Bacterial flora of the conjunctiva in healthy mules (Equus mulus)

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florfenicol, flumequine and gentamicin was identified against 90.82, 70.64 and 67.88 % of isolates respectively. Bacterial species isolated in the current.
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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

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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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