Saudi Journal of Ophthalmology (2015) 29, 255258

Original Article

Is there is an association between the presence of Staphylococcus

species and occurrence of vernal keratoconjunctivitis?
Ahmed M. Al-Hakami a,; Abdulrahaman Al-Amri b; Ihab Abdulrahim a; Mohamed E. Hamid a

Abstract
Purpose: The aim of this study was to identify the association of normal bacterial flora with vernal keratoconjunctivitis (VKC) occurrence in VKC and non-VKC groups.
Methods: Conjunctival specimens were collected from 18 VKC patients and 22 healthy controls, cultured and identified following
standard methods. The association between the presence of bacteria and occurrence of VKC was analyzed using Chi square
statistic.
Results: Comparable bacterial growth was observed in VKC (77.8%) as well as control group (77.2%) (p = 0.970). Analysis of individual bacterial revealed that Staphylococcus aureus was detected more frequently in VKC (27.78% vs. 4.55% in control, p = 0.041)
and Staphylococcus epidermidis was found much more commonly in the control eyes (45.45% in control vs. 5.56% in VKC,
p = 0.005).
Conclusions: An aggravating role of S. aureus colonization in the occurrence of VKC, and a possible role of S. epidermidis against
the occurrence of VKC were concluded.
Keywords: Allergy, Conjunctivitis, Eyes, Staphylococci, Vernal keratoconjunctivitis
2015 The Authors. Production and hosting by Elsevier B.V. on behalf of Saudi Ophthalmological Society, King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
http://dx.doi.org/10.1016/j.sjopt.2015.05.002

Introduction
Vernal keratoconjunctivitis (VKC) is a bilateral allergic conjunctivitis, most commonly observed in young patients living
in warm dry climate as in Saudi Arabia.1 It is characterized
by recurrent seasonal episodes of itching, tearing, burning,
mucous stringy discharge, severe photophobia, blepharospasm and foreign body sensation.2,3 Although in early
phase of the allergic reaction, antihistamines are effective
treatment options, and severe allergic reaction necessitates
cytotoxic and immunosuppressant treatment for a long time.4
The pathophysiology of VKC seems to be multifactorial, as
several different mechanisms involving immune, nervous, and

endocrine systems have been proposed. Earlier, it was

believed that the expression of a classical type I
IgE-mediated hypersensitivity reaction at the conjunctival
level is involved in the immunopathogenesis of VKC.5
However, since nearly half of VKC cases are not associated
with positive RAST or skin prick test, it is unlikely to be solely
an IgE-mediated disease.6 Other studies demonstrated the
involvement of neural factors such as substance P (a
neuropeptide) and nerve growth factor in the pathogenesis
of VKC. The over-expression of estrogen and progesterone
receptors in the conjunctiva of VKC patients points toward
the possible involvement of endocrine system.5 Detection
of Toll-like receptor (TLR) expression at the ocular surface

Received 12 September 2014; received in revised form 17 April 2015; accepted 7 May 2015; available online 27 May 2015.
a
b

Department of Microbiology and Clinical Parasitology, College of Medicine, King Khalid University, Saudi Arabia
Department of Surgery, College of Medicine, King Khalid University, Saudi Arabia

Corresponding author.
e-mail address: amhakami@gmail.com (A.M. Al-Hakami).
Peer review under responsibility
of Saudi Ophthalmological Society,
King Saud University

Production and hosting by Elsevier

Access this article online:

www.saudiophthaljournal.com
www.sciencedirect.com

256
during VKC led to the suggestions about possible role of
TLRs in VKC.7 As such, the etiology and pathophysiology of
VKC remain unclear.
Conjunctival sac is colonized by different species of gram
positive as well as gram negative normal bacterial flora,8 of
which staphylococci form the majority.8,9 There is evidence
to indicate the possible role of normal bacterial flora, particularly Staphylococcus aureus in triggering and exacerbation
of different forms of allergies. Staphylococcal enterotoxin A
(SEA) and B (SEB)-specific IgE antibodies have been detected
in the tears of patients with allergic conjunctival disorders,
particularly during exacerbation.10,11 Staphylococcal enterotoxins have also been linked to disease severity of skin allergy
and asthma as well.12,13 In this study, we aim to find out the
association between the presence of normal bacterial flora
(particularly S. aureus and S. epidermidis) and occurrence
of VKC.

Materials and methods

The study included 18 VKC patients (13 males, 5 females)
and 22 healthy volunteers (13 males and 9 females) as controls. Healthy volunteers were selected from subjects who
came to have refractive examinations to receive spectacles
and contact lenses and who consented to cultures and to
the protocol of the study. The study was approved by
Institutional Review Board and written informed consent
was obtained from all adult subjects and in the case of children from their parents.

Sample collection and transportation

Upper tarsal conjunctiva, lower conjunctival sac, and
upper lid margin skin were swabbed for bacterial cultures
in all subjects using sterile swabs under strict aseptic conditions. Each swab was immediately inserted in a tube containing Brain Heart infusion broth (Oxoid, Basingstoke,
Hampshire, England) which served as a transport medium.
Inoculated specimens were transported to the laboratory
for bacteriological analysis.

A.M. Al-Hakami et al.

amount of bacterial colony to a surface of clean, dry glass
slide using a loop or sterile wooden stick. A drop of 3%
H2O2 was paced on to the slide and the preparation was
mixed. A positive result is the rapid evolution of oxygen
(within 510 s.) as evidenced by bubbling. A negative result
is shown when no bubbles are evident. Oxidase test was
done by soaking a small piece of filter paper in 1% Kovcs
oxidase reagent which was left for to dry. A loopful of a
well-isolated bacterial colony from a fresh bacterial plate
was picked and rubbed onto the filter paper. Oxidase positive organisms produced show color changes to dark purple
within 510 s.
Identities of organism were then confirmed using Vitek II
automated identification system following procedures
described by manufacturer (BioMrieux SA, Marcy, France).
The VITEK card contains 64 wells which comprise many fluorescent biochemical tests: 20 of which are carbohydrate
assimilation; 4 are phosphatase, urea, nitrate, and actidione
tests. When a test outcome is documented as low discrimination, this indicates that the result is doubtful. In these
cases, the previously mentioned morphological and biochemical tests were repeated to come to a decision on such
uncertain results. The VITEK 2 scheme managed card automatically from filling, sealing and then transferring them into
the connected incubator (35 C). The cards are filled automatically every 15 min by a fluorescence system. Each resulting profile is decoded according to a precise algorithm. The
acquired results were compared to the ID-GP (identification
of gram positive bacteria) database. In the majority of the
cases the recognized gram positive bacteria are identified
with high percentages of certainties.

Statistical analysis
Collected patients data and results of cultured samples
were analyzed, using Statistical Package for Social Sciences
program (SPSS; Version 16). The association between the
presence of bacteria and occurrence of VKC was analyzed
using Chi square statistic. P-values < 0.05 were considered
statistically significant.

Isolation of bacterial agents

A swab from each specimen was streaked onto a blood
agar plate and MacConkey agar plates. Inoculated plates
were incubated aerobically at 37 C for 24 h. Plates were
examined for bacterial growth. Number and types of colonies
were labeled and recorded till further identification.

Identification of bacterial agents

Bacterial growth was subjected to an identification
scheme using morphological and biochemical tests following
standard procedures.14 Isolates were first examined microscopically using Gram stained slides to look for gram positive
cocci that usually arranged in clusters. Isolates were then
examined for hemolysis after overnight incubation at 37 C
on sheep blood agar. DNase production was tested on
DNase test agar as per the manufacturers recommendations
(Difco). Coagulase test was done with rabbit plasma following the procedure described by the manufacturer (BioMerieux). Catalase test was done by transferring a small

Results
Table 1 demonstrates the bacterial growth of samples
from both vernal keratoconjunctivitis (VKC) group and control group. Fourteen out of 18 VKC samples (77.8%) vs. 17
out of 22 control samples (77.27%) revealed bacterial growth
(p = 0.970). Staphylococcus species were the most common
bacterial isolates representing 50% (9/18) of the VKC samples
and 54.55% (12/22) of the control samples (p = 0.775).
Streptococcus species were the next most common bacteria
constituting 11.11% (2/18) of the bacterial isolates in VKC
samples and 18.18% (4/22) in controls (p = 0.533).
Staphylococci detected in this study (50%) grew aerobically on blood agar. All strains produced catalase, which differentiated them from the catalase-negative streptococci. S.
aureus was coagulase positive; this test distinguished it from
the other staphylococci. S. aureus produced opaque,
smooth, circular, colonies that were yellow (golden) to white
with beta hemolysis whereas S. epidermidis were white in
color with alpha- or no hemolysis.

257

Staphylococcus species and vernal keratoconjunctivitis

Table 1. Bacteria isolates recovered from vernal keratoconjunctivitis (VKC) cases (n = 18) compared to control cases without VKC (n = 22).

No growth
Klebsiella pneumoniae
Pseudomonas fluorescens
Staphylococcus epidermidis
Staphylococcus aureus
Staphylococcus hominis
Staphylococcus warneri
Staphylococcus sp.
Streptococcus spp.

VKC patient (n = 18)

VKC negative individuals (n = 22)

4
1
1
1
5
1
1
1
2

22.22
5.56
5.56
5.56
27.78
5.56
5.56
5.56
11.11

5
1
0
10
1
0
0
1
4

22.73
4.55
0.00
45.45
4.55
0.00
0.00
4.55
18.18

Among VKC samples, S. aureus (27.8%, 5/18) was the

most common staphylococci isolate and among the controls,
S. epidermidis (45.5%, 10/22) was the most common staphylococci isolate. Analysis of individual bacterial revealed that
S. aureus represented 27.78% (5/18) of the VKC samples
compared to 4.55% (1/22) of the controls (p = 0.041)
(Fig. 1). S. epidermidis was isolated in 5.56% (1/18) of the
VKC samples and 45.45% (10/22) of the controls (p = 0.005).

Discussion
Vernal keratoconjunctivitis (VKC) is a potentially serious
seasonal illness that is characterized by bilateral inflammation
of the conjunctiva. It is frequently encountered in the hot and
dry climates such as in Saudi Arabia.1 It is logical to make
attempts at identifying the trigging factors involved and prevent them, if possible. Data from the previous literature seem
to suggest the possible role of S. aureus in causing exacerbation of VKC and AKC. This study was aimed to evaluate the
presence of any association between the type of conjunctival
bacterial flora and occurrence of VKC.
Overall, the growth of any bacterial flora between the VKC
and controls was similar (p = 0.970). Similarly, there was no
statistically significant difference in the number of
Staphylococcal and Streptococcal species isolated from
VKC vs. control eyes. S. aureus, which has previously been
linked to exacerbation of VKC was isolated more frequently
in VKC eyes compared to the controls (27.78% vs. 4.55%),
the difference in frequencies being statistically significant
(p = 0.041). In contrast, S. epidermidis, the most common
bacterial isolate in our study, was found much more frequently in the controls compared to the VKC (45.45% vs.
5.56%), the difference in frequencies being statistically highly
significant (p = 0.005).

Figure 1. Association of Staphylococcus epidermidis with VKC negative

control and Staphylococcus aureus with increased VKC rates.

A review of literature reveals the potential role of S. aureus

and its secretions staphylococcal enterotoxins A and B in the
exacerbation of VKC. Shoji et al. investigated the presence of
staphylococcal enterotoxin A (SEA) and B (SEB)-specific IgE
antibodies in tears from patients with allergic conjunctival
disorders and found high frequency of VKC eyes to be positive for SEA-specific IgE antibodies and high frequency of
AKC eyes to be positive for SEB-specific IgE antibodies.10
Titers for antibodies were higher in patients with more severe
clinical findings. In contrast, the normal controls were negative for both antibodies.10 Similarly, Tabuchi et al. found that
IgE antibodies to staphylococcal enterotoxin A and/or B
were present in the serum of almost 60% (13/22) of the
VKC/AKC patients that had exacerbation in the form of atopic dermatitis; in contrast none of eyes in the control group
(allergic conjunctivitis without AD) had IgE antibodies to
SEA/SEB detected in their tears.11 The statistically significant
higher frequency of S. aureus isolates in VKC eyes (compared
to the controls) complements the findings by Shoji et al. and
Tabuchi et al. As such, the findings of our study point toward
the potentially aggravating role of S. aureus colonization in
the occurrence of VKC.
Another important finding in our study was the statistically
significant higher detection of S. epidermidis in the controls.
To the best of our knowledge, such a finding has never been
reported earlier in VKC patients. However, in dermatology,
higher frequency of S. epidermidis has been reported from
the skin of healthy persons in contrast to atopic dermatitis
patients who had high frequency of S. aureus detection from
their skin.15 Apparently, there could be a protective role of S.
epidermidis against the growth of S. aureus and potentially,
the occurrence of VKC. Likewise, studies investigating bacterial interference have found that a serine protease Esp, produced by S. epidermidis inhibits the growth of S. aureus.16
The findings of this study seem to point toward the possible association of S. aureus and VKC occurrence. Data of the
study also suggest possible association between the presence of S. epidermidis and defense against VKC. It is important to observe that high frequency of S. aureus in VKC
patients may also be due to higher potential of infection in
such patients, possibly due to transmittance of infecting bacteria due to frequent touch or rubbing the eyes.15
Alternatively, compromised immunity due to treatment with
steroids may explain the higher frequency of S. aureus in
VKC patients; however, most of the VKC patients in our study
were not using steroids. In addition, contrary to the
expected, a previous paper studying S. aureus colonization
in the skin of atopic dermatitis patients found a decline in
S. aureus colonization from 100% at the time of exacerbation
to 30% following 412 weeks of treatment with antihistamines and steroids (and no antibiotics), ruling out steroid

258
induced compromised immunity as a cause of S. aureus
colonization.17
Identifying triggers, especially the modifiable ones is an
important aspect of the research aimed at improving the
management of VKC. Future studies with larger sample size
along with experimental animal models would confirm the
role of S. aureus colonization and its secreted staphylococcal
enterotoxins A and B in the occurrence and exacerbation of
VKC and at determining ways to modify such triggers.

Conflict of interest
The authors report no conflicts of interest.

Acknowledgments
The research was funded by the Deanship of Scientific
Research, King Khalid University, Abha, Saudi Arabia (project
number: 1433/235).

References
1. Kumar S. Vernal keratoconjunctivitis: a major review. Acta
Ophthalmol. 2009;87:13347.
2. Al-Akily SA, Bamashmus MA. Ocular complications of severe vernal
keratoconjunctivitis (VKC) in Yemen. Saudi J Ophthalmol
2011;25:2914.
3. Tabbara KF. Ocular complications of vernal keratoconjunctivitis. Can
J Ophthalmol 1999;34:8892.
4. Pucci N, Caputo R, Mori F, et al. Long-term safety and efficacy of
topical cyclosporine in 156 children with vernal keratoconjunctivitis.
Int J Immunopathol Pharmacol 2010;23:86571.

A.M. Al-Hakami et al.

5. Bonini S, Coassin M, Aronni S, et al. Vernal keratoconjunctivitis. Eye
(Lond) 2004;18:34551.
6. Stahl JL, Barney NP. Ocular allergic disease. Curr Opin Allergy Clin
Immunol 2004;4:4559.
7. Bonini S, Micera A, Iovieno A, et al. Expression of Toll-like receptors
in healthy and allergic conjunctiva. Ophthalmology 2005;112:1528,
discussion 489.
8. Hsu HY, Lind JT, Tseng L, et al. Ocular flora and their antibiotic
resistance patterns in the midwest: a prospective study of patients
undergoing cataract surgery. Am J Ophthalmol 2013;155(3644):e2.
9. Capriotti JA, Pelletier JS, Shah M, et al. Normal ocular flora in healthy
eyes from a rural population in Sierra Leone. Int Ophthalmol
2009;29:814.
10. Shoji J, Kato H, Kitazawa M, et al. Evaluation of staphylococcal
enterotoxin-specific IgE antibody in tears in allergic keratoconjunctival
disorders. Jpn J Ophthalmol 2003;47:60911.
11. Tabuchi K, Inada N, Shoji J, et al. The relationship between
Staphylococcus aureus and atopic keratoconjunctivitis. Nihon
Ganka Gakkai Zasshi 2004;108:397400.
12. Bachert C, van Steen K, Zhang N, et al. Specific IgE against
Staphylococcus aureus enterotoxins: an independent risk factor for
asthma. J Allergy Clin Immunol 2012;130(37681):e8.
13. Nomura I, Tanaka K, Tomita H, et al. Evaluation of the staphylococcal
exotoxins and their specific IgE in childhood atopic dermatitis. J
Allergy Clin Immunol 1999;104:4416.
14. Forbes B, Sahm DF, Weissfeld AS. Bailey and Scotts diagnostic
microbiology. 11 ed. Mosby, Toronto: St. Louis; 2002.
15. Nakata K, Inoue Y, Harada J, et al. A high incidence of
Staphylococcus aureus colonization in the external eyes of patients
with atopic dermatitis. Ophthalmology 2000;107:216771.
16. Vandecandelaere I, Depuydt P, Nelis HJ, et al. Protease production
by Staphylococcus epidermidis and its effect on Staphylococcus
aureus biofilms. Pathog Dis 2014.
17. Adamek-Guzik T, Guzik T, Czerniawska-Mysik G, et al. Effects of
combined therapy with oral antihistamines and topical
corticosteroids on Staphylococcus aureus colonization in atopic
dermatitis. Alergia Astma Immunologia 2002;7:3343.