Oct 15, 1984 - The isolates were obtained from pigs with proliferative enteritis and included 10 strains eachof .... C. c-o/i (n = 10). Range. 50%. 90%. Range.
Vol. 27, No. 1
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1985, p. 55-59
0066-4804/85/01055-05$02.00/0 Copyright © 1985, American Society for Microbiology
In Vitro Activities of 47 Antimicrobial Agents Against Three Campylobacter spp. from Pigst CONNIE J. GEBHART,* GILBERT E. WARD ANb HAROLD J. KURTZ Department of Veterinary Diagnostic Investigation, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108 Received 7 May 1984/Accepted 15 October 1984 The in vitro activities of 47 antimicrobial agents against 30 isolates of Campylobacter species from pigs were determined by the agar dilution technique. The isolates were obtained from pigs with proliferative enteritis and included 10 strains each of Campylobacter coli, Campylobacter sputorum subsp. mucosalis, and "Campylobacter hypointestinalis Gebhart et al." (this name is not on the Approved Lists). Carbadox, furazolidone, nitrofurantoin, gentamicin, and dimetridazole were the most active drugs, inhibiting all three Campylobacter species with a MIC for 50% of the isolates of 2 ,ug/ml or less. Trimethoprim-sulfamethoxazole, cefazolin, sulfachloropyridazine, novobiocin, vancomycin, sulfathiazole, cyclohexamide, bacitracin, p-arsanilic acid, and colistin were the least active, with MICs for 50% of the isolates ranging from 16 to .128 ,ug/ml.
isolates were identified by using previously described criteria (6, 9, 24). The other six isolates tested were obtained from the American Type Culture Collection, They itcluded ATCC 27374 C. fetus subsp. fetus Vdron and Chatelain, ATCC 29428 C. fetus subsp. jejuni Smibert, ATCC 15296 C. fetus Sebald and Vdron, ATCC 19438 C. fetus subsp. venerealis Veron and Chatelain, ATCC 33246 C. fetus subsp. intestinalis Smibert, and ATCC 25936 C. fetus subsp. fetus biotype 2 Sebald and Veron. All isolates were stored in sheep blood at -70°C and maintained for up to 4 days on Mueller-Hinton agar (Difco Laboratories, Detroit, Mich.) before use. Antimicrobial agents. The antimicrobial agents examined in this study and their sources are listed in Table 1. Antimicrobial agents were constituted in an appropriate diluent (2) so that each stock solution contained 5,120 pLg or U of active agent per ml. Stock solutions were prepared fresh or kept frozen at -20°C until used. Further, twofold dilutions were prepared in sterile distilled water for media preparation. Media. Organisms were grown and maintained on Mueller-Hinton agar with 5% defibrinated sheep blood. MuellerHinton broth (Difco) was used for preparing inocula for susceptibility tests. Antimicrobial susceptibility tests were performed in duplicate on Mueller-Hinton agar (without blood) and Wilkins-Chalgren agar (Difco). Agar was prepared in samples of 18 ml in tubes autoclaved at 121°C for 15 min. After the agar had cooled to 50°C, 2 ml of the concentration of antimicrobial agent was added to the tubes of agar, mixed, and poured into petri plates (15 by 100 mm) and allowed to solidify. Plates were dried overnight at room temperature. After excess surface moisture was evaporated, the plates were held in sealed bags at 4°C for no longer than 48 h before use. Incubation. For growth, plates were incubated at 37°C in a microaerophilic atmosphere obtained by using an evacuation replacement system without a catalyst. The air was evacuated twice to 400 mm of mercuty, and the jar was refilled each time with a gas mixture of 10% carbon dioxide, 10% hydrogen, and 80% nitrogen. The plates were examined after 48 h. MIC determinations. MICs of 47 ahtimicrobial agents were determined by agar dilution methods (5, 12). Plates were prepared with Mueller-Hinton or Wilkins-Chalgren agar,
Several Campylobacter species have been isolated from the intestines of swine. Although C. coli is isolated from normal swine and is generally not associated with a disease, it is recognized as a cause of diarrhea in human beings (17). C. sputorum subsp. mucosalis (9) and "C. hyointestinalis Gebhart et al." (6) have been isolated from swine with proliferative enteritis. Swine proliferative enteritis is a disease complex characterized by proliferative lesions of the intestine with intracellular Campylobacter-shaped organisms in the proliferating enterocytes (14) and affects weaned pigs of all ages. Limited information is available on the antimicrobial susceptibility patterns of swine Campylobacter isolates. The purpose of this study was to assay the in vitro effects of various antimicrobial agents, especially those currently in medicinal and veterinary use; to determine the agents to which C. sputorum subsp. mucosalis and "C. hyointestinalis" are generally susceptible and which, therefore, might be useful in the treatment of swine proliferative ileitis; to identify antimicrobial agents to which some or all swine Campylobacter species are resistant and which might be used to develop a selective medium for culturing swine isolates; and to identify patterns in antimicrobial susceptibility which might be useful in differentiating the three swine
Campylobacter species. In the present study, in vitro susceptibility patterns of 30 recent clinical isolates of "C. hyointestinalis," C. sputorum subsp. mucosalis, and C. coli were determined by an agar dilution technique against 47 antimicrobial agents. MATERIALS AND METHODS Bacterial isolates. A total of 36 isolates of Campylobacter species were tested. Thirty were individually isolated on non-antibiotic-containing media from ileal mucosal scrapings from pigs with naturally occurring proliferative enteritis. Prophylactic and therapeutic regimens for the enteric disease of the pigs from which isolates were obtained werd unknown. These 30 isolates consisted of 10 strains each of C. coli, "C. hyointestinalis," and C. sputorum subsp. mucosalis. All 30 * Corresponding author. t Paper no. 14,026 in the scientific journal series of the Minnesota Agricultural Experiment Station. 55
56
ANTIMICROB. AGENTS CHEMOTHER.
GEBHART, WARD, AND KURTZ
Antimicrobial agent
Nitroimidazoles Metronidazolea Dimetridazoleb
Nitrofurans Furazolidone' Nitrofurantoin'
Carbadoxd Lincomycins Lincomycina
Clindamycinf Penicillins Penicillin G' Ampicillina
Carbenicillin" Chloramphenicola
TABLE 1. Susceptibilities of Campylobacter spp. from swine to antimicrobial agents MIC (p.g/ml) required to inhibit: C. c-o/i (n = 10) C. sputorum subsp. mucosalis (n = 10) "C. hyointestinalis" (n = 10) 50% 50% 90% 50% 90% Range Range Range 0.5 0.5
1 0.5
4-64 0.25-16
0.125-0.25 1-2
0.25 1
0.25 2
.0.125-0.25
0.125
128 >128
.128
.128
2-16 2128I 16-32e 16-32
64->128 32->128 16-64
.0.125 32 0.5 2 0.25
0.25 64 0.5 8 1
16
16
-1286
128
16e
32e
16
32
>128
.128 64 64
32 16
.128 8-16
2128 2128 16-32
2 128e 2 128e 64-2 128 .128 .128 .128
128e 128
16 .128 .128
128
.128 .128 .128
.128 .128 .128
32 16 32 4 4
.128 128 64 .128 .128
methoxazolea.i
Macrolides Tylosina
Spiramycina Rifampina Erythromycina Oleandomycind
Arsenicals Phenylarsonic acidb p-Arsanilic acida
32 16 16 .0.125 2
>128 >128
64 16 32 0.125 2
16-.128 4-.:128 32-64 1->128 2->128
.128 .128
.128 .128
-128 .128
.128 .128
.128' 8 1 4
.128e 8-32 4-.128 2-. 128
.128e
.128e
Aminoglycosides Streptomycina Spectinomycina Kanamycina Neomycina Tobramycina Amikacina Gentamicina
.128e
16-32
4-_128 4-_128 4-8 2-8 1-2
128e 16 8 8 4 2 2
128 32 16 .128 8 8 2
0. 5->128e 4-8 0.25-1 2-8
le
8 1 4
.0.125-0.5
0.5
0.5
0.25-1 0.5-4
1 0.5
1 2
1-4 1-4
s0.125-1
8 4 4 2 2 0.5
Continued on
16 .128 .128 2 2 1
following page
VOL. 27, 1985
IN VITRO SUSCEPTIBILITIES OF SWINE CAMPYLOBACTERS
57
TABLE 1-Continued MIC (,ug/ml) required to inhibit:
Antimicrobial agent
Others Novobiocina Vancomycina Nalidixic acida Trimethoprima Cyclohexamidea
Ipronidazole' Flavomycink
"C. hyointestinalis" (n = 10) Range 50% 90%
32-64 .128 .128 64->-128 .128 s0.125-1 8
32 .128 .128 .128 .128 0.5 8
64 .128 .128 .128 .128 1 8
C. sputorum subsp. mucosalis (n = 10) 50% 90% Range
16-64 64-=128 16-64 32-64 .128 s0.125-1 0.5-1
32 64 32 64 .128 0.5 0.5
32 .128 64 64 .128 1 1
Range
C. coli (n = 10) 50%
16-32 .128 4-32 64-.128 .128 0.25-32 2-8
32 .128 8 64 .128 0.5 4
90%
32 .128 32 .128 .128 2 4
a Sigma Chemical Co., St. Louis, Mo. bSalisbury Laboratories, Inc., Charles City, Iowa. C Smith Kline Animal Health Products, West Chester, Pa. d Pfizer Inc., Groton, Conn. e Units per milliliter. f The Upjohn Co., Kalamazoo, Mich. 9 Merck Sharp & Dohme, West Point, Pa. hE. R. Squibb & Sons, Inc., New Brunswick, N.J. 1.25 ,ug of trimethoprim and 23.75 ,ug of sulfamethoxazole. Hoffmann-La Roche Inc., Nutley, N.J. kAmerican Hoechst Corp., Somerville, N.J.
containing serial twofold dilutions of each antimicrobial agent from 128 to 0.125 pLg or U per ml. To prepare inocula, loopfuls of test organisms were taken from pure 24-h cultures and emulsified in Mueller-Hinton broth to a turbidity equal to a no. 1 McFarland standard. The suspension was then spot inoculated onto each plate containing an antimicrobial agent with a replicator multipoint inoculating device (Cathra International, Inc., St. Paul, Minn.) that simultaneously inoculates up to 36 test organisms to the surface of plated agar media. The final inocula contained ca. 104 CFU of organisms per spot. Plates were inoculated from lowest to highest concentration of antimicrobial agent. Plates without antimicrobial agents were inoculated at the beginning of each series of dilutions as a control for viability of the test organisms. Also, both aerobic and anaerobic reference strains obtained from the American Type Culture Collection were included, as suggested by the National Committee for Clinical Laboratory Standards (12, 13), and incubated either aerobically or anaerobically. After 2 days of incubation, MICs were read as the lowest concentration of antimicrobial agent yielding no growth, one discrete colony, or a barely perceptible haze. When the MICs obtained on Mueller-Hinton agar differed by more than 2 concentrations from those obtained on Wilkins-Chalgren agar, the test was repeated. Also, when the MICs for the aerobic and anaerobic reference strains fell outside of 2 log dilutions of the anticipated results, the test was repeated with freshly prepared antimicrobial agents and media. RESULTS Table 1 shows the activities of the drugs tested against the three Campylobacter species, expressed as the 50% MIC, 90% MIC, and range of each drug. Of all the antimicrobial agents tested, the nitroimidazoles, nitrofurans, carbadox, and gentamicin displayed the highest activity against all three Campylobacter species. The MICs of carbadox were the lowest of all agents tested. The lincomycins were active against C. sputorum subsp. mucosalis but showed a wide range of activity against "C. hyointestinalis" and C. coli. Clindamycin had somewhat lower MICs than did lin-
comycin. Penicillin G had only moderate activity against the three Campylobacter species. Ampicillin and carbenicillin were highly active against "C. hyointestinalis" and C. sputorum subsp. mucosalis but were not as active against C. coli. Chloramphenicol showed high activity against C. sputorum subsp. mucosalis only. The tetracyclines were only moderately active against any of the species and demonstrated wide ranges of variation. Oxytetracycline and chlortetracycline showed somewhat more activity than did tetracycline. The cephalosporins displayed different activities, depending on which Campylobacter species was being tested. Most cephalosporins showed activity against "C. hyointestinalis" and C. sputorum subsp. mucosalis but not against C. coli. All peptides tested showed low activity against "C. hyointestinalis" and C. coli and moderate to low activity against C. sputorum subsp. mucosalis. The sulfonamides as a group and trimethoprim-sulfamethoxazole showed little activity against any of the three Campylobacter species. The macrolides generally showed moderate to low activity, except erythromycin and oleandomycin, which had high activity against C. sputorum subsp. mucosalis and a wide range of activity against "C. hyointestinalis" and C. coli. The arsenicals tested showed little activity. Streptomycin, spectinomycin, kanamycin, and neomycin showed wide ranges of activity, especially against "C. hyointestinalis" and C. coli. Among the aminoglycosides tested, gentamicin was the most active. Novobiocin, vancomycin, nalidixic acid, trimethoprim, and cyclohexamide all showed low activity, with the exception of nalidixic acid, which showed some activity when tested against C. coli. Ipronidazole and flavomycin showed relatively high activity against all three Campylobacter species. DISCUSSION In this study, carbadox proved to be the most active drug in vitro, inhibiting all three Campylobacter species at
