Evaluation of etiology and antibiogram in otitis--a clinical study of 40 dogs.
Otitis is a common medical terminology for inflammation of ear. Primary causes of otitis include parasites, hypersensitivity, keratinization disorder, foreign bodies, ear gland disorder and autoimmune diseases. These conditions are responsible for altering the ear canal allowing abnormal colonization of microorganisms. Treatment of these underlying conditions cannot be overlooked for eventual resolution of otitis.
Perpetuating factors include bacteria (Staphylococcus spp. and Pseudomonas spp.), yeast (Malassezia spp.) and pathologic changes such as glandular hyperplasia, epithelial folds, neoplasia, edema, mineralization and fibrosis. Secondary otitis media often perpetuates otitis externa. These conditions are frequently seen as etiology of ear infection, when in actuality they are sequelae to an underlying primary cause (Ettinger and Feldman 2000). The present study was planned to confirm the etiology and evaluate efficacy of different therapeutic regimen.
Material and Methods
In the present investigation, dogs suffering from ear infection and presented with the complaint of head shaking with itching in the ear were considered for the study. Thorough clinical examination of ear was carried out with cytological, mycological and bacteriological culture examination of otitic exudates for isolation of fungi, yeasts and bacteria. Antibiotic sensitivity test was carried out in case of bacterial otitis to appropriate proper line of treatment.
Otitic discharges were collected from affected ears using sterile cotton swabs for isolation of bacteria. Swab material collected was inoculated into nutrient broth and incubated at 370C for 6 hrs. Later on loopful of broth was streaked on Nutrient agar and MacConkey agar plate and incubated at 37[degrees]C for 24 hrs.
The characteristics of distinctly looking colonies were studied and isolated colonies were picked up and subjected to Grams staining to study morphological characters and staining reaction of organisms. Colonies of different types of bacteria were picked up and sub-cultured for further identification.
For isolation of yeast and fungi, the otitic exudate was inoculated on Sabouraud's dextrose agar (SDA) with Chloramphenicol slants, incubated at 25[degrees]C and examined every 4-6 days. The slants were held for at least 2 weeks before considering as negative.
Identification of isolates was carried out on basis of various biochemical tests as described by Markey et al. (2013).
Antibiogram of Bacterial Isolates
In-vitro antibiotic sensitivity of isolates was studied by disc diffusion method (Bauer et al., 1965). Briefly a small quantity of 6 hrs culture grown in nutrient broth at 37[degrees]C was taken with the help of Pasteur pipette and transferred on to Muller Hinton Agar plate. Using a sterile spreader a homogenous lawn was made and plates were allowed to dry for a brief period. The antibiotic disc were then placed 20 mm apart from each other and gently pressed on surface of agar. The plates were incubated at 37[degrees]C for 18-24 hrs.
The diameter of zone of inhibition including disc diameter was measured and results were interpreted as sensitive, intermediate and resistant based on recommendations of the manufacturers.
A total of 10 antibiotic discs--Ampicillin (A-10 mcg,) Amoxycillin (Am-30mcg), Cefalexin (Cn 30mcg), Norfloxacin (Nx-10mcg), Chloramphenicol (C-30 mcg), Ceftriaxone (Ctr--30mcg), Azithromycin (Azm- 30 mcg), Ciprofloxacin (Cip- 1 mcg), Ampicilin-Cloxacilin (Ax- 10 mcg) and Erythromycin (E-30 mcg) supplied by Hi-Media laboratories were used in the present study.
Exfoliative Cytological Examination
Otitic exudate swab was used for preparing smears and microscopic examination for presence of bacteria, yeast/fungi. The smears were examined after staining with Gram's stain/ Giemsa stain/ Lactophenol cotton blue stain.
Skin scraping examinations
Skin was scraped with a scalpel to collect skin tissue scraps. Skin scraps were gently mixed with potassium hydroxide 10% solution and processed for microscopic examination to detect mites or dermatophytes.
Results and Discussion
The microbial flora of ear canal of canines suffering from otitis has been studied by several workers and results differ widely. Similarly the present investigation revealed different etiological agents, Details of recovered isolates are presented in (Table 1).
In all 20 bacterial isolates were recovered, of these Staphylococci spp. were 8 (40%) followed by E. coli 5 (25%), Pseudomonas spp. 4 (20%), (Fig. 1 and 2) Proteus spp. 2 (10 %) and Bacillus spp. 1 (5%) respectively. Dakshinkar et al. (1992) recovered 25.93% of Staphylococcus aureus, 14.8% Staphylococcus epidermidis, 14.8% Streptococci spp., Proteus spp., 5.7% and 5.7% E. coli and Klebsiella spp.
In the present study, mycological investigation was carried out in which 12 cases of fungal otitis were observed on exfoliative cytology. Of these Malessezia pachydermatis was recovered in 9 dogs and Aspergillus spp. 3 dogs (Table 1) were confirmed on SDA agar with chloramphenicol slant. Similarly Blanco et al. (2014) used Sabouraud's dextrose agar medium for isolation of yeasts especially M. pachydermatis in which they observed growth after 24 hours of incubation.
Exfoliative cytology is the inexpensive method and less time consuming to assess presence of etiology in number and type of etiology whether bacterial, fungal or parasitic. Cytology revealed presence of gram positive cocci, gram negative rods and Malessezia spp. on grams staining (Fig. 3 and 4). It helped in diagnosis prior to start effective treatment. Fernandez et al. (2006) found cytology as the most efficient method for identification of Malassezia pachydermatis. Also Murphy (2001) expressed that cytological examinations of otitic exudate were the most appropriate method for determining type and number of microorganisms present.
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Similarly, the parasitological study was carried out to explore parasitic etiology. Total eight dogs with otitis were found to be positive for parasitic otitis. Of these, Sarcoptic mites (Fig. 5) were recorded in 4 cases, Demodex mites in 2 dogs (Fig.6) and Rhipicephalus ticks in 2 dogs were recovered.
Antibiotic sensitivity test
Antibiotic sensitivity tests were conducted to evaluate the sensitivity of causative organisms for antibiotics was evaluated. The antibiotic sensitivity tests were carried out by collecting swab of otitic discharge and fallowing standard laboratory procedure The antibiogram of bacterial isolates is presented in Table 2.
In-vitro antibiogram of Staphylococcus spp. indicated highest 62.5% (8) sensitivity to Ceftriaxone and least sensitivity to Chloramphenicol 12.5% (1). Next in order of effectiveness were Ciprofloxacin 25%, Norfloxacin 25% and Azithromycin 25%. In-vitro antibiogram of E. coli indicated highest 100% (5) sensitivity to
Ceftriaxone and least sensitivity to Norfloxacin (20%). Next in order of sensitivity was Ciprofloxacin 80% (4) followed by Chloramphenicol (40%).
In-vitro antibiogram of Pseudomonas spp. indicated highest 100% (4) sensitive to Ceftriaxone and next to Ciprofloxacin 80% sensitivity (Fig. 7). The in-vitro antibiogram of Proteus spp. indicates highest 2 (100%) sensitive to Ceftriaxone and Bacillus spp. were sensitivity to Norfloxacin, Chloramphenicol, Cephalexin, Azithromycin and Ciprofoxacin equally. These findings are in agreement with Chaudhary et al. (2003) who observed similar sensitivity pattern of antibacterial agents to different bacterial isolates as Ciprofloxacin 86.67%, Norfloxacin 56.67% and Chloramphenicol 38%. Baksi et al. (2004) also observed that out of 8 cases of Staphylococci spp. 2 were sensitive to Ciprofloxacin and 3 were sensitive to Chloramphenicol.
In-vitro antibiotic sensitivity pattern of different bacterial isolates was evaluated and presented in Table 3.
Ceftriaxone is found to be effective against almost all bacterial isolates viz. Staphylococcus spp., E. coli, Pseudomonas spp. Proteus organism followed by ciprofloxacin which found effective against Staphylococci, E. coli, Pseudomonas and Bacillus organism. Other antibiotics under study showed sensitivity to one or two bacterial isolates only. However, Ampicillin-Cloxacilin and Ampicillin has not shown sensitivity to any bacterial isolates justifying the rationality of Ceftriaxone and Ciprofloxacin in the treatment of canine otitis. This finding is in akin with the finding of Kumar and Rao (1997) Chaudhary et al. (2003) and Senthil Kumar (2010) who also recorded nearly same degree of sensitivity to Ceftriaxone and Ciprofloxacin to isolates from otitic dogs.
The present study indicates that there may be different etiological agents causing otitis in dogs. Confirmation of etiology causing otitis is necessary for correct selection of antibiotic and related therapy, instead of going for haphazard treatment, which may lead to complications and frustration for clinician also.
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(1.) Corresponding author E-mail: firstname.lastname@example.org
M.J. Umale, A.M. Rode (1), N.P. Dakshinkar, C.G Panchbhai and A.A. Sanghai
Department of Veterinary Clinical Medicine, Ethics and Jurisprudence
Nagpur Veterinary College
Maharashtra Animal and Fishery Science University (MAFSU)
Table 1 : Etiological agents isolated from otitic dogs No.of No.of Etiology samples isolates (%) screened Malessezia spp. 92 09 22.5 Staphylococci spp. 08 20.0 E. coli 05 12.5 Pseudomonas spp. 04 10.0 Sarcoptic mites 04 10.0 Aspergillus spp. 03 7.5 Proteus spp. 02 5.0 Demodex mites 02 5.0 Rhipicephalus ticks 02 5.0 Bacillus spp. 01 2.5 Total 40 100 Table 2: Antibiogram of bacterial isolates in canine otitis Bacteria Isolates Antibiotic Sensitivity (%) Staphylococci spp. 08 Ciprofloxacin Norfloxacin 2 (25%) 2 (25%) E. coli 05 Norfloxacin Ceftriaxone 1 (20%) 5 (100%) Pseudomonas spp. 04 Ceftriaxone Ciprofloxacin 4 (100%) 3 (75%) Proteus spp. 02 Ceftriaxone -- 2 (100%) Bacillus spp. 01 Norfloxacin Chloramphenicol 1 (100%) 1 (100%) Bacteria Antibiotic Sensitivity (%) Staphylococci spp. Ceftriaxone Azithromycin 6 (62.50%) 3 (25%) E. coli Ciprofloxacin Chloram- 4 (80%) phenicol 2 (40%) Pseudomonas spp. -- -- Proteus spp. -- -- Bacillus spp. Cephalexin Azithromycin 1 (100%) 1 (100%) Bacteria Antibiotic Sensitivity (%) Staphylococci spp. Chloramphenicol 1 (12.50%) E. coli -- Pseudomonas spp. -- Proteus spp. -- Bacillus spp. Ciprofloxacin 1 (100%) Table 3: In-vitro antibiotic sensitivity patterns of different bacterial isolates in canine otitis Antibiotic Staphy E-coli Pseudo- Proteus Bacillus lococci monas spp. spp. spp. spp. Ciprofloxacin 2 4 3 0 1 Cephalexin 1 0 0 0 1 Chloramphenicol 1 2 0 0 1 Norfloxacin 2 1 0 0 1 Ampicilin+ Cloxacilin 0 0 0 0 0 Amoxycilin 1 2 0 0 0 Ampicilin 0 0 0 0 0 Ceftriaxone 6 5 4 2 0 Azithromycin 3 1 0 0 1 Erythromycin 0 2 0 0 0
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|Title Annotation:||Clinical Article|
|Author:||Umale, M.J.; Rode, A.M.; Dakshinkar, N.P.; Panchbhai, C.G; Sanghai, A.A.|
|Date:||Jul 1, 2015|
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