending to corneal ulceration was
noted in 30(2.88%) patients. 26(2.49%) patients had
conjunctivitis, 24(2.3%) had dry eye syndrome and other
complications such as bullous keratopathy, pre-existing
viral keratitis, lid abnormalities, Bell’s palsy,
lagophthalmos, trichiasis and adherent leucoma were
seen to be co-existing in 73(7%) patients.
A history of corneal trauma associated with bacterial
keratitis was documented in 292(28%) patients. Corneal
injury with soil, sand and / or stone resulting in bacterial
keratitis was significantly more than with any other
material (p<0.0001). Those eyes that had an injury
where soil was the agent had 5.89(95% CI: 4.54% -
7.62%) times the odds of bacterial keratitis than those
that had trauma by other agents. Gopinathan et al found
vegetative matter as the principal traumatic agent for the
development of fungal keratitis.22 Bacterial keratitis
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Vol.21, No.4
associated with contact lens wear was documented in
33(3.16%) patients and all 33(100%) had gram negative
bacilli growth. Similarly Houang et al reported the
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ulcers of contact lens wearers.17 Prolonged use of topical
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