Increased Resistance of Staphylococcus pseudintermedius to the Commonly Used Antibiotics in Canine Dermatology
December 12, 2011 —
admin
Filed under:
| Attachment | Size |
|---|---|
| increased_resistance_of_staphylococcus_pseudintermedius_from_december_book_en.pdf | 452.7 KB |
Embedded Scribd iPaper - Requires Javascript and Flash Player
Increased Resistance of Staphylococcus pseudintermedius to the Commonly Used Antibiotics in Canine Dermatology
Zur, G.,1* Elad, D.,2 and Sterenzy-Agiv, N.1
1
Veterinary Teaching Hospital. The Koret School of Veterinary Medicine. The Robert H. Smith Faculty of Agriculture, Food and Environment. The Hebrew University of Jerusalem, P.O. Box 12, Rehovot, 76100, Israel 2 Department of Bacteriology. The Kimron Veterinary Institute, P.O. Box 12, Beit Dagan 50250, Israel
* Corresponding author: Dr. Gila Zur; Tel +972-3-9688547; e-mail: zurgila@agri.huji.ac.il
Increased resistance of Staphylococcus spp. is a growing problem in veterinary medicine. This study was performed to examine antimicrobial susceptibility of Staphylococcus pseudintermedius and other bacteria isolated from canine skin and ear infections over a 7 year period: 2000-2006. Eighty-six dogs which met predetermined inclusion criteria were examined. Bacterial isolates were examined for susceptibility to four commonly used antibiotics: cephalothin (first generation cephalosporin), amoxicillin-clavulanate, potentiated sulfonamide and enrofloxacin. The degree of susceptibility and the percentage of resistant strains of the bacteria to the antibiotics were compared between two periods: 2000-2002 and 2003-2006. Comparison between sample sites and association between susceptibility and signalment were examined. Resistance of S. pseudintermedius and Proteus spp. to cephalosporins and amoxicillin-clavulanate increased over the years and it was statistically significant for S. pseudintermedius (P=0.018). Susceptibility of S. pseudintermedius to cephalosporins and amoxicillin-clavulanate decreased over the years (P= 0.009). Susceptibility of Proteus spp. to amoxicillin-clavulanate and fluoroquinolones decreased as well (P=0.037, P=0.055 respectively). More skin than ear isolates of S. pseudintermedius and Proteus were resistant to cephalosporins and amoxicillinclavulanate. More ear isolates of Proteus were resistant to potentiated sulfonamides (P= 0.039). All isolates of Proteus from mix breed dogs, but not from German shepherds were resistant to cephalosporins (P=0.032). This study showed an increased resistance and decreased susceptibility of two common bacteria found in dogs' skin and ear infections. These findings should encourage clinicians to submit samples for culture and susceptibility before commencing antibacterial therapy, so as to provide a more accurate therapeutic decision and avoid unnecessary antibiotic use. Key words: dogs, bacteria, Staphylococcus pseudintermedius, antibiotics
AB ST RAC T
INTRODUCTION
Bacterial skin and ear infections are very common in dogs. In most of the cases the proliferation of bacteria is secondary to another dermatological problem such as allergy, ectoparasites or systemic diseases such as hypothyroidism, hyperadrenocorticism and others. Staphylococcus pseudintermedius (formerly S. intermedius) is the most common microorganism isolated from skin and ear infections (1, 2). It is also the
Israel Journal of Veterinary Medicine Vol. 66 (4) December 2011
most common commensal isolate from healthy skin and ears (3, 4, 5). Treatment of canine pyoderma and otitis externa are usually empirical and are based on clinical experience. Samples are sent for culture and susceptibility tests usually when more than one type of microorganism is identified in a cytological examination, or when there is a failure in response to the commonly used antibiotics (1, 5). Bacteria can easily acquire and transfer multiple resistant genes which accelerate the emergence of antibacterial resistance. It occurs parIncreased bacterial resistance in dogs
143
Research Articles
ticularly in animal species and increases the risk of spread of resistance to other species, including humans (6). The emerging problem of methicillin resistant Staphylococcus aureus (MRSA) also affects veterinary medicine and pet dogs have been found to be carriers (7). Furthermore, an increased resistance of some other types of Staphylococci has been found over the last few years (8). Although the occurrence of S. pseudintermedius in humans is uncommon, it is considered as a zoonotic microorganism and its resistance can cause a serious problem to dog owners (7). The increased resistance of S. pseudintermedius isolates from pyoderma and otitis externa to multiple antimicrobials has been previously reported (9). The aim of this study was to examine the antimicrobial susceptibility of S. pseudintermedius and other bacteria isolated from skin and ear infections in dogs in Israel, and to examine whether the susceptibility to commonly used antibiotics has changed over a period of 7 years. The different isolates from skin and ears were compared and the susceptibility to antimicrobials checked for their possible correlation with epidemiological parameters.
Samples from ears were collected from the distal horizontal canals from cases of otitis externa by using sterile cottontipped applicators.
Method of culture and susceptibility
Samples were inoculated directly onto sheep blood agar (Columbia agar base supplemented with 5% sheep blood), and onto MacConkey agar in order to detect any gram negative bacteria. Identification of the bacteria was carried out based on colony and microscopic morphology, plasma coagulase and biochemical characteristics by standard methods (10). Antimicrobial susceptibility to cephalothin, amoxicillineclavulanate, potentiated sulfonamides and enrofloxacin was assessed by disk-diffusion, performed and interpreted according to CLSI standards (11). Quantitative results of susceptibility were assessed by the sensitivity index as described by Elad (12).
Statistical analysis
MATERIALS AND METHODS
Data collection
Medical records of dogs diagnosed with pyoderma and/or otitis externa that were examined at the dermatology department of the Veterinary Teaching Hospital, Koret School of Veterinary Medicine, The Hebrew University of Jerusalem (D-K-VMTH) between the years 2000-2006 were examined retrospectively. Inclusion criteria were the presence of data of bacterial culture in which S. pseudintermedius was isolated; signalment; the site from which the sample was obtained for bacteriology; results of susceptibility tests and results of cytological examinations. The absence of any of the above data was considered as exclusion criterion. Dogs that were treated with antibiotics for up to a week prior to obtaining the samples for culture and susceptibility were excluded. Furthermore where any other bacterial isolates were made their susceptibility test results were recorded and included in this study.
Data collected from the 7 year period were divided to two segments: 2000-2002 and 2003-2006, and resistance was compared between the 2 periods (the years were divided in such a way as to obtain equal number of cases in each group). The following parameters were examined from the collected data: y Association between the resistance of S. pseudintermedius to the signalment. y Association between the resistance of S. pseudintermedius to the site from which samples were obtained. y Association between cytology results and those of the culture y Association between resistances of S. pseudintermedius to those of other bacteria to the same antibiotics. y Association between the resistance of S. pseudintermedius to one antibiotic and its resistance to other antibiotics. All statistical tests were conducted using Statistical Package for the Social Sciences (SPSS), version 14. In order to assess the association between two qualitative variables, the Chi-square and the Fisher’s exact tests were applied. The paired association between qualitative variables was analyzed using the McNemar test. The comparison of quantitative variables between two independent groups was
Israel Journal of Veterinary Medicine Vol. 66 (4) December 2011
Samples collection
Samples from skin were obtained from intact pustules and/ or epidermal collarettes in cases of superficial pyoderma of various etiologies using sterile cotton-tipped applicators.
144
Zur, G.
Research Articles
Table 1: Percentages of resistances of various bacteria to the commonly used antibiotics
There were 24.1% isolates from skin Antibiotic: Bacteria: Cephalosporin Amoxicillin- Potentiated Fluoroquinolone and 75.9% isolates from ears. In clavulanate sulfonamide 41% of cases an additional type of S. pseudintermedius 7 12 15 7 bacteria was isolated and in 12% of Proteus spp 33 29 67 0 cases two more additional species of Pseudomonas aeruginosa NP NP NP 11 bacteria were isolated. Proteus spp. E.coli 14 29 0 0 were most frequently isolated in Streptococcus 14 0 0 0 addition to S. pseudintermedius folα-haemolytica lowed by Pseudomonas aeruginosa. Streptococcus 14 0 14 0 The latter were isolated only from β-haemolytica ear samples. Other bacteria that Corynebacterium spp 0 0 80 0 were isolated appear in the folPasteurella multocida 0 0 0 0 lowing order: E.coli, α-haemolytic Klebsiellaa 100 100 100 100 Streptococcus spp., β-haemolytic Bacillus sppa 100 100 0 0 Streptococcus spp., Corynebacterium NP – not performed spp., Pasteurella multocida, Klebsiella a only one isolate spp. and Bacillus spp. The latter 7 carried out using the independent samples t-test, as well as types of bacteria were not statistically analyzed for susceptithe non-parametric Mann-Whitney test. When comparing bility due to small numbers in each group. quantitative variables between three or more independent Resistance of the bacterial isolates to the groups, ANOVA and the non-parametric Kruskal-Wallis different antibiotics tests were applied. The strength of the linear association beFour commonly used antibiotics were analyzed: cephalothin, tween two quantitative variables was estimated by calculatamoxicillin-clavulanate, potentiated sulfonamides and enroing the Pearson correlation coefficient. All tests applied were floxacin. The resistance of the various bacteria isolates to the two-tailed, and a p-value of 5% or less was considered stafour common antibiotics is presented in Table 1. tistically significant.
RESULTS
Eighty six dogs met the inclusion criteria and entered into the study.
Signalment
Out of the 86 dogs, 64% were males and 36% were females, 74.4% were intact. Age of onset of skin or ear infections ranged between 2 months and 10.5 years, with a mean of 3.28 years, standard deviation (SD) of 2.98 and median of 2 years. Breeds: 25 dogs (29.1%) were mix breeds, 12 (14%) were German shepherd dogs, 7 (8.1%) – Shar pei, 7 (8.1%) Labrador retriever and the rest were other breeds with 3 or less dogs in each breed.
S. pseudintermedius: seven isolates (8%) were resistant to more than one antibiotic. one isolate (1.2%) was resistant to all 4 antibiotics, 2 isolates (2.3%) were resistant to 3 antibiotics and 4 isolates (4.6%) were resistant to two antibiotics. Proteus spp: two isolates (13%) were resistant to 2 antibiotics and 3 isolates (20%) were resistant to 3 different antibiotics. One isolate of E. coli and one of Bacillus spp. were resistant to 2 antibiotics. One isolate of Klebsiella was resistant to all 4 antibiotics.
Resistance to more than one antibiotic
Comparison of antibiotic resistance between two periods: 2000-2002 and 2003-2006
Bacteriology data
Cocci were detected in 97% of cytology samples and rods in 78%. Rods in general were isolated in 47% of the cultures.
Israel Journal of Veterinary Medicine Vol. 66 (4) December 2011
The results of a comparison between the two periods are presented in Table 2. Resistance of S. pseudintermedius to cephalothin and to amoxicillin-clavulanate was statistically significantly higher in the latter years (P=0.018). Between 2000-2002 none of the
Increased bacterial resistance in dogs
145
Research Articles
Table 2: Percentages of resistances of the common bacteria: comparison between two periods 2000-2002(I) and 2003-2006(II) Bacteria: Antibiotic: Cephalosporin I II P value amoxicillin-clavulanate I II P value potentiated sulfonamide I II P value fluoroquinolone I II P value NP – not performed Table 3: Susceptibility degree of the common bacteria: comparison between two periods 2000-2002(I) and 2003-2006(II) Bacteria: Antibiotic: Cephalosporin I II P value Amoxicillin-clavulanate I II P value Potentiated sulfonamide I II P value Fluoroquinolone I II P value NP – not performed S. Pseudomonas pseudintermedius Proteus spp. aeruginosa 2.18 0.81 NP 1.74 0.54 NP 0.009 0.31 1.87 1.20 NP 1.47 0.65 NP 0.009 0.037 1.0 0.54 NP 0.9 0.53 NP 0.534 0.97 1.33 1.57 1.36 1.24 1.20 0.72 0.365 0.055 0.38 S. Pseudomonas pseudintermedius Proteus spp. aeruginosa 0 17 NP 13 44 NP 0.018 0.264 3 0 NP 20 44 NP 0.019 0.078 12.5 67 NP 17 67 NP 0.528 1.0 2.5 0 0 11 0 50 0.129 0.047
44%, almost achieving statistical significance (P =0.078). The resistance of Pseudomonas aeruginosa to fluoroquinolones was found only in the second period (P=0.047).
Antibiotic resistance and the site of infection
Twenty percent of S. pseudintermedius isolates from skin infections were resistant to cephalothin while only 3% of ear samples were resistant (P=0.011). Similar results were found for S. pseudintermedius resistance to amoxicillin-clavulanate (25% of skin samples whereas 8% of ear samples were resistant. P=0.051). The same tendency but without statistical significance was found for Proteus spp. resistance to cephalosporins (50% of skin samples and 27% of ear samples, P=0.409) and to amoxicillin-clavulanate (50% of skin samples and 20% of ear samples, P=0.262). Contrary results were found for Proteus spp. resistance to potentiated sulfonamides: 82% of ear isolates vs. 25% of skin isolates were resistant (P=0.039).
isolates was resistant to cephalothin and 3% were resistant to amoxicillin-clavulanate, while between 2003-2006 13% were resistant to cephalothin and 20% to amoxicillin-clavulanate. No differences were found among resistances to potentiated sulfonamides and enrofloxacin between the two periods. Resistance of Proteus spp. to cephalosporins increased from 17% in the first period to 44% in the second period but this was not statistically significant (P =0.264). No resistance of the Proteus isolates to amoxicillin-clavulanate was noticed in the first period, and in the second period it increased to
No association was found between resistances of S. pseudintermedius to one antibiotic and its resistance to other antibiotics. A negative correlation was found between susceptibility of S. pseudintermedius and Proteus spp. to potentiated sulfonamides when isolated from the same sample: when S. pseudintermedius was susceptible to this antibiotic Proteus spp. was resistant (P= 0.04). No other differences were found between other bacteria in their susceptibility or resistance to a particular antibiotic.
Association between resistances of S. pseudintermedius to different antibiotics, and between different bacteria to the same antibiotic
Antibiotics resistances and signalment
A greater number of cases of S. pseudintermedius resistance to potentiated sulfonamides were seen in females compared to males: 29% vs. 7% (P=0.011). No differences were found between males and females in the resistance of S. pseudintermedius to other antibiotics or resistance of other bacteria to the examined antibiotics. There was no difference regardIsrael Journal of Veterinary Medicine Vol. 66 (4) December 2011
146
Zur, G.
Research Articles
ing antibiotic resistance of the various isolated bacteria in relation to the age of onset of the infection in the dogs in this study. Resistance of S. pseudintermedius to potentiated sulfonamides and enrofloxacin was found in 27% of the isolates obtained from Labrador retrievers, however none of the isolates was resistant to cephalexin. On the other hand, 14% of the S. pseudintermedius isolates from Shar peis were resistant to cephalexin whereas none were resistant to amoxicillin-clavulanate or enrofloxacin. Resistance to amoxicillinclavulanate was found in 21% of S. pseudintermedius bacteria isolated from mix breed dogs. All the above differences between breeds were not statistically significant. All the isolates of Proteus spp. from mix breeds were resistant to cephalothin. None of the German shepherd dog isolates was resistant to cephalexin (P=0.032). No differences were found between the different breeds in respect to resistance of Pseudomonas aeruginosa to enrofloxacin.
to potentiated sulfonamides was 0.29 and from skin was 1.19 (P=0.063). No differences were found between susceptibility tests results of these bacteria to the other antibiotics.
No differences were found between susceptibility tests results and the sex of the dogs or the age of onset of the infections. Susceptibility of S. pseudintermedius to enrofloxacin was lower in the Labrador retriever than in other breeds (0.81 vs. 1.3, P=0.067). No other differences were found among the various breeds and the degree of susceptibility of the other bacteria to the various antibiotics.
Association between susceptibility tests results and signalment
DISCUSSION
This study demonstrates increased resistance and decreased susceptibility of S. pseudintermedius and Proteus spp. to various kinds of antibiotics during a period of 7 years. S. intermedius isolated from dogs has been recently recognized and re-named as S. pseudintermedius. However, this study was preformed when the bacteria was isolated as S. intermedius. Reports of increased resistance to antibiotics of Staphylococci in Europe between the1980's and the 1990's are summarized by Lloyd (8). However, in the majority of reports different antibiotics were evaluated. An increased resistance of Staphylococci from dogs with pyoderma in Sweden to penicillinase-stable β-lactam antimicrobials was reported by Holms (13) over a period of 5 years in the late 1990's. Furthermore, S. pseudintermedius lacking susceptibility to β-lactamase–resistant antimicrobials is now being recognized in both infected dogs and cats in Europe (14) and the United States (15). In the first period of our study (2000-2002) no resistance of S. pseudintermedius to cephalosporins was observed, which is in agreement with results of other studies (3, 16-18). However during the second period of our study (2003-2006), 20% of the isolates were resistant. The results presented in our study are lower than those reported in a recent study (14), but much higher than other studies which were conducted during the same period, in which results showed that almost no resistant strains were detectable (4, 17, 19). The overall resistance to amoxicillin-clavulanate in our study was observed in 12% of S. pseudintermedius isolates. This contradicts other studies in which no resistance to this antibiotic was found (3, 4, 17-19). In our study an increased
Increased bacterial resistance in dogs
Comparison of the degree of susceptibility between the periods: 2000-2002 and 2003-2006
Comparison of the degree of susceptibility between the periods – 2000-2002 and 2003-2006 is presented in Table 3. The average degree of susceptibility of S. pseudintermedius to cephalosporins during the period 2000-2002 was 2.18, and in the period 2003-2006, 1.74 (P= 0.009). The average degree of susceptibility of S. pseudintermedius to amoxicillinclavulanate during the period 2000-2002 was 1.87, and in the period 2003-2006 it reached 1.47 (P= 0.009). No differences were found in the degrees of susceptibility of S. pseudintermedius to potentiated sulfonamides or to enrofloxacin between the two periods. The average degree of susceptibility of Proteus spp. to amoxicillin-clavulanate during the period 2000-2002 was 1.20 compared to 0.65 during the period 2003-2006 (P= 0.037). The average degree of susceptibility of Proteus spp. to enrofloxacin was 1.57 during the period 2000-2002 and was 1.2 during the period 2003-2006 (P= 0.055).
Association between susceptibility tests results and the site of infection
The average degree of susceptibility of S. pseudintermedius isolated from ear samples to amoxicillin-clavulanate was 1.76, and from skin samples was 1.29 (P=0.014). The average degree of susceptibility of Proteus spp. isolated from ears
Israel Journal of Veterinary Medicine Vol. 66 (4) December 2011
147
Research Articles
resistance of Proteus spp. to amoxicillin-clavulanate was also noted. This also contradicts other results in which most of the bacteria examined, excluding Pseudomonas showed low resistance to this antibiotic (17). The highest frequency of S. pseudintermedius and Proteus spp. resistance in our study was to potentiated sulfonamides (15% and 67%, respectively) and this pattern was not different between the two periods that were examined. The resistance of S. pseudintermedius is similar to that of another study (4) however in regard to Proteus our results were higher than reported elsewhere (4, 17). Although the resistance of S. pseudintermedius and Proteus spp. to potentiated sulfonamides was found to be twice the frequency than that to cephalosporins, a progressive time related increase in resistance was noticed only to cephalosporin. Furthermore, although these two microorganisms showed similar patterns of resistance to these antibiotics, they had conflicting behavior towards potentiated sulfonamides where one was susceptible while the other was resistant. This finding does not support theories of transfer of resistance between different kinds of microorganisms colonizing the same site. This could be due to the lack of plasmids found within this bacterial population (21). Although plasmids are commonly found within strains of S. aureus, this has not been demonstrated within strains of S. pseudintermedius (5). S. pseudintermedius resistance to fluoroquinolones was stable throughout the two periods of our study and was detected in 7% of the isolates, results which were similar to that of cephalosporins. It is possible that some of these bacteria were multi-drug resistant, but no molecular analysis was performed to confirm this possibility. Multi-drug resistant Staphylococci have been recovered from dogs receiving many antibiotics previously, including fluoroquinolones (5). S. pseudintermedius resistance to fluoroquinolones is considered very rare (16) and its frequency in this study is higher than in most reports (3, 4, 16-20, 22), but similar or lower than a report from Sweden where resistance, ranging from 8% to 12%, were reported between 1992 and 2002 (23). All the Proteus spp. and the other gram negative bacteria in this study were susceptible to enrofloxacin except for Pseudomonas, which was resistant in 11% of the isolates. It is important to note that the Pseudomonas in this study was isolated only from ears. It is possible that topical application of fluoroquinolones can over-come the in vitro susceptibil-
ity tests results and be effective against Pseudomonas otitis (24). In one study of Pseudomonas otitis, resistance to different fluoroquinolones was much higher and ranged between 25-57% (25). A very high resistance rate (16-52%) to various fluoroquinolones was found also in another study in which isolates from ears and skin were examined (26). In our study no attempt was made to differentiate between the different fluoroquinolones. Only enrofoxacin was examined, and the results were then extended to other fluoroquinolones. This was also suggested by Lloyd and Noble (27). In another two reports (25, 26) the authors showed that this application is not necessarily true and may lead to treatment failure or a missed opportunity for successful treatment. It can also be concluded that skin infection with gram negative bacteria can be successfully managed with fluoroquinolones or at least can be treated until results of culture and susceptibility are obtained. A complete susceptibility of gram negative rods to fluoroquinolones was reported by others (4). However in another report 12/59 strains of E.coli were resistant (20). The high resistance rates that were found in this study in comparison to other reports can be explained by the fact that we examined bacterial isolates from diseased skin and ears only, while in other reports bacterial isolates from healthy skin and ears were also examined (3, 16, 19, 22). However, in another two reports no differences in antimicrobial susceptibility were found between isolates from healthy dogs and dogs with otitis externa (3, 22). Furthermore some of the reported resistances are from isolates obtained from infections other than pyoderma or otitis externa (16, 19, 20, 26). In our study there was a significant reduction over time in the degree of susceptibility of S. pseudintermedius to cephalosporins and to amoxicillin-clavulanate and of Proteus spp. to amoxicillin-clavulanate and to fluoroquinolones. The susceptibility in our study was examined by the disc-diffusion method which may be less sensitive than MIC tests (28). A decrease in the susceptibility of S. pseudintermedius to amoxicillin-clavulanate between the years 1982-1991 and 2004-2007 was also found in another report that used the MIC method. Similar to our results, no difference in the susceptibility to fluoroquinolones were detected (20). The reasons for increased resistances and decreased antibiotics susceptibilities were beyond the aims of this study. However, one possible explanation for increasing antimiIsrael Journal of Veterinary Medicine Vol. 66 (4) December 2011
148
Zur, G.
Research Articles
crobial resistance is the wide usage of antibiotics (29, 30). Exposure to antimicrobial agents promotes emergence of resistance by facilitating the survival of resistant strains or inducing the expression of existing antimicrobial resistance genes (6). The dogs in our study suffered from ear and skin infections which are usually chronic. It is possible that previous antibiotic treatments increased resistance of the infective agents. However this cannot explain the increased resistance and decreased susceptibility between the two periods that were examined. Furthermore, in a survey in Europe no change in the susceptibility of S. pseudintermedius or Pseudomonas from canine skin and ear infection was noticed following the introduction of marbofloxacin (31). Another explanation for the differences found in resistance pattern between this study and other reports is the possible variability among geographic origins (3). Furthermore, the authors plan a future study in which the association between previous use of antibiotics and the development of bacterial resistance will be investigated. The importance of these results is to encourage clinicians to use the most appropriate antibiotics following culture and susceptibility test results and also to discourage clinicians from the wide use of these antibiotics due to increased resistance and potential zoonosis, which may cause severe infections in both species (32). It is also important to mention the limitation of susceptibility tests, because in vitro results not always correlate with the antibiotic activity in vivo (3) and also different results can be obtained from different laboratories (33).
ACKNOWLEDGEMENT
The authors thank Mrs. Tali Bdolah-Abram for the statistical analysis.
REFERENCES
1. Scott, D. W., Miller, W. H. and Griffin, C. E.: Bacterial skin diseases. In: Miller and Kirk’s Small Animal Dermatology. W.B. Saunders, Philadelphia. pp 274-335, 2001. 2. Rosser, E. J.: Causes of otitis externa. Vet. Clin. Sm. Anim. 34:459–468, 2004. 3. Lyskova, P., Vydrzalova, M. and Mazurova, J.: Identification and antimicrobial susceptibility of bacteria and yeasts isolated from healthy dogs and dogs with otitis externa. J. Vet. Med. A 54:559– 563, 2007. 4. Hariharan, H., Coles, M., Poole, D., Lund, L. and Page, R.: Update on antimicrobial susceptibilities of bacterial isolates from canine and feline otitis externa. Can. Vet. J. 47:253–255, 2006.
Israel Journal of Veterinary Medicine Vol. 66 (4) December 2011
5. May, E. R.: Bacterial Skin Diseases: Current thoughts on pathogenesis and management. Vet. Clin. Sm. Anim. 36:185–202, 2006. 6. Clarke, C. R.: Antimicrobial resistance. Vet. Clin. Sm. Anim. 36:987–1001, 2006. 7. Tomlin, J., Pead, M. J. Lloyd, D. H., Howell, S., Hartmann, F., Jackson, H.A. and Muir, P.: Methicillin-resistant Staphylococcus aureus infections in 11 dogs. Vet Rec. 144:60–64, 1999. 8. Lloyd, D. H.: Reservoirs of antimicrobial resistance in pet animals. J. Clin. Infect. Dis. 45(Suppl 2):148-52, 2007. 9. Petersen, A. D., Walker, R. D., Bowman, M. M., Schott, H. C. and Rosser Jr., E. J.: Frequency of isolation and antimicrobial susceptibility patterns of Staphylococcus intermedius and Pseudomonas aeruginosa isolates from canine skin and ear samples over a 6-year period (1992– 1997). J. Am. Anim. Hosp. Assoc. 38:407–413, 2002. 10. Quinn, P. J., Carter, M. E., Markey B. and Carter, G. R.: Clinical Veterinary Microbiology. Wolfe Publishing, London, 1994. 11. CLSI. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; Approved Standard – 3rd ed. CLSI document M31-A3. Wayne, PA: Clinical and Laboratory Standards Institute, 2008. 12. Elad, D.: The Sensitivity Index, a calculation to improve the choice of antibacterial therapy. Antimicrob. Chemother. 27:250, 1991. 13. Holm, B. R., Petersson, U., Mörner, A., Bergstrom, K., Franklin, A. and Greko, C.: Antimicrobial resistance in staphylococci from canine pyoderma: a prospective study of first-time and recurrent cases in Sweden. Vet. Rec. 151:600–605, 2002. 14. Loeffler, A., Linek, M., Moodley, A., Guardabassi, L., Sung, J. M. L., Winkler, M., Weiss, R. and Lloyd, D. H.: First report of multiresistant, mecA-positive Staphylococcus intermedius in Europe: 12 cases from a veterinary dermatology referral clinic in Germany. Vet. Dermatol. 18:412-421, 2007. 15. Morris, D. O., Rook, K. A., Shofer, F. S. and Rankin, S. C.: Screening of Staphylococcus aureus, Staphylococcus intermedius, and Staphylococcus schleiferi isolates obtained from small companion animals for antimicrobial resistance:a retrospective review of 749 isolates (2003–04). Vet. Dermatol. 17:332–337, 2006. 16. Futagawa-Saito, K. Ba-Thein, B. and Fukuyasu, T.: High occurrence of multi-antimicrobial resistance in Staphylococcus intermedius isolates from healthy and diseased dogs and domesticated pigeons. Res. Vet. Sci. 83:336–339, 2007. 17. Pedersen, K, Pedersen,K., Jensen, H., Finster, K., Jensen, V. F. and Heuer, O. E.: Occurrence of antimicrobial resistance in bacteria from diagnostic samples from dogs. Antimicrob. Chemother. 60:775–781, 2007. 18. Ganiere, J., Medaille, C.and Mangion, C.: Antimicrobial drug susceptibility of Staphylococcus intermedius clinical isolates from canine pyoderma. J Vet Med B Infect Dis Vet Public Health. 52:25–31, 2005. 19. Vanni, M., Tognetti, R., Pretti, C., Crema, F., Soldani, G., Meucci, V. and Intorre, L.: Antimicrobial susceptibility of Staphylococcus intermedius and Staphylococcus schleiferi isolated from dogs. Res. Vet. Sci. 87:192–195, 2009. 20. bGottlieb, G., Wigney, D. I., Martin, P. A., Norris, J. M., Malik, R. and Govendir, M.: Susceptibility of canine and feline Escherichia coli and canine Staphylococcus intermedius isolates to fluoroquinolones. Austral. Vet. J. 86:147-152, 2008. Increased bacterial resistance in dogs
149
Research Articles
21. Kloos, W. E., Orban, B. S. and Walker, D. D.: Plasmid composition of Staphylococcus species. Can. J. Microbiol. 27:271–278, 1981. 22. Tejedor Junco, M. T. and Martín Barrasa, J. L.: Identification and antimicrobial susceptibility of coagulase positive Staphylococci isolated from healthy dogs and dogs suffering from otitis externa. J. Vet. Med. B 49, 419–423, 2002. 23. Swedish Veterinary Antimicrobial Resistance Monitoring (SVARM).SVARM 2002. Uppsala: The National Veterinary Institute, 2003. Available at: http://www.sva.se/en/Startpage/Engelsk-malgruppsnavigering/animalhealth/Antibiotic-Resistence/ Monitoring-/SVARM-reports/ 24. Morris, D. O.: Medical therapy of otitis externa and otitis media. Vet. Clin. North Am. Small Anim. Pract. 34:541–555, 2004 25. Everett Wildermuth, B., Griffin, C.E., Rosenkrantz, W. S and Boord, M. J.: Susceptibility of Pseudomonas isolates from the ears and skin of dogs to enrofloxacin, marbofloxacin, and ciprofloxacin. J. Am. Anim. Hosp. Assoc. 2007;43:337-341 26. Rubin, J., Walker, R. D., Blickenstaff, K., Bodeis-Jones, S. and Zhao, S.: Antimicrobial resistance and genetic characterization of fluoroquinolone resistance of Pseudomonas aeruginosa isolated from canine infections. Vet. Microbiol. 131:164–172, 2008. 27. Lloyd, D. H. and Noble, W. C.: Use and abuse of antibiotics in veterinary dermatology. Vet. Dermatol. 10:161, 1999
28. McKay, L., Schuman Rose, C. D., Matousek, J. L., Schmeitzel, L. S., Gibson, N.M. and Gaskin, J. M.: Antimicrobial testing of selected fluoroquinolones against Pseudomonas aeruginosa isolated from canine otitis. J. Am. Anim. Hosp. Assoc. 43:307-312, 2007 29. Normand, E. H., Gibson, N. R, Reid, S. W. J., Carmichael, S. and Taylor, D. J.: Antimicrobial-resistance trends in bacterial isolates from companion-animal community practice in the UK. Prev. Vet. Med. 46:267–78, 2000 30. Lloyd, D. H.: Antimicrobial resistance. In: Harrison BA, editor. 20th Proceedings of the North American Veterinary Dermatology Forum. Sarasota, FL. pp. 33–36, 2005. 31. Meunier, D., Acar, J. F., Martel, J. L., Kroemer, S. and Valle, M.: A seven-year survey of susceptibility to marbofloxacin of pathogenic strains isolated from pets. Int. J. Antimicrob. Agents. 24:592– 598, 2004. 32. Guardabassi, L., Schwarz, S. and Lloyd, D. H.: Pet animals as reservoirs of antimicrobial-resistant bacteria. J Antimicrob Chemother. 54:321–332, 2004 33. Schick, A. E., Angus, J. C. and Coyner, K. S.: Variability of laboratory identification and antibiotic susceptibility reporting of Pseudomonas spp. isolates from dogs with chronic otitis externa. Vet. Dermatol. 18:120–126, 2007.
150
Zur, G.
Israel Journal of Veterinary Medicine Vol. 66 (4) December 2011
Increased Resistance of Staphylococcus pseudintermedius to the Commonly Used Antibiotics in Canine Dermatology
Zur, G.,1* Elad, D.,2 and Sterenzy-Agiv, N.1
1
Veterinary Teaching Hospital. The Koret School of Veterinary Medicine. The Robert H. Smith Faculty of Agriculture, Food and Environment. The Hebrew University of Jerusalem, P.O. Box 12, Rehovot, 76100, Israel 2 Department of Bacteriology. The Kimron Veterinary Institute, P.O. Box 12, Beit Dagan 50250, Israel
* Corresponding author: Dr. Gila Zur; Tel +972-3-9688547; e-mail: zurgila@agri.huji.ac.il
Increased resistance of Staphylococcus spp. is a growing problem in veterinary medicine. This study was performed to examine antimicrobial susceptibility of Staphylococcus pseudintermedius and other bacteria isolated from canine skin and ear infections over a 7 year period: 2000-2006. Eighty-six dogs which met predetermined inclusion criteria were examined. Bacterial isolates were examined for susceptibility to four commonly used antibiotics: cephalothin (first generation cephalosporin), amoxicillin-clavulanate, potentiated sulfonamide and enrofloxacin. The degree of susceptibility and the percentage of resistant strains of the bacteria to the antibiotics were compared between two periods: 2000-2002 and 2003-2006. Comparison between sample sites and association between susceptibility and signalment were examined. Resistance of S. pseudintermedius and Proteus spp. to cephalosporins and amoxicillin-clavulanate increased over the years and it was statistically significant for S. pseudintermedius (P=0.018). Susceptibility of S. pseudintermedius to cephalosporins and amoxicillin-clavulanate decreased over the years (P= 0.009). Susceptibility of Proteus spp. to amoxicillin-clavulanate and fluoroquinolones decreased as well (P=0.037, P=0.055 respectively). More skin than ear isolates of S. pseudintermedius and Proteus were resistant to cephalosporins and amoxicillinclavulanate. More ear isolates of Proteus were resistant to potentiated sulfonamides (P= 0.039). All isolates of Proteus from mix breed dogs, but not from German shepherds were resistant to cephalosporins (P=0.032). This study showed an increased resistance and decreased susceptibility of two common bacteria found in dogs' skin and ear infections. These findings should encourage clinicians to submit samples for culture and susceptibility before commencing antibacterial therapy, so as to provide a more accurate therapeutic decision and avoid unnecessary antibiotic use. Key words: dogs, bacteria, Staphylococcus pseudintermedius, antibiotics
AB ST RAC T
INTRODUCTION
Bacterial skin and ear infections are very common in dogs. In most of the cases the proliferation of bacteria is secondary to another dermatological problem such as allergy, ectoparasites or systemic diseases such as hypothyroidism, hyperadrenocorticism and others. Staphylococcus pseudintermedius (formerly S. intermedius) is the most common microorganism isolated from skin and ear infections (1, 2). It is also the
Israel Journal of Veterinary Medicine Vol. 66 (4) December 2011
most common commensal isolate from healthy skin and ears (3, 4, 5). Treatment of canine pyoderma and otitis externa are usually empirical and are based on clinical experience. Samples are sent for culture and susceptibility tests usually when more than one type of microorganism is identified in a cytological examination, or when there is a failure in response to the commonly used antibiotics (1, 5). Bacteria can easily acquire and transfer multiple resistant genes which accelerate the emergence of antibacterial resistance. It occurs parIncreased bacterial resistance in dogs
143
Research Articles
ticularly in animal species and increases the risk of spread of resistance to other species, including humans (6). The emerging problem of methicillin resistant Staphylococcus aureus (MRSA) also affects veterinary medicine and pet dogs have been found to be carriers (7). Furthermore, an increased resistance of some other types of Staphylococci has been found over the last few years (8). Although the occurrence of S. pseudintermedius in humans is uncommon, it is considered as a zoonotic microorganism and its resistance can cause a serious problem to dog owners (7). The increased resistance of S. pseudintermedius isolates from pyoderma and otitis externa to multiple antimicrobials has been previously reported (9). The aim of this study was to examine the antimicrobial susceptibility of S. pseudintermedius and other bacteria isolated from skin and ear infections in dogs in Israel, and to examine whether the susceptibility to commonly used antibiotics has changed over a period of 7 years. The different isolates from skin and ears were compared and the susceptibility to antimicrobials checked for their possible correlation with epidemiological parameters.
Samples from ears were collected from the distal horizontal canals from cases of otitis externa by using sterile cottontipped applicators.
Method of culture and susceptibility
Samples were inoculated directly onto sheep blood agar (Columbia agar base supplemented with 5% sheep blood), and onto MacConkey agar in order to detect any gram negative bacteria. Identification of the bacteria was carried out based on colony and microscopic morphology, plasma coagulase and biochemical characteristics by standard methods (10). Antimicrobial susceptibility to cephalothin, amoxicillineclavulanate, potentiated sulfonamides and enrofloxacin was assessed by disk-diffusion, performed and interpreted according to CLSI standards (11). Quantitative results of susceptibility were assessed by the sensitivity index as described by Elad (12).
Statistical analysis
MATERIALS AND METHODS
Data collection
Medical records of dogs diagnosed with pyoderma and/or otitis externa that were examined at the dermatology department of the Veterinary Teaching Hospital, Koret School of Veterinary Medicine, The Hebrew University of Jerusalem (D-K-VMTH) between the years 2000-2006 were examined retrospectively. Inclusion criteria were the presence of data of bacterial culture in which S. pseudintermedius was isolated; signalment; the site from which the sample was obtained for bacteriology; results of susceptibility tests and results of cytological examinations. The absence of any of the above data was considered as exclusion criterion. Dogs that were treated with antibiotics for up to a week prior to obtaining the samples for culture and susceptibility were excluded. Furthermore where any other bacterial isolates were made their susceptibility test results were recorded and included in this study.
Data collected from the 7 year period were divided to two segments: 2000-2002 and 2003-2006, and resistance was compared between the 2 periods (the years were divided in such a way as to obtain equal number of cases in each group). The following parameters were examined from the collected data: y Association between the resistance of S. pseudintermedius to the signalment. y Association between the resistance of S. pseudintermedius to the site from which samples were obtained. y Association between cytology results and those of the culture y Association between resistances of S. pseudintermedius to those of other bacteria to the same antibiotics. y Association between the resistance of S. pseudintermedius to one antibiotic and its resistance to other antibiotics. All statistical tests were conducted using Statistical Package for the Social Sciences (SPSS), version 14. In order to assess the association between two qualitative variables, the Chi-square and the Fisher’s exact tests were applied. The paired association between qualitative variables was analyzed using the McNemar test. The comparison of quantitative variables between two independent groups was
Israel Journal of Veterinary Medicine Vol. 66 (4) December 2011
Samples collection
Samples from skin were obtained from intact pustules and/ or epidermal collarettes in cases of superficial pyoderma of various etiologies using sterile cotton-tipped applicators.
144
Zur, G.
Research Articles
Table 1: Percentages of resistances of various bacteria to the commonly used antibiotics
There were 24.1% isolates from skin Antibiotic: Bacteria: Cephalosporin Amoxicillin- Potentiated Fluoroquinolone and 75.9% isolates from ears. In clavulanate sulfonamide 41% of cases an additional type of S. pseudintermedius 7 12 15 7 bacteria was isolated and in 12% of Proteus spp 33 29 67 0 cases two more additional species of Pseudomonas aeruginosa NP NP NP 11 bacteria were isolated. Proteus spp. E.coli 14 29 0 0 were most frequently isolated in Streptococcus 14 0 0 0 addition to S. pseudintermedius folα-haemolytica lowed by Pseudomonas aeruginosa. Streptococcus 14 0 14 0 The latter were isolated only from β-haemolytica ear samples. Other bacteria that Corynebacterium spp 0 0 80 0 were isolated appear in the folPasteurella multocida 0 0 0 0 lowing order: E.coli, α-haemolytic Klebsiellaa 100 100 100 100 Streptococcus spp., β-haemolytic Bacillus sppa 100 100 0 0 Streptococcus spp., Corynebacterium NP – not performed spp., Pasteurella multocida, Klebsiella a only one isolate spp. and Bacillus spp. The latter 7 carried out using the independent samples t-test, as well as types of bacteria were not statistically analyzed for susceptithe non-parametric Mann-Whitney test. When comparing bility due to small numbers in each group. quantitative variables between three or more independent Resistance of the bacterial isolates to the groups, ANOVA and the non-parametric Kruskal-Wallis different antibiotics tests were applied. The strength of the linear association beFour commonly used antibiotics were analyzed: cephalothin, tween two quantitative variables was estimated by calculatamoxicillin-clavulanate, potentiated sulfonamides and enroing the Pearson correlation coefficient. All tests applied were floxacin. The resistance of the various bacteria isolates to the two-tailed, and a p-value of 5% or less was considered stafour common antibiotics is presented in Table 1. tistically significant.
RESULTS
Eighty six dogs met the inclusion criteria and entered into the study.
Signalment
Out of the 86 dogs, 64% were males and 36% were females, 74.4% were intact. Age of onset of skin or ear infections ranged between 2 months and 10.5 years, with a mean of 3.28 years, standard deviation (SD) of 2.98 and median of 2 years. Breeds: 25 dogs (29.1%) were mix breeds, 12 (14%) were German shepherd dogs, 7 (8.1%) – Shar pei, 7 (8.1%) Labrador retriever and the rest were other breeds with 3 or less dogs in each breed.
S. pseudintermedius: seven isolates (8%) were resistant to more than one antibiotic. one isolate (1.2%) was resistant to all 4 antibiotics, 2 isolates (2.3%) were resistant to 3 antibiotics and 4 isolates (4.6%) were resistant to two antibiotics. Proteus spp: two isolates (13%) were resistant to 2 antibiotics and 3 isolates (20%) were resistant to 3 different antibiotics. One isolate of E. coli and one of Bacillus spp. were resistant to 2 antibiotics. One isolate of Klebsiella was resistant to all 4 antibiotics.
Resistance to more than one antibiotic
Comparison of antibiotic resistance between two periods: 2000-2002 and 2003-2006
Bacteriology data
Cocci were detected in 97% of cytology samples and rods in 78%. Rods in general were isolated in 47% of the cultures.
Israel Journal of Veterinary Medicine Vol. 66 (4) December 2011
The results of a comparison between the two periods are presented in Table 2. Resistance of S. pseudintermedius to cephalothin and to amoxicillin-clavulanate was statistically significantly higher in the latter years (P=0.018). Between 2000-2002 none of the
Increased bacterial resistance in dogs
145
Research Articles
Table 2: Percentages of resistances of the common bacteria: comparison between two periods 2000-2002(I) and 2003-2006(II) Bacteria: Antibiotic: Cephalosporin I II P value amoxicillin-clavulanate I II P value potentiated sulfonamide I II P value fluoroquinolone I II P value NP – not performed Table 3: Susceptibility degree of the common bacteria: comparison between two periods 2000-2002(I) and 2003-2006(II) Bacteria: Antibiotic: Cephalosporin I II P value Amoxicillin-clavulanate I II P value Potentiated sulfonamide I II P value Fluoroquinolone I II P value NP – not performed S. Pseudomonas pseudintermedius Proteus spp. aeruginosa 2.18 0.81 NP 1.74 0.54 NP 0.009 0.31 1.87 1.20 NP 1.47 0.65 NP 0.009 0.037 1.0 0.54 NP 0.9 0.53 NP 0.534 0.97 1.33 1.57 1.36 1.24 1.20 0.72 0.365 0.055 0.38 S. Pseudomonas pseudintermedius Proteus spp. aeruginosa 0 17 NP 13 44 NP 0.018 0.264 3 0 NP 20 44 NP 0.019 0.078 12.5 67 NP 17 67 NP 0.528 1.0 2.5 0 0 11 0 50 0.129 0.047
44%, almost achieving statistical significance (P =0.078). The resistance of Pseudomonas aeruginosa to fluoroquinolones was found only in the second period (P=0.047).
Antibiotic resistance and the site of infection
Twenty percent of S. pseudintermedius isolates from skin infections were resistant to cephalothin while only 3% of ear samples were resistant (P=0.011). Similar results were found for S. pseudintermedius resistance to amoxicillin-clavulanate (25% of skin samples whereas 8% of ear samples were resistant. P=0.051). The same tendency but without statistical significance was found for Proteus spp. resistance to cephalosporins (50% of skin samples and 27% of ear samples, P=0.409) and to amoxicillin-clavulanate (50% of skin samples and 20% of ear samples, P=0.262). Contrary results were found for Proteus spp. resistance to potentiated sulfonamides: 82% of ear isolates vs. 25% of skin isolates were resistant (P=0.039).
isolates was resistant to cephalothin and 3% were resistant to amoxicillin-clavulanate, while between 2003-2006 13% were resistant to cephalothin and 20% to amoxicillin-clavulanate. No differences were found among resistances to potentiated sulfonamides and enrofloxacin between the two periods. Resistance of Proteus spp. to cephalosporins increased from 17% in the first period to 44% in the second period but this was not statistically significant (P =0.264). No resistance of the Proteus isolates to amoxicillin-clavulanate was noticed in the first period, and in the second period it increased to
No association was found between resistances of S. pseudintermedius to one antibiotic and its resistance to other antibiotics. A negative correlation was found between susceptibility of S. pseudintermedius and Proteus spp. to potentiated sulfonamides when isolated from the same sample: when S. pseudintermedius was susceptible to this antibiotic Proteus spp. was resistant (P= 0.04). No other differences were found between other bacteria in their susceptibility or resistance to a particular antibiotic.
Association between resistances of S. pseudintermedius to different antibiotics, and between different bacteria to the same antibiotic
Antibiotics resistances and signalment
A greater number of cases of S. pseudintermedius resistance to potentiated sulfonamides were seen in females compared to males: 29% vs. 7% (P=0.011). No differences were found between males and females in the resistance of S. pseudintermedius to other antibiotics or resistance of other bacteria to the examined antibiotics. There was no difference regardIsrael Journal of Veterinary Medicine Vol. 66 (4) December 2011
146
Zur, G.
Research Articles
ing antibiotic resistance of the various isolated bacteria in relation to the age of onset of the infection in the dogs in this study. Resistance of S. pseudintermedius to potentiated sulfonamides and enrofloxacin was found in 27% of the isolates obtained from Labrador retrievers, however none of the isolates was resistant to cephalexin. On the other hand, 14% of the S. pseudintermedius isolates from Shar peis were resistant to cephalexin whereas none were resistant to amoxicillin-clavulanate or enrofloxacin. Resistance to amoxicillinclavulanate was found in 21% of S. pseudintermedius bacteria isolated from mix breed dogs. All the above differences between breeds were not statistically significant. All the isolates of Proteus spp. from mix breeds were resistant to cephalothin. None of the German shepherd dog isolates was resistant to cephalexin (P=0.032). No differences were found between the different breeds in respect to resistance of Pseudomonas aeruginosa to enrofloxacin.
to potentiated sulfonamides was 0.29 and from skin was 1.19 (P=0.063). No differences were found between susceptibility tests results of these bacteria to the other antibiotics.
No differences were found between susceptibility tests results and the sex of the dogs or the age of onset of the infections. Susceptibility of S. pseudintermedius to enrofloxacin was lower in the Labrador retriever than in other breeds (0.81 vs. 1.3, P=0.067). No other differences were found among the various breeds and the degree of susceptibility of the other bacteria to the various antibiotics.
Association between susceptibility tests results and signalment
DISCUSSION
This study demonstrates increased resistance and decreased susceptibility of S. pseudintermedius and Proteus spp. to various kinds of antibiotics during a period of 7 years. S. intermedius isolated from dogs has been recently recognized and re-named as S. pseudintermedius. However, this study was preformed when the bacteria was isolated as S. intermedius. Reports of increased resistance to antibiotics of Staphylococci in Europe between the1980's and the 1990's are summarized by Lloyd (8). However, in the majority of reports different antibiotics were evaluated. An increased resistance of Staphylococci from dogs with pyoderma in Sweden to penicillinase-stable β-lactam antimicrobials was reported by Holms (13) over a period of 5 years in the late 1990's. Furthermore, S. pseudintermedius lacking susceptibility to β-lactamase–resistant antimicrobials is now being recognized in both infected dogs and cats in Europe (14) and the United States (15). In the first period of our study (2000-2002) no resistance of S. pseudintermedius to cephalosporins was observed, which is in agreement with results of other studies (3, 16-18). However during the second period of our study (2003-2006), 20% of the isolates were resistant. The results presented in our study are lower than those reported in a recent study (14), but much higher than other studies which were conducted during the same period, in which results showed that almost no resistant strains were detectable (4, 17, 19). The overall resistance to amoxicillin-clavulanate in our study was observed in 12% of S. pseudintermedius isolates. This contradicts other studies in which no resistance to this antibiotic was found (3, 4, 17-19). In our study an increased
Increased bacterial resistance in dogs
Comparison of the degree of susceptibility between the periods: 2000-2002 and 2003-2006
Comparison of the degree of susceptibility between the periods – 2000-2002 and 2003-2006 is presented in Table 3. The average degree of susceptibility of S. pseudintermedius to cephalosporins during the period 2000-2002 was 2.18, and in the period 2003-2006, 1.74 (P= 0.009). The average degree of susceptibility of S. pseudintermedius to amoxicillinclavulanate during the period 2000-2002 was 1.87, and in the period 2003-2006 it reached 1.47 (P= 0.009). No differences were found in the degrees of susceptibility of S. pseudintermedius to potentiated sulfonamides or to enrofloxacin between the two periods. The average degree of susceptibility of Proteus spp. to amoxicillin-clavulanate during the period 2000-2002 was 1.20 compared to 0.65 during the period 2003-2006 (P= 0.037). The average degree of susceptibility of Proteus spp. to enrofloxacin was 1.57 during the period 2000-2002 and was 1.2 during the period 2003-2006 (P= 0.055).
Association between susceptibility tests results and the site of infection
The average degree of susceptibility of S. pseudintermedius isolated from ear samples to amoxicillin-clavulanate was 1.76, and from skin samples was 1.29 (P=0.014). The average degree of susceptibility of Proteus spp. isolated from ears
Israel Journal of Veterinary Medicine Vol. 66 (4) December 2011
147
Research Articles
resistance of Proteus spp. to amoxicillin-clavulanate was also noted. This also contradicts other results in which most of the bacteria examined, excluding Pseudomonas showed low resistance to this antibiotic (17). The highest frequency of S. pseudintermedius and Proteus spp. resistance in our study was to potentiated sulfonamides (15% and 67%, respectively) and this pattern was not different between the two periods that were examined. The resistance of S. pseudintermedius is similar to that of another study (4) however in regard to Proteus our results were higher than reported elsewhere (4, 17). Although the resistance of S. pseudintermedius and Proteus spp. to potentiated sulfonamides was found to be twice the frequency than that to cephalosporins, a progressive time related increase in resistance was noticed only to cephalosporin. Furthermore, although these two microorganisms showed similar patterns of resistance to these antibiotics, they had conflicting behavior towards potentiated sulfonamides where one was susceptible while the other was resistant. This finding does not support theories of transfer of resistance between different kinds of microorganisms colonizing the same site. This could be due to the lack of plasmids found within this bacterial population (21). Although plasmids are commonly found within strains of S. aureus, this has not been demonstrated within strains of S. pseudintermedius (5). S. pseudintermedius resistance to fluoroquinolones was stable throughout the two periods of our study and was detected in 7% of the isolates, results which were similar to that of cephalosporins. It is possible that some of these bacteria were multi-drug resistant, but no molecular analysis was performed to confirm this possibility. Multi-drug resistant Staphylococci have been recovered from dogs receiving many antibiotics previously, including fluoroquinolones (5). S. pseudintermedius resistance to fluoroquinolones is considered very rare (16) and its frequency in this study is higher than in most reports (3, 4, 16-20, 22), but similar or lower than a report from Sweden where resistance, ranging from 8% to 12%, were reported between 1992 and 2002 (23). All the Proteus spp. and the other gram negative bacteria in this study were susceptible to enrofloxacin except for Pseudomonas, which was resistant in 11% of the isolates. It is important to note that the Pseudomonas in this study was isolated only from ears. It is possible that topical application of fluoroquinolones can over-come the in vitro susceptibil-
ity tests results and be effective against Pseudomonas otitis (24). In one study of Pseudomonas otitis, resistance to different fluoroquinolones was much higher and ranged between 25-57% (25). A very high resistance rate (16-52%) to various fluoroquinolones was found also in another study in which isolates from ears and skin were examined (26). In our study no attempt was made to differentiate between the different fluoroquinolones. Only enrofoxacin was examined, and the results were then extended to other fluoroquinolones. This was also suggested by Lloyd and Noble (27). In another two reports (25, 26) the authors showed that this application is not necessarily true and may lead to treatment failure or a missed opportunity for successful treatment. It can also be concluded that skin infection with gram negative bacteria can be successfully managed with fluoroquinolones or at least can be treated until results of culture and susceptibility are obtained. A complete susceptibility of gram negative rods to fluoroquinolones was reported by others (4). However in another report 12/59 strains of E.coli were resistant (20). The high resistance rates that were found in this study in comparison to other reports can be explained by the fact that we examined bacterial isolates from diseased skin and ears only, while in other reports bacterial isolates from healthy skin and ears were also examined (3, 16, 19, 22). However, in another two reports no differences in antimicrobial susceptibility were found between isolates from healthy dogs and dogs with otitis externa (3, 22). Furthermore some of the reported resistances are from isolates obtained from infections other than pyoderma or otitis externa (16, 19, 20, 26). In our study there was a significant reduction over time in the degree of susceptibility of S. pseudintermedius to cephalosporins and to amoxicillin-clavulanate and of Proteus spp. to amoxicillin-clavulanate and to fluoroquinolones. The susceptibility in our study was examined by the disc-diffusion method which may be less sensitive than MIC tests (28). A decrease in the susceptibility of S. pseudintermedius to amoxicillin-clavulanate between the years 1982-1991 and 2004-2007 was also found in another report that used the MIC method. Similar to our results, no difference in the susceptibility to fluoroquinolones were detected (20). The reasons for increased resistances and decreased antibiotics susceptibilities were beyond the aims of this study. However, one possible explanation for increasing antimiIsrael Journal of Veterinary Medicine Vol. 66 (4) December 2011
148
Zur, G.
Research Articles
crobial resistance is the wide usage of antibiotics (29, 30). Exposure to antimicrobial agents promotes emergence of resistance by facilitating the survival of resistant strains or inducing the expression of existing antimicrobial resistance genes (6). The dogs in our study suffered from ear and skin infections which are usually chronic. It is possible that previous antibiotic treatments increased resistance of the infective agents. However this cannot explain the increased resistance and decreased susceptibility between the two periods that were examined. Furthermore, in a survey in Europe no change in the susceptibility of S. pseudintermedius or Pseudomonas from canine skin and ear infection was noticed following the introduction of marbofloxacin (31). Another explanation for the differences found in resistance pattern between this study and other reports is the possible variability among geographic origins (3). Furthermore, the authors plan a future study in which the association between previous use of antibiotics and the development of bacterial resistance will be investigated. The importance of these results is to encourage clinicians to use the most appropriate antibiotics following culture and susceptibility test results and also to discourage clinicians from the wide use of these antibiotics due to increased resistance and potential zoonosis, which may cause severe infections in both species (32). It is also important to mention the limitation of susceptibility tests, because in vitro results not always correlate with the antibiotic activity in vivo (3) and also different results can be obtained from different laboratories (33).
ACKNOWLEDGEMENT
The authors thank Mrs. Tali Bdolah-Abram for the statistical analysis.
REFERENCES
1. Scott, D. W., Miller, W. H. and Griffin, C. E.: Bacterial skin diseases. In: Miller and Kirk’s Small Animal Dermatology. W.B. Saunders, Philadelphia. pp 274-335, 2001. 2. Rosser, E. J.: Causes of otitis externa. Vet. Clin. Sm. Anim. 34:459–468, 2004. 3. Lyskova, P., Vydrzalova, M. and Mazurova, J.: Identification and antimicrobial susceptibility of bacteria and yeasts isolated from healthy dogs and dogs with otitis externa. J. Vet. Med. A 54:559– 563, 2007. 4. Hariharan, H., Coles, M., Poole, D., Lund, L. and Page, R.: Update on antimicrobial susceptibilities of bacterial isolates from canine and feline otitis externa. Can. Vet. J. 47:253–255, 2006.
Israel Journal of Veterinary Medicine Vol. 66 (4) December 2011
5. May, E. R.: Bacterial Skin Diseases: Current thoughts on pathogenesis and management. Vet. Clin. Sm. Anim. 36:185–202, 2006. 6. Clarke, C. R.: Antimicrobial resistance. Vet. Clin. Sm. Anim. 36:987–1001, 2006. 7. Tomlin, J., Pead, M. J. Lloyd, D. H., Howell, S., Hartmann, F., Jackson, H.A. and Muir, P.: Methicillin-resistant Staphylococcus aureus infections in 11 dogs. Vet Rec. 144:60–64, 1999. 8. Lloyd, D. H.: Reservoirs of antimicrobial resistance in pet animals. J. Clin. Infect. Dis. 45(Suppl 2):148-52, 2007. 9. Petersen, A. D., Walker, R. D., Bowman, M. M., Schott, H. C. and Rosser Jr., E. J.: Frequency of isolation and antimicrobial susceptibility patterns of Staphylococcus intermedius and Pseudomonas aeruginosa isolates from canine skin and ear samples over a 6-year period (1992– 1997). J. Am. Anim. Hosp. Assoc. 38:407–413, 2002. 10. Quinn, P. J., Carter, M. E., Markey B. and Carter, G. R.: Clinical Veterinary Microbiology. Wolfe Publishing, London, 1994. 11. CLSI. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; Approved Standard – 3rd ed. CLSI document M31-A3. Wayne, PA: Clinical and Laboratory Standards Institute, 2008. 12. Elad, D.: The Sensitivity Index, a calculation to improve the choice of antibacterial therapy. Antimicrob. Chemother. 27:250, 1991. 13. Holm, B. R., Petersson, U., Mörner, A., Bergstrom, K., Franklin, A. and Greko, C.: Antimicrobial resistance in staphylococci from canine pyoderma: a prospective study of first-time and recurrent cases in Sweden. Vet. Rec. 151:600–605, 2002. 14. Loeffler, A., Linek, M., Moodley, A., Guardabassi, L., Sung, J. M. L., Winkler, M., Weiss, R. and Lloyd, D. H.: First report of multiresistant, mecA-positive Staphylococcus intermedius in Europe: 12 cases from a veterinary dermatology referral clinic in Germany. Vet. Dermatol. 18:412-421, 2007. 15. Morris, D. O., Rook, K. A., Shofer, F. S. and Rankin, S. C.: Screening of Staphylococcus aureus, Staphylococcus intermedius, and Staphylococcus schleiferi isolates obtained from small companion animals for antimicrobial resistance:a retrospective review of 749 isolates (2003–04). Vet. Dermatol. 17:332–337, 2006. 16. Futagawa-Saito, K. Ba-Thein, B. and Fukuyasu, T.: High occurrence of multi-antimicrobial resistance in Staphylococcus intermedius isolates from healthy and diseased dogs and domesticated pigeons. Res. Vet. Sci. 83:336–339, 2007. 17. Pedersen, K, Pedersen,K., Jensen, H., Finster, K., Jensen, V. F. and Heuer, O. E.: Occurrence of antimicrobial resistance in bacteria from diagnostic samples from dogs. Antimicrob. Chemother. 60:775–781, 2007. 18. Ganiere, J., Medaille, C.and Mangion, C.: Antimicrobial drug susceptibility of Staphylococcus intermedius clinical isolates from canine pyoderma. J Vet Med B Infect Dis Vet Public Health. 52:25–31, 2005. 19. Vanni, M., Tognetti, R., Pretti, C., Crema, F., Soldani, G., Meucci, V. and Intorre, L.: Antimicrobial susceptibility of Staphylococcus intermedius and Staphylococcus schleiferi isolated from dogs. Res. Vet. Sci. 87:192–195, 2009. 20. bGottlieb, G., Wigney, D. I., Martin, P. A., Norris, J. M., Malik, R. and Govendir, M.: Susceptibility of canine and feline Escherichia coli and canine Staphylococcus intermedius isolates to fluoroquinolones. Austral. Vet. J. 86:147-152, 2008. Increased bacterial resistance in dogs
149
Research Articles
21. Kloos, W. E., Orban, B. S. and Walker, D. D.: Plasmid composition of Staphylococcus species. Can. J. Microbiol. 27:271–278, 1981. 22. Tejedor Junco, M. T. and Martín Barrasa, J. L.: Identification and antimicrobial susceptibility of coagulase positive Staphylococci isolated from healthy dogs and dogs suffering from otitis externa. J. Vet. Med. B 49, 419–423, 2002. 23. Swedish Veterinary Antimicrobial Resistance Monitoring (SVARM).SVARM 2002. Uppsala: The National Veterinary Institute, 2003. Available at: http://www.sva.se/en/Startpage/Engelsk-malgruppsnavigering/animalhealth/Antibiotic-Resistence/ Monitoring-/SVARM-reports/ 24. Morris, D. O.: Medical therapy of otitis externa and otitis media. Vet. Clin. North Am. Small Anim. Pract. 34:541–555, 2004 25. Everett Wildermuth, B., Griffin, C.E., Rosenkrantz, W. S and Boord, M. J.: Susceptibility of Pseudomonas isolates from the ears and skin of dogs to enrofloxacin, marbofloxacin, and ciprofloxacin. J. Am. Anim. Hosp. Assoc. 2007;43:337-341 26. Rubin, J., Walker, R. D., Blickenstaff, K., Bodeis-Jones, S. and Zhao, S.: Antimicrobial resistance and genetic characterization of fluoroquinolone resistance of Pseudomonas aeruginosa isolated from canine infections. Vet. Microbiol. 131:164–172, 2008. 27. Lloyd, D. H. and Noble, W. C.: Use and abuse of antibiotics in veterinary dermatology. Vet. Dermatol. 10:161, 1999
28. McKay, L., Schuman Rose, C. D., Matousek, J. L., Schmeitzel, L. S., Gibson, N.M. and Gaskin, J. M.: Antimicrobial testing of selected fluoroquinolones against Pseudomonas aeruginosa isolated from canine otitis. J. Am. Anim. Hosp. Assoc. 43:307-312, 2007 29. Normand, E. H., Gibson, N. R, Reid, S. W. J., Carmichael, S. and Taylor, D. J.: Antimicrobial-resistance trends in bacterial isolates from companion-animal community practice in the UK. Prev. Vet. Med. 46:267–78, 2000 30. Lloyd, D. H.: Antimicrobial resistance. In: Harrison BA, editor. 20th Proceedings of the North American Veterinary Dermatology Forum. Sarasota, FL. pp. 33–36, 2005. 31. Meunier, D., Acar, J. F., Martel, J. L., Kroemer, S. and Valle, M.: A seven-year survey of susceptibility to marbofloxacin of pathogenic strains isolated from pets. Int. J. Antimicrob. Agents. 24:592– 598, 2004. 32. Guardabassi, L., Schwarz, S. and Lloyd, D. H.: Pet animals as reservoirs of antimicrobial-resistant bacteria. J Antimicrob Chemother. 54:321–332, 2004 33. Schick, A. E., Angus, J. C. and Coyner, K. S.: Variability of laboratory identification and antibiotic susceptibility reporting of Pseudomonas spp. isolates from dogs with chronic otitis externa. Vet. Dermatol. 18:120–126, 2007.
150
Zur, G.
Israel Journal of Veterinary Medicine Vol. 66 (4) December 2011
Journal:
