Antimicrobial Resistance in Bacterial Pathogens of Canine Otitis

Corresponding Auhtor: Sheila Rezler Wosiacki Department of Veterinary Medicine, State University of Maringa, Estrada da Paca, s/n, 87507-190, Umuarama, Parana, Brazil Tel: +55 44 3621-9412 Email: srwosiacki@uem.br Abstract: Otitis is one of the most frequent infections in dogs. This is attributed to the misuse of drugs enabling generation of multi-resistant micro-organisms. The emergence of multiresistant bacterial strains in veterinary medicine is a reality that must be studied and evaluated by the professionals. The objective of this study was to isolate and evaluate the antimicrobial susceptibility of bacterial pathogens of otitis in dogs. Otologic swabs were collected from 36 dogs with clinical otitis. 41 bacterial strains were isolated and antimicrobial susceptibility tests were performed by disk diffusion method with 34 antimicrobial agents. Presence of the resistance gene mecA of Staphylococcus was examined for 22 strains of staphylococci by PCR. A total of 1108 ratings antimicrobial agents were performed. The percentage of drug resistance was 34.66% (n = 384) of the assessments with partial or total resistance. Major bacterial pathogens were Staphylococcus spp. (65.85%), Pseudomonas spp. (12.19%) and Enterobacteria species (19.51%). 53.66% of the isolates were considered multiresistant. Antimicrobial agents considered most resistant in the strains studied were penicillin (75.00%), tetracyclin (50.00%), amoxicillin (48.78%), trimethoprimsulfamethoxazole (46.15%), clindamycin and rifampicin (43.24%). 11 strains were phenotypically characterized as MRS, 4 genotypically as MRS, 2 as MLSB-MRS and 2 as gram negative ESBL-producing.


Introduction
Otitis is one of the most frequent infections in dogs. It is a disease of multifactorial causes and can manifest acutely or chronically. Predisposing factors such as anatomical conformations of both the ear canal and the ears, changes in microflora and immune suppression are some of the main observed.
The normal ear canal microflora is varied consisting of coagulase-positive and coagulase-negative Staphylococcus spp., β-haemolytic Streptococcus spp., Proteus spp., Escherichia coli and Pseudomonas spp. (Hariharan et al., 2006;Petrov et al., 2013). Changes to this microflora can lead to the development of opportunistic and also pathogenic bacteria (Oliveira et al., 2012).
According to (Oliveira et al., 2012;Oliveira et al., 2005;2006b) main bacterial pathogens of canine otitis are Staphylococcus and Pseudomonas species. Other bacterial species found in smaller percentages are Streptococcus, Escherichia coli, Proteus and Enterococcus species (Hariharan et al., 2006).
Frequently observed failure of treatment of ear infections. This is attributed to the misuse of drugs enabling generation of multi-resistant micro-organisms, that result in reduced efficacy of the drugs. This occurrence has increased concomitantly with routine use of antibiotics, especially broad-spectrum drugs that are able to act on a large number of bacterial species. Not performing tests for agent identification and susceptibility favors the generation of resistant strains, making it essential to carry out these (Ishii et al., 2011).
The emergence of multiresistant bacterial strains in veterinary medicine is a reality that must be studied and evaluated by the professionals.
The objective of this study was to isolate and evaluate the antimicrobial susceptibility of bacterial pathogens of otitis in dogs.

Sample Collection
Otologic swabs were collected from 36 dogs with clinical otitis in the Veterinary Hospital of State of Maringá, Brazil between March, 2012 andSeptember, 2014. The samples were initially incubated in Brain Heart Infusion broth -BHI (OXOID ® ) at 36°C for 2 to 18 hours, then plated on Blood agar (5% sheep blood defibrillated in Nutrient Agar-OXOID ® ) and MacConkey agar (OXOID ® ), incubated at 36°C for 24/48 h. The isolates were identified based on colony morphology and biochemical reaction.
The Multiple Antibiotic Resistance (MAR) index was calculated by the number of resistant ratings over the total tested according (Krumperman, 1983).

Staphylococcus spp. mecA Detection
Presence of the resistance gene mecA of Staphylococcus spp. was examined for 22 strains of staphylococci by PCR. The DNA extraction was performed by (Doyle et al., 1987). 200 µL of Tris-EDTA (TE) was added in to microtubes with isolated colonies and centrifuged at 8000×G for 5 min. The supernatant was discarded and the precipitation was suspended in 600 µL of CTAB (Cetyl Trimethyl Ammonium Bromide) and 40 µL of CIA (Chloroform/isoamyl alcohol), heated in a water bath at 65°C for 30 min. Over 800 µL of CIA was added and centrifuged at 12000×G for 5 min. 600 µL of the supernatant was transferred to another microtube and was added 600 µL of cold isopropanol. The mixture was incubated at -20°C for 16 h and centrifuged at 13,500×g for 20 min at 4°C. The supernatant was discarded and the DNA was dried in laminar flow. The purified DNA was eluted in 200 µL TE.

Statistical Analysis
The results were submitted to descriptive analysis to calculate the absolute and relative frequencies.
A total of 1108 ratings antimicrobial agents were performed. The percentage of drug resistance was 28.70% (n = 318) and intermediate resistant, 5.95% (n = 66) totaling 34.66% (n = 384) of the assessments with partial or total resistance (Fig. 1).
The resistance to antimicrobial agents is contained in Table 2 and Fig. 2.
The MAR index medium was 0.293. Of the 41 strains evaluated, 53.66% (22/41) showed ≥0.2 MAR and were considered multiresistant by (Krumperman, 1983) (Table 1).  About 4 of 22 Staphylococcus spp. strains were positive for mecA detection by PCR, genotypically characterized as MRS. Only one of these was resistant to oxacillin by disk diffusion, the others three were susceptible.

Discussion
The main micro-organisms found in this study were Staphylococcus spp. and Pseudomonas spp., with frequency of 65.85% and 12.19 respectively. In 1990, (Oliveira et al., 2006a) also found similar etiology, being isolated 61.02% of Staphylococcus spp., 12.71% of Pseudomonas spp., still having 14.41% of Streptococcus spp. and other agents with low prevalence. (Oliveira et al., 2005) isolated 53.04% of Staphylococcus spp. and 25.05% of Pseudomonas spp. (Oliveira et al., 2006a) isolated 55% of Staphylococcus spp. and 10% of Pseudomonas spp. (Oliveira et al., 2012) identified 46.75% of Staphylococcus spp. and 8.8% of Pseudomonas spp. Therefore it is observed that bacterial etiology of dog's ear canal infections is very similar, not depending on geographic region nor conditions such as animal race, age, or sex.
Data obtained in this study corroborate with most of other studies performed with the profile and etiology of resistance to antimicrobials, being found a low prevalence of resistance in all of them to most of antimicrobials drugs to Staphylococcus and greater resistance to Pseudomonas justified by the high intrinsic resistance of this microorganism. 13 antimicrobial classes were tested in this study, totalizing 34 antimicrobial agents of tested.
β-lactam penicillins drugs (penicillin R=75.00%) and aminopenicillin (ampicillin R= 53.66% and amoxacillin R=48.78%) showed high level of resistance, however when associated with β-lactamase inhibitors (sulbactan with ampicillin R=22.86% and clavulonic acid with amoxacillin R=26.83%) it was noted the improvement in sensitivity to drugs in 57.41% and 45% of the cases respectively. So this study recommends the association between aminopenicillin with β-lactamase inhibitors.
The semi synthetic oxacillin β-lactam drug is, according to CLSI (2008), a drug for pre-editting of resistance in Staphylococcus spp. to all β-lactam, associated to resistance to cefoxitin. According to Kim et al. (2012) and Cartwright et al. (2013) resistance to oxacillin and cefoxitin shows phenotypically the presence of mecA gene. On the other hand resistance to cefoxitin with sensitivity to oxacillin, shows the presence of mecC gene, which is responsible for the production of a protein that links additional penicillin (PBP2a) that provides low affinity of linking to β-lactam drugs. Staphylococcus spp. that carries this gene is called MRS. Several studies show the existence of MRS in Medicine Veterinary.
In this study 11 (40.74%) of 27 staphylococci strains were resistant to oxacillin. However, only 4 carried the mecA gene of which 3 were oxacillin-susceptible. Nevertheless, all four will be reported as MRS, making 14.81% (4/27). These results show that the phenotypic detection of oxacillin-resistance in not totally related to the presence of mecA gene as well as there are possible other genes of resistance to β-lactam agents that not only the mecA, gene being so important as this.
Tested β-lactam cephalosporins agents showed low resistance level, ranging from 35.00% to cephalothin, 35.71% cephalexin and 16.22% to ceftriaxone. However they showed great resistance in gram negatives (cephalexin and cephalothin).
Carbapenems have a chemical structure similar to penicillin, but with chemical characteristics which give them greater affinity to PBPs, showing greater potency and an expanded antibacterial spectrum. Resistance to carbapenems is already considered a problem to public health in many countries (Ribeiro, 2013). Carbapenems evaluated in this study have shown 2.63% of resistance to meropenem (only in gram positive). Although there are no rules that forbid the use of carbapenems in Veterinary Medicine, these antimicrobials must be used very carefully in order to avoid pressure in selecting resistant clones and resistance transmission to other bacteria, potentially some contact with humans.
After the discovery of multiresistant gram positive bacteria, specially the MRS, antimicrobial class of glycopeptides vancomycin and teicoplamin have been the last alternative for the treatment against these micro-organisms for many years in Medicine (WHO, 2009). In Veterinary Medicine the resistance to glycopeptides has been hardly studied due to little use. According to Haenni et al. (2010)  Aminoglycoside class showed one of the best level of susceptibility in this study, ranging from 7.69% to 25.81%, as well as phenicols.
Macrolides, Lincosamides and Streptogramin B form MLSB group of antibiotics, although having different formulas, they present the same mechanism action, inhibiting the protein synthesis through the link to the 23S receptor of rRNA that takes part of the 50S subunity of the bacterial ribosome. Since 1956, soon after the introduction of erythromycin in the market, Staphylococcus aureus resistance to MLSb group (Leclercq, 2002) has already been seen. In Medicine Veterinary, clindamycin is largely used, also indicated to infections caused by Staphylococcus, mainly the MRS (Fiebelkorn et al., 2013). However, Kim et al. (2004) while analysing the presence of resistance to MLSb group in Staphylococcus aureus found out that 97% of MRSA (Meticillin-Resistant Staphylococcus aureus) showed resistance to at least one of the antibiotics of this group. Epidemiologically, the crossed-resistance among these 3 classes of antimicrobials is very important Dipersio and Dipersio (2005) once they are largely used in Veterinary Medicine taking to the increase of resistance of animal origin.
The tested macrolides showed resistance of 39.39% to erythromycin and 30.77% to azithromycin. Resistance to clindamycin was found in 43.24% of the studied samples. 11.11% (03/27) of Staphylococcus spp. resistant to MLSb group were detected with the two tested drugs, two of them being characterized as MRS.
The class of fluoroquinolones, according to CLSI (2008) must be reported together, where resistance to a drug indicates the resistance of the whole class. Percentages of resistance of 10.81% to cyprofloxacin, 19.51 % to norfloxacin, 23.68% to enrofloxacin and 17.07% to levofloxacin were found, showing greater significant resistance in gram positives, being these drugs of great value for the empirical treatment by gram negative bacteria.