334
TAMARZADEH (A.) AND ARAGHI-SOOREH (A.)
Bacterial flora of the conjunctiva in healthy mules
(Equus mulus)
A. TAMARZADEH1, A. ARAGHI-SOOREH2*
1
2
Graduated from Faculty of Veterinary Medicine, Urmia Branch, Islamic Azad University, 57135-969 Urmia, IRAN.
Department of Clinical sciences, Faculty of Veterinary Medicine, Urmia Branch, Islamic Azad University, 57135-969 Urmia, IRAN.
* Corresponding author: [email protected]
SUMMARY
RESUME
This study was aimed to identify the bacterial isolates of eye surface in healthy
mules and to determine the sensitivity of isolates to antimicrobial agents. A
total of 100 conjunctival swabs were taken from 50 mules, ranging in age from
5 to 12 years, and without any signs of ophthalmic problems. Samples were
cultured for aerobic bacteria and susceptibility assessed to 12 antibiotics.
Bacterial growth was positive in 85/100 (85 %) of sampled eyes. Most of
the isolated bacteria (97/109: 89%) were Gram-positive pathogens. Isolates
in order of frequency were Staphylococcus epidermidis, Corynebacterium
spp., Bacillus cereus, Streptococcus equi subsp. zooepidemicus, Escherichia
coli, Citrobacter diversus and Yersinia enterocolitica. Antibacterial efficacy of
florfenicol, flumequine and gentamicin was identified against 90.82, 70.64
and 67.88 % of isolates respectively. Bacterial species isolated in the current
study were comparable with those previously reported for the ocular surface
of other equine species.
Flore bactérienne conjonctivale chez les mules en bonne santé (Equus
mulus)
Keywords: bacterial flora, eye, mule, Staphylococcus
spp., antibiotic sensitivity
Cette étude visait à identifier la flore bactérienne de la surface oculaire de
mules en bonne santé et à déterminer la sensibilité de ces germes à certains
antibiotiques. Un total de 100 prélèvements conjonctivaux a été réalisé à partir
de 50 mules, âgées de 5 à 12 ans, et ne présentant aucun signe de problèmes
oculaires. Les échantillons ont été cultivés pour les bactéries aérobies et la
sensibilité à 12 antibiotiques a été évaluée. Une croissance bactérienne a été
obtenue chez 85 échantillons sur 100 (85%). La plupart des bactéries isolées
(97/109: 89%) étaient Gram positif. Les agents bactérien identifiés étaient
par ordre de fréquence Staphylococcus epidermidis, Corynebacterium spp.,
Bacillus cereus, Streptococcus equi subsp. zooepidemicus, Escherichia coli,
Citrobacter diversus et Yersinia enterocolitica. L’efficacité antibactérienne
du florfenicol, de la fluméquine et de la gentamicine s’est manifestée
respectivement sur 90,82, 70,64 et 67,88% des souches isolées. Les espèces
bactériennes identifiées dans la présente étude étaient comparables à celles
rapportées pour la surface oculaire d’autres équidés.
Mots-clés : flore bactérienne, oeil, mule, Staphylococcus
spp., sensibilité aux antibiotiques
Introduction
Ulcerative keratitis is a common disease in horses
and can be potentially sight threatening if not diagnosed
early and treated properly. A prominent cornea and its
susceptibility to trauma, a relatively slow rate of corneal
healing, and insufficient defense mechanisms of ocular
surface are considered for frequency of corneal infections
in horses [3]. Most resident bacteria of the eye surface
have a role in maintenance of health cornea by decreasing
ability of invasive bacteria to attach and colonize the ocular
surface [24]. However, these microorganisms can become
potentially pathogen if corneal ulcers/erosions due to trauma
occur [3]. Hence identification of normal flora of eyes
permits the clinician to anticipate the presence of certain
organisms on the eye surface and, in the event of trauma,
to initiate proper preventive antibacterial treatment. Many
studies have described normal conjunctival bacterial flora of
horses [1,10,11,28] and donkeys [9], but there is no report
in the literature regarding normal ocular microflora of
mules. Genera of Staphylococcus, Streptococcus, Bacillus and
Corynebacterium are the most frequently isolated organisms
of normal equine ocular surface [1,9,10,14]. The purposes
of present study were to document of bacterial flora of
conjunctiva in the healthy mules and to assess antimicrobial
susceptibility of isolates for the recommendation of
appropriate antibiotics for preventive treatment of corneal
ulcers in mules.
Material and Methods
ANIMALS AND AREA OF STUDY
The study population included 50 healthy mules, 29 (58 %)
females and 21(42 %) males, ranging in age from 5 to 12 years,
and living in mountainous districts of Urmia (2,120 m above
sea level), located West-Azerbaijan province, northwestern
Iran with temperate climate. The study was approved by the
ethics committee for animal experimentation by the Islamic
Azad University-Urmia Branch (Serial No. 1683/2011).
SAMPLE COLLECTION
From July to August 2011, the lower cul-de-sac of 100
eyes were swabbed using a dry sterile cotton swab per eye,
taking care not to contaminate the swab by contact with the
eyelashes or skin of eyelids. Swabs were placed in test tubes
containing sterile transport medium (peptone water; Merck,
Darmstadt, Germany), and were transported immediately to
Revue Méd. Vét., 2014, 165, 11-12, 334-337
NORMAL CONJUNCTIVAL BACTERIAL FLORA IN MULES
335
the microbiology laboratory in a chilled thermal box.
Results
BACTERIOLOGY
ISOLATES
In the laboratory, samples were inoculated on 5% ovine
blood (Merck, Darmstadt, Germany) and MacConkey
(Merck, Darmstadt, Germany) plates and incubated at 37 °C
for 48 h in an aerobic condition. Isolates were subjected to
identification using standard microbiological methods [21].
Out of the 100 samples, 85 (85%) were found to be positive
for bacterial growth. A total of 109 bacteria belonging to 7
genera were isolated (Table I), with Gram-positive bacteria
(4 genera) predominating (97/109: 89 %). Staphylococcus
epidermidis was the most commonly isolated organism,
accounting for 29.35 % of isolates. Among the Gram-negative
bacteria (3 genera), the most frequently isolated species was
Escherichia coli (7.33 % of isolates).
ANTIMICROBIAL SENSITIVITY TEST
Antibiotic sensitivity test of all the bacterial isolates
was conducted using disc diffusion technique on MuellerHinton agar (Merck, Darmstadt, Germany) at 37 °C for 24
h. The results of sensitivity testing with the 12 antibiotic
disks including chloramphenicol 30 μg (C), tylosin 30
μg (TY), enrofloxacin 5μg (NFX), trimethoprim 5 μg
(TMP), amoxicillin 25 μg (AMX), furazolidone 100 μg
(FR), flumequine 30 μg (FM), ceftiofur 30 μg (CEF),
gentamicin 10 μg (GM), colistin 10 μg (CL), lincomycin
15 μg/ spectinomycin 200 μg (LS) (Padtan Teb, Tehran,
Iran) and florfenicol 30 μg (FFC) (Iran Daru, Tehran, Iran)
were interpreted and recorded to the Clinical Laboratory
Standards Institute (CLSI) recommendations [18].
ANTIBIOTIC SENSITIVITY OF ISOLATES
Table II exhibits the antibiotic sensitivity profile of all
bacterial isolates from healthy mule’s conjunctiva. Six out
of the 12 antimicrobials including florfenicol, flumequine,
enrofloxacin, trimetoprim, chloramphenicol and ceftiofur
were effective against all isolates. Only 14 of the 109
(12.84 %) isolates were sensitive to all antibiotics tested.
Florfenicol, flumequine and gentamicin were the most
efficient antimicrobials with efficacy rate of 90.82, 70.64 and
67.88 % against the isolates, respectively.
Bacteria
No. of isolates
% of isolates
Staphylococcus epidermidis
32
29.35
Corynebacterium spp.
27
24.77
Bacillus cereus
24
22.01
Streptococcus equi subsp. zooepidemicus
14
12.84
Escherichia coli
8
7.33
Citrobacter diversus
3
2.75
Yersinia enterocolitica
1
0.91
Table I: Bacteria recovered from normal conjunctival sac of mules
Isolates
FFC
LS
NFX
C
CEF
CL
FR
TY
AMX
GM
TMP
FM
Staph. epidermidis
100
75.00
75.00
50.00
12.50
0
0
0
46.87
84.37
50.00
68.75
Corynebacterium spp.
92.59
74.07
25.92
29.62
37.02
0
0
0
11.11
88.88
37.03
74.07
Bacillus cereus
83.33
58.33
25.00
33.33
29.16
0
0
0
0
0
70.83
83.33
Strep. equi subsp.
zooepidemicus
85.71
78.57
42.85
50.00
42.00
14.28
7.14
7.14
35.71
85.71
42.00
57.14
Escherichia coli
75.00
0
62.50
62.50
12.50
37.50
0
0
62.50
87.50
62.50
62.50
Citrobacter diversus
100
66.66
66.66
66.66
33.33
0
33.33
0
0
100
66.66
33.33
Yersinia enterocolitica
100
0
100
100
100
100
0
0
0
100
100
100
90.82
65.13
46.78
43.11
27.52
5.50
1.83
0.91
25.68
67.88
52.29
70.64
Table II: Percentage distribution of antibiotic sensitivity pattern among bacteria recovered from conjunctival sac of healthy mules
Revue Méd. Vét., 2014, 165, 11-12, 334-337
336
Discussion
To our knowledge, this is the first published report of the
conjunctival bacterial isolates in healthy mules. In the current
study Gram-positive bacteria (89% of all isolates) were
predominant in conjunctival sac of mules. This finding is in
agreement with those reported for horses (52-77%) [11,14]
and donkeys (65%) [9]. Isolation of a higher incidence of
Gram-negative bacteria compared to Gram-positive ones
[5], was reported in only one study of normal conjunctival
flora in Italian horses, which may reflect geographic, seasonal
or environmental differences [14].
In our study, Staphylococcus spp. was found to be the
most frequently isolated bacterial species. Similar result has
been found in studies of horses [28], donkeys [9] and other
animal species such as water buffalos [27], dogs [19], cats
[8], domestic rabbits [4], Asian elephants [26], mule deers
[7], raccoons [22], opossums [20], iguanas [25] and birds
[6]. It seems that the genus Staphylococcus is an important
component of conjunctival bacterial flora in many domestic
and wild animal species.
We found a higher number of opportunistic bacteria, i.e.,
coagulase-negative Staphylococcus, Corynebacterium and
B. cereus, than previously reported from both normal [1,14]
and diseased eyes of horses [13,16,29].
Additionally, a low prevalence of potential pathogens,
such as beta-hemolytic Streptococcus [2,12,15,23,29] and
Enterobacteriace genera [13,16] was identified in our study.
Potential pathogens have also been isolated in low frequency
in other studies of normal equine eye flora [1,16].
Because pathogenic bacteria are ubiquitous in the
environment, the low isolation rates of these organisms from
healthy ocular surface suggest that the normal flora may
control or suppress colonization of pathogens.
Superficial ulcers generally heal quickly without
complications with application of broad spectrum antibiotics
and some other drugs. However, infected ulcers may progress
to corneal perforation in as little as 24 h [3]. Hence, immediate
use of antimicrobial is recommended for treatment of corneal
ulcers to prevent the establishment of infection following the
trauma. Recommended antimicrobials for the preventive
treatment of corneal ulcers in horses include topical
gentamicin and chloramphenicol [11,14]. In the current
study, high efficacy (more than 90% of isolates sensitive) was
displayed by florfenicol, but this drug is not licensed for use
in equine species. Flumequine (with efficacy on more than
70% of the isolates) has toxic effects on corneal epithelial
cells [17]. Antimicrobials gentamicin and chloramphenicol
(available for ophthalmic topical use) were effective on only
67.88% and 43.11% of isolates, respectively. It is clear that
none of the assessed antimicrobials are recommendable for
preventive treatment of corneal ulcers in mules and further
studies are needed for finding of appropriate antibiotics.
TAMARZADEH (A.) AND ARAGHI-SOOREH (A.)
Genera of bacteria identified in this study were similar
to genera isolated from other studies of normal equine
eyes. Many of these isolates were non-pathogenic or weakly
pathogenic but among them highly pathogens also could be
found.
Acknowledgments
The authors wish to thank Dr. Ebrahim BANIHASSAN
and staff of the microbiology laboratory at the Urmia Branch,
Islamic Azad University for their assistance.
Grant support
This manuscript has not been supported with any
organization.
Conflict of interest
The authors acknowledge no conflict of interest in this
study.
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