MIC90 of >64.0 ,ug/ml, but subsequent testing with thymidine phosphorylase-supplemented medium yielded an. MIC90 of 0.06 ,ug/ml. Both lincomycin and ...
Vol. 32, No. 3
JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1994, p. 793-795 0095-1 137/94/$04.00+0 Copyright C) 1994, American Society for Microbiology
Antimicrobial Susceptibility of Staphylococcus hyicus Isolated from Exudative Epidermitis in Pigs HENRIK C. WEGENER,' JEFFREY L. WATrS,2* SARAH A. SALMON,2 AND ROBERT J. YANCEY, National Veterinary Laboratory, Copenhagen, Denmark, and Animal Health Discovery Research, The Upjohn Companiy, Kalamazoo, Michigan 490012
JR.2
Received 24 September 1993/Returned for modification 18 November 1993/Accepted 14 December 1993
Exudative epidermitis or greasy pig syndrome is caused by the coagulase-variable staphylococcal species Staphylococcus hyicus. Treatment of this disease is problematic because of the limited number of antimicrobial agents available for this purpose. Thirteen antimicrobial agents were evaluated for their activities against 100 S. hyicus strains isolated from pigs with exudative epidermitis. Novobiocin was the most active compound tested, with an MIC for 90% of the strains tested (MIC90) of .0.06 ,ug/ml. Enrofloxacin, ampicillin, and ceftiofur were the next most active compounds, with MIC90s of 0.25, 0.5, and 1.0 ,ug/ml, respectively. However, 41.4% of the 99 strains tested were positive for ,-lactamase production. The MIC90s of erythromycin, tetracycline, and streptomycin were >32.0 ,ug/ml. Initial testing with sulfadiazine-trimethoprim yielded an MIC90 of >64.0 ,ug/ml, but subsequent testing with thymidine phosphorylase-supplemented medium yielded an MIC90 of 0.06 ,ug/ml. Both lincomycin and spectinomycin were relatively inactive against the S. hyicus strains tested, with MIC90s of >64.0 and > 128.0 ,ug/ml, respectively. However, the combination of the two compounds at ratios of 1:2 (lincomycin to spectinomycin) and 1:8 were more active, with MICgs of 16.0 and 4.0 ,ug/mi, respectively. These results indicate that novobiocin and sulfadiazine-trimethoprim were the most active compounds tested against the S. hyicus strains isolated from pigs with exudative epidermitis. Furthermore, the combination of lincomycin and spectinomycin was more active than the individual compounds against the strains tested. stored on nutrient agar slants (Difco Laboratories, Detroit, Mich.) until further use. MIC determinations were conducted by using either a commercial, dehydrated 96-well MIC panel and a semiautomated inoculation system (Sensititre Systems Group, Radiometer America Inc., Westlake, Ohio) or a manual broth microdilution method (4). The inoculum was prepared and MICs were determined according to the manufacturer's instructions. The activity of sulfadiazine-trimethoprim (TBR) was tested with Mueller-Hinton broth (BBL Microbiology Systems, Cockeysville, Md.) supplemented with 200 U of thymidine phosphorylase per liter. The following antimicrobial agents were included in the commercial broth microdilution MIC panel: ceftiofur, ampicillin, erythromycin, tetracycline, enrofloxacin, spectinomycin, and TBR. The following compounds were tested by the manual broth microdilution MIC method: lincomycin, lincomycin-spectinomycin, novobiocin, and streptomycin. The lincomycin-spectinomycin combination was tested at ratios of 1:2 and 1:8. The following quality control organisms were included in the study: Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853 (4). The quality control ranges specified by the manufacturer for the various antimicrobial agents were used in the study. ,-Lactamase production was determined by an acidometric method (Beta Lactam Diagnostic tablets; Rosco Diagnostica, Taastrup, Denmark). Prior to testing, ,B-lactamase production was induced by inoculating each isolate radially on a blood agar plate, a penicillin tablet was placed in the center of the plate, and the plate was incubated aerobically for 18 to 24 h at 35°C. 1-Lactamase production was determined by removing the colony material adjacent to the penicillin disk with a sterile loop, preparing a turbid bacterial suspension in 0.25 ml of saline, adding a Beta Lactam disk to the suspension, incubating the suspension for 4 h at 35°C, and observing the suspension
Exudative epidermitis (EE) is an acute generalized disease of suckling and weaned pigs caused by Staphylococcus hyicus (8). The clinical picture of EE is characterized by exfoliation of the skin, excessive sebaceous secretion, and the formation of a brownish coat of exudate that may cover the entire body. Morbidity can be up to 90% in infected herds. The infection is frequently treated with antimicrobial agents (5), but treatment is a problem because of the frequent occurrence of antimicrobial resistance in S. hyicus from pigs and subsequent treatment failure (11). Previous studies on the antimicrobial susceptibility of S. hyicus were conducted by the agar disk diffusion method (6, 11). Furthermore, the numbers of antimicrobial agents tested have been limited, and a number of antimicrobial agents which have recently been approved for veterinary use were not included in those studies. The purpose of the investigation described here was to determine the MICs of various antimicrobial agents for S. hyicus isolates from pigs with EE and to compare the activities of antimicrobial agents traditionally used to treat EE with those of antimicrobial agents not previously investigated for this purpose.
MATERLALS AND METHODS A total of 100 S. hyicus strains isolated from the skin of piglets with EE during 1991 and 1992 were used in the study. The strains were collected from piglets from 100 different herds from all regions of Denmark. Isolation and identification were performed as described by Wegener (9). One S. hyicus strain from each piglet was selected for the study. Strains were * Corresponding author. Mailing address: Animal Health Discovery Research, 7923-190-MR, The Upjohn Company, Kalamazoo, MI 49001. Phone: (616) 385-6605. Fax: (616) 384-2347.
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WEGENER ET AL. TABLE 1. MICs for 100 S. hyicus strains isolated from pigs with EE MIC (,g/ml) Antimicrobial agent
TBRa TBRb Novobiocin Ampicillin
Tetracycline Ceftiofur Enrofloxacin
Erythromycin Lincomycin Spectinomycin Lincomycin-spectinomycin (1:2) Lincomycin-spectinomycin (1:8) Streptomycin
50%
90%
Mode
Range
> 64.0 0.03 64.0 0.06 C0.06 0.5 >32.0 1.0 0.25 >32.0 >64.0 > 128.0 16.0 4.0 >64.0
> 64.0
2.0->64.0 0.015-0.25 .0.06-8.0 C0.03-2.0 0.13->32 0.13-1.0 0.13-0.5 0.25->32 0.25->64.0 32.0-> 128.0 0.5->64.0 0.06-> 16.0 1.0->64.0
64.0 4.0 0.25 4.0
0.03 C0.06 0.25 0.25 1.0 0.13 0.5 >64.0 64.0 2.0 0.25 2.0
Tested in unsupplemented Mueller-Hinton broth. h Tested in Mueller-Hinton broth supplemented with 200 U of thymidine phosphorylase per liter.
a
for a color change. A yellow or brownish color in the test was interpreted as a positive test result for j-lactamase production, while no change in color (violet) was considered to be a negative test result. RESULTS AND DISCUSSION
Previous studies of the antimicrobial susceptibility of S. hyicus from pigs by the agar disk diffusion technique have categorized those isolates as resistant or susceptible on the basis of the criteria developed for human bacterial isolates (6, 11). However, the extrapolation of interpretive criteria on basis of the pharmacokinetics of humans or other domestic animals may not be valid for pigs. Since valid interpretive criteria based on the pharmacokinetics of the tested antimicrobial agents in pigs and the in vivo efficacies of the antimicrobial agents against S. hyicus are not available, we could not categorize the isolates as resistant or susceptible, and so only the MIC data are reported here. In the present study, novobiocin was the most active compound against the S. hyicus strains tested, with an MIC for 90% of isolates tested (MIC90) of .0.06 ,ug/ml (Table 1). On the basis of this in vitro activity, novobiocin may have potential for the treatment of EE in pigs. However, novobiocin is currently approved in the United States only as an infusion product for the treatment of bovine mastitis and as a feed additive for the treatment of staphylococcal infections in chickens and turkeys. Because of its good in vivo efficacy, TBR is widely used to treat EE in Denmark. A previous study (11) performed by a tablet agar diffusion method suggested that none of the 100 S. hyicus strains isolated from Danish swine with EE were resistant to TBR. In the present study, initial testing of TBR by a broth microdilution method yielded high MICs (MIC90, >64.0 ,ug/ml) (Table 1). However, the addition of thymidine phosphorylase to the medium at 200 U/liter reduced the MIC90 of TBR for these strains to .0.06 ,ug/ml. This appears to indicate that the batch of Mueller-Hinton broth used in the present study contained levels of thymidine in excess of normally acceptable levels. However, the E. faecalis quality control strain yielded acceptable values with TBR, indicating that the medium did not contain excessive thymidine levels. Thus, it appears that S. hyicus is much more sensitive to thymidine than the quality control organism, which may result in an indication of false resistance on routine in vitro testing. Thus, the interpretive criteria for TBR when testing S. hyicus
isolated from pigs with EE need to be developed by using thymidine phosphorylase-supplemented medium. Until such interpretive criteria are developed, veterinary diagnostic laboratories should include S. hyicus ATCC 11249 as an additional quality control strain and supplement the growth medium with thymidine phosphorylase when testing S. hyicus strains with the sulfonamides. However, studies of experimentally induced EE in pigs have indicated that TBR is less effective than expected in vivo, as determined by its in vitro activity (10). This may indicate that the organism is capable of scavenging thymidine from the host and suggests that in vivo correlations with in vitro testing of this pathogen need to be developed. The fluoroquinolones are a class of antimicrobial agents which exhibit moderate to good activity against the staphylococci (2). Enrofloxacin was included in the present study as a representative of the fluoroquinolones. Enrofloxacin exhibited good activity (MIC90, 0.25 p.g/ml) against S. hyicus from pigs with EE. No fluoroquinolones are approved for use in the treatment of EE in pigs in the United States but are approved for use in swine, cattle, and poultry in Denmark. Ampicillin demonstrated good activity, with an MIC90 of 0.5 ,ug/ml for the 100 S. hyicus strains tested. However, staphylococci capable of producing 1-lactamases may demonstrate ampicillin MICs of as low as 0.5 ,ug/ml because of the inducible nature of the enzyme (3). In the present study, we determined ,B-lactamase production after culturing the organism in the presence of low levels of penicillin to induce enzyme production. Of 99 strains tested, 41.4% were postive for ,-lactamase production. These findings are in agreement with those of a recent study (11) that indicated that 44.0% of 100 clinical isolates of S. hyicus from pigs were resistant to penicillin by the agar diffusion method. However, the MIC90s for f-lactamasenegative and -positive strains were 0.25 and 0.5 .Lg/ml, respectively. Further studies are needed to determine the in vivo efficacy of ampicillin against 3-lactamase-producing S. hyicus strains. Ceftiofur, an extended-spectrum cephalosporin (12), has recently been approved for use in treating swine respiratory disease in most European countries including Denmark. The activity of ceftiofur was relatively good, with an MIC90 of 1.0 ,ug/ml. No differences in the activity of ceftiofur against ,-lactamase-producing strains was noted. Lincomycin and spectinomycin are currently marketed as a combination product for the treatment of swine respiratory disease. Separately, lincomycin and spectinomycin were inac-
SUSCEPTIBILITY OF S. HYICUS FROM PIGS
VOL. 32, 1994
tive against the S. hyicus strains tested, with MIC9(s of >64.0 and >128.0 ,ug/ml, respectively. However, the combination of the two compounds at ratios of 1:2 and 1:8 (lincomycin to spectinomycin) were much more active, with MIC90s of 16.0 and 4.0 jig/ml, respectively (Table 1), suggesting a synergistic interaction between the two compounds. Furthermore, the M'C50 of lincomycin-spectinomycin (1:8) was 0.25 ,ug/ml. It is interesting that lincomycin-spectinomycin at a ratio of 1:8, the ratio of concentrations achieved in pig plasma, yielded lower MICs than the 1:2 combination (the ratio of lincomycinspectinomycin in the product). This suggests that, when testing the susceptibilities of organisms to combinations of different antimicrobial compounds in vitro, the concentrations achieved in the host animal should be tested. None of the remaining antimicrobial agents tested demonstrated good activity against the majority of S. hyicus isolates from pigs with EE. The MIC90s of erythromycin, tetracycline, and streptomycin were >32.0 ,ug/ml (Table 1). The range of MICs of all of these antimicrobial agents was large, indicating that resistant as well as susceptible populations of S. hyicus existed. Only 43.0, 63.0, and 58.0% of the strains investigated could be regarded as susceptible to erythromycin, tetracycline, and streptomycin, respectively, on the basis of the breakpoints established in humans (1). This is in accordance with previous investigations and confirms that these drugs should be used for the treatment of EE only following antimicrobial susceptibility testing of isolates from the affected herd (11). Plasmid-mediated macrolide-lincosamide resistance has been reported to occur and to be frequently present in S. hyicus from pigs (7, 11). In the present investigation, the MICs of erythromycin were high (>32.0 jig/ml) for 39 isolates (39.0%), and for 30 of these isolates, the lincomycin MIC was also high (>64.0 pLg/ml). For only nine isolates were macrolide MICs high but the MICs of the lincosaminides were not. In conclusion, the in vitro results presented here indicate that novobiocin and TBR were the most active compounds tested against S. hyicus. S. hyicus strains were also determined to be extremely susceptible to the thymidine levels in the growth medium, an important consideration in testing the sulfonamides. The addition of thymidine inhibitors to the growth medium, e.g. thymidine phosphorylase, resolved this problem. Ceftiofur and enrofloxacin also demonstrated good activities against S. hyicus, and these antimicrobial agents
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might be considered for use in the treatment of EE in pigs. Ampicillin showed good in vitro activity, but its in vivo efficacy against ,B-lactamase-producing S. hyiclus strains needs to be established. Furthermore, lincomycin and spectinomycin tested in combination were more active than the compounds tested individually against the strains.
1. 2.
3. 4.
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6.
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