In vitro antimicrobial susceptibility of
Pasteurella haemolytica and Pasteurella multocida
recovered from cattle with bovine
respiratory disease complex
Karen W. Post, N. Andy Cole, Russell H. Raleigh Abstract. The antimicrobial susceptibilities of 421 Pasteurella haemolytica and 158 P. multocida isolates recovered from cattle with respiratory disease were determined with a microdilution minimal inhibitory con- centration test system. Isolates were analyzed for patterns of resistance to ampicillin, ceftiofur, erythromycin, gentamicin, penicillin, spectinomycin, sulfachlorpyridazine, sulfadimethoxine, tetracycline, and tylosin. All isolates tested were found susceptible to ceftiofur and sulfachlorpyridazine. Pasteurella haemolytica isolates were resistant to ampicillin, penicillin, sulfadimethoxine, tetracycline, and tylosin. Pasteurella multocida isolates were resistant to sulfadimethoxine, tetracycline, and tylosin.
Antimicrobial susceptibility patterns are important [cfu]/ml). The adjusted culture was then diluted in Mueller- in determining appropriate therapy against bacterial Hinton broth so that after inoculation each well contained pathogens. Information regarding minimal inhibitory approximately 5 x 105 cfu/ml. Plates were sealed and in- concentration (MIC) of antimicrobials to cattle isolates cubated at 37 C for 16-20 hr.
of Pasteurella species is limited to a few studies.3-6,9 Plates were read with a reflective viewer, and the MIC was The purpose of this study was to determine the anti- recorded as the lowest concentration of antimicrobial that microbial susceptibility patterns and MICs of P. hae- completely inhibited all visible bacterial growth. For the sul- fonamides, the MIC was recorded as the well where bacterial molytica and P. multocida isolated from cattle with growth was reduced by ≥ 80% when compared with the growth control well. The antimicrobial agents, concentrations, and Materials and methods
criteria used to determine susceptibility, moderate suscep- tibility, or resistance are presented in Table 1. Susceptibility Bacterial cultures. Pasteurella haemolytica and P. mul- interpretations were based on those described in previous tocida isolates were selected from nasal swabs and tissue studies or on available pharmacokinetic data1,4,6 (Halstead specimens submitted from cattle with respiratory disease to SL, Walker RD, Baker RE, et al.: 1989, Abstr Conf Res the Texas Veterinary Medical Diagnostic Laboratory, Am- Workers Animal Dis #70, p. 32A). In addition, the MIC arillo, between February and June 1989. These samples were concentrations that inhibited 50% (MIC ) and 90% (MIC ) from cases where MIC testing was requested. The isolates of the isolates were determined? Each lot of plates was sub- jected to quality control using reference strains Staphylococcus Minimal inhibitory concentration testing. Antimicrobial aureus (ATCC 29213), Streptococcusfaecalis (ATCC 29212), powders used in the microdilution MIC procedure were am- Pseudomonas aeruginosa (ATCC 27853), and Escherichia picillin, a ceftiofur, b erythromycin, c gentamicin, c penicillin c spectinomycin,c sulfachlorpyridazine, d sulfadimethoxine, c tetracycline, a and tylosine. A standard microdilution tech- nique was used to determine MICs.7 Antimicrobials were Tables 2 and 3 list the range of MIC (mcg/ml) of diluted either in phosphate-buffered saline (PBS), pH 8.0, or each antimicrobial agent against the isolates of P. hae- sterile water, and microtiter plates were prepared using a molytica and P. multocida, respectively, and list the dispenser.gEach of the Pasteurella isolates was inoculated into 3.5 ml of Mueller-Hinton brothf and incubated 4-6 hr cumulative MIC and MIC values of the Pasteurella at 37 C to obtain a final concentration equal to a 0.5 Mc- isolates. For the reference strains, the susceptibility Farland nephelometer standard (108 colony-forming units patterns (MIC) were within established limits.
Ninety percent of P. haemolytica isolates were mark- edly susceptible to ceftiofur, gentamicin, and sulfa- From the Texas Veterinary Medical Diagnostic Laboratory, 6610 chlorpyridazine. The MIC90 values for erythomycin Amarillo Blvd. West, Amarillo, TX 79106 (Post, Raleigh), and theUS Department of Agriculture, Conservation and Production Re- and spectinomycin were 4 µg/ml and 16 µg/ml, re- search Laboratory, PO Drawer 10, Bushland, TX 79012 (Cole).
spectively, indicating moderate susceptibility. The Revised for publication March 14, 1990.
MIC values for ampicillin, penicillin, sulfadimethox- Susceptibilities of Pasteurella from cattle Table 1. Dilution ranges of antimicrobial agents used for min- Table 3. Antimicrobial minimal inhibitory concentration sus-
imum inhibitory concentration (MIC) testing against Pasteurella ceptibility determinations (MIC and MIC ) of 158 Pasteurella haemolytica and P. multocida. multocida isolates recovered from cattle.
ine, tetracycline, and tylosin were all in the resistantcategory.
Ninety percent of the P. multocida isolates indicated mine if changes in resistance are developing. This is a marked susceptibility to ceftiofur and sulfachlorpyr- particularly important with the more newly released idazine. Ninety percent of the isolates were moderately antimicrobial agents, i.e., ceftiofur, because resistance susceptible to ampicillin, erythromycin, gentamicin, commonly increases in proportion to the frequency of penicillin, and spectinomycin. As with P. haemolytica, use. Apparently, the incidence of resistance is increas- P. multocida was resistant to sulfadimethoxine, tet- ing among the Pasteurella species.3,5,9
Ceftiofur and sulfachlorpyridazine appear to be very effective against both P. haemolytica and P. multocida.
However, these in vitro observations should only be Studies involving food animal pathogens4,6 have used as a guide for the clinician in the selection of the demonstrated that MIC testing is preferable over the most appropriate antimicrobial agent. Resistance to disk diffusion method for susceptibility testing because these antimicrobials may develop through increased it provides quantitative information. The MIC value use, and continued surveillance is warranted.
can provide the necessary information for the calcu- lation of effective dosage regimens.
Sources and manufacturers
It is important to periodically monitor the antimi- crobial susceptibilities of Pasteurella species to deter- a. American Research Products Co., S. Euclid, OH.
Table 2. Antimicrobial minimal inhibitory concentration sus-
d. E. R. Squibb and Sons, New Brunswick, NJ.
ceptibility determinations (MIC and MIC ) of 421 Pasteurella
e. Eli Lilly and Co., Indianapolis, IN.
haemolytica isolates recovered from cattle.
g. Autodiluter II and SRD II semi-automaic single reagent dis- penser, Dynatech Laboratories, Alexandria, VA.
h. American Type Culture Collection, Rockville, MD.
1. Brander GC: 1977, Tissue distribution of an antibiotic: amox- icillin. Vet Med Small Anim Clin 72:744-751.
2. Carter GR, ed.: 1984, Diagnostic procedures in veterinary bac- teriology and mycology, 4th ed., pp. 111-121. Charles C Thomas 3. Chang WH, Carter GR: 1976, Multiple drug resistance in Pas- teurella multocida and Pasteurella haemolytica from cattle and swine. J Am Vet Med Assoc 169:710-712.
4. Fales WH, Berg JN, Morehouse LG: 1986, Use and comparison of minimal inhibitory concentration and disk diffusion antimi-crobial susceptibility testing with bovine isolates of Pasteurella haemolytica type 1 and Pasteurella multocida recovered from Missouri cattle with bovine respiratory disease complex. Proc 7. National Committee for Clinical Laboratory Standards: 1985, Annu Meet Am Assoc Vet Lab Diagn 29: 1-8.
Methods for dilution antimicrobial susceptibility tests for bacteria 5. Fales WH, Selby LA, Webber JJ, et al.: 1982, Antimicrobial that grow aerobically, vol. 5, no. 22. National Committee for resistance among Pasteurella spp. recovered from Missouri and Clinical Laboratory Standards, Villanova, PA.
Iowa cattle with bovine respiratory disease complex. J Am Vet 8. Prescott JF, Baggot JD: 1988, Antimicrobial therapy in veteri- nary medicine. Blackwell Scientific Publications, Boston, MA.
6. Libal MC: 1985, Comparison of minimal inhibitory concentra- 9. Shoo MIS: 1989, Comparing different isolates of Pasteurella hae- tion and disk-difIusion antimicrobic sensitivity testing of bacte- molytica from beef calves using their in vitro antimicrobial sen- rial pathogens isolated from food animals. Am J Vet Res 46: sitivity patterns. Vet Microbiol 20:73-78.

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