ANTIMICROBIAL AGENTS AND CHEMoTHERAPY, July 1979, p. 37-39
0066-4804/79/07-0037/03$02.00/0
Vol. 16, No. 1
Susceptibility of Campylobacter fetus subsp. jejuni to Twenty Antimicrobial Agents MATS WALDER
Department of Clinical Bacteriology, University of Lund, The General Hospital, Malm6, Sweden Received for publication 24, April 1979
One hundred recent clinical isolates of Campylobacter fetus subsp. jejuni were tested by an agar dilution technique for susceptibility to each of 20 antimicrobial agents. Doxycycline and gentamicin were the most active of the drugs examined, inhibiting all strains at concentrations achievable in serum. Although the median minimal inhibitory concentration of erythromycin was low, 8% of the isolates were highly resistant. All isolates of Campylobacter fetus subsp. jejuni were relatively resistant to the f,-lactam antibiotics. Some strains were highly resistant to metronidazole and tinidazole. Culture media and antibiotics. The medium used was Mueller-Hinton agar (Difco) enriched with 5% human blood. Test compounds and sources were: benzylpenicillin, lot 19351, Kabi, Sweden; ampicillin, batch 184, Astra, Sweden; mecillinam, batch G A 36375, Leo, Denmark; mezlocillin, Pt 3813C, Bayer, West Germany; carbenicillin, Al 21, Astra, Sweden; cephalothin, YV 0162, Lilly; cephalexin, lot 454-226-20, Lilly; cefuroxime, 1894-A4, Glaxo; cefotaxime, Ch-B-24, Hoechst AG, West Germany; trimethoprim, A 327 119, Roche, Switzerland; sulfamethoxazole, batch 390 060, Roche, Switzerland; clindamycin, A 7800 153, Upjohn; lincomycin, R 7700028, Upjohn; erythromycin, lot 4805CD, Abbot; tetracycline, lot 706-2364, Pfizer, doxycycline, lot GS 3065/A/3, Pfizer, gentamicin, 225 99009100, Schering Corp.; chloramphenicol, lot 668125 A, Parke Davis; metronidazole, 104300, Leo, Sweden; and tinidazole, Pfizer. Standard solutions of these drugs were prepared (with correction for compound impurities) and subjected to twofold serial dilution prior to incorporation of the latter in Mueller-Hinton agar. Susceptibility testing. The inoculum was derived
Campylobacter fetus subsp. intestinalis (14) has occasionally been recovered from blood, joint fluid, cerebrospinal fluid, and placenta of patients, especially those with compromised host defences (9, 10, 17). Using a special technique, Dekeyser et al. (4) isolated a related vibrio (9) from patients with diarrhea. With the development and application of a selective culture medium (13), isolation of C. fetus subsp. jejuni from the feces of enteritis patients (14) has been a frequent occurrence in many areas of the world (5, 12). Campylobacter enteritis may result from aninal contacts or ingestion of contaminated food and water (14). The illness is usually characterized by an acute diarrhea, with watery or even blood-stained stools, of several days duration, with or without fever (11). Apart from diarrhea, the outstanding symptom is abdominal pain, which often persists after diarrhea has ceased. Isolation of C. fetus subsp. jejuni from stools is not in itself an indication for antibiotic treatment, unless the patient is in acute distress. However, the organisms sometimes enter the bloodstream and then must be eradicated with antimicrobial agents. At the moment, there is comparatively little published informnation to guide the choice of drug for therapy (2, 3). The aim of the present investigation was to determine the susceptibility of C. fetus subsp. jejuni to commonly available antibiotics.
from a 48-h culture of C fetus subsp. jejuni grown on peptone blood agar plates and harvested in a medium containing, per liter of distilled water: Trypticase, 170 g; Phytone, 30 g; sodium chloride, 40 g; potassium nitrate, 10 g; sodium formate, 10 g; and hemin, 30 mg. The suspension was then diluted in this medium so that there were 106 to 107 organisms per ml. With a multipoint inoculator (15), 2 to 3 pd of the diluted bacterial suspension was applied to the surface of the antibiotic agar plates. These were incubated for 48 h at 37°C in gas jars containing at most 5% 02 in addition to a gas mixture of N2, H2, and CO2. Minimal inhibitory concentrations (MICs) were recorded as the lowest antibiotic concentrations that gave no visible growth.
MATERIALS AND METHODS Bacterial strains. Ninety-eight isolates were obtained in Malmo from patients with acute diarrhea; two reference strains were kindly provided by M. B. Skirrow, Worchester, England. After identification by standard procedures (8, 9), these strains were stored in Stuart medium at -20°C until tested.
RESULTS The range of MIC values for each of the 20 antimicrobial agents tested and the concentrations of these drugs required to inhibit 90 (MIC90) and 50% of the test strains are presented 37
38
WALDER
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Susceptibility of C. fetus subsp. jejuni to various antimicrobial drugs Inhibitory concn (pg/ml of medium) For % of
Drug
strain
Range
50
Benzylpenicillin Ampicillin Mecillinam Mezlocillin Carbenicillin Cephalothin Cephalexin Cefurosime Cefotaxime Trimethoprim Sulfamethoxazole Clindamycin Lincomycin Erythromycin
Tetracycline Doxycycline Gentamicin Chloramphenicol Metronidazole Tinidazole
2.5 2.5 2.5 20 5
40 40 40 160 80 >160 40 - >160 40 - >160 5 - 40 >160 5 - 80 0.1 - >50 1.6 - >50 0.2 - >50 0.4 6.2 3.1 0.05 3.1 0.4 3.1 - 12.5 0.4 - >25 0.4 - >25 -
5 5 5 40 20 >160 160 80 10 >160 20 0.4 6.2 1.6 0.8 0.2 0.8 6.2 3.1 1.6
90 20 20 20 80 40 >160 >160 >160 40 >160 40 0.8 12.5 3.1 3.1 1.6 1.6 12.5 >25 >25
in Table 1. These data show that, as a group, the f8-lactam antibiotics had low activity; MICso concentrations of 20 ,Ag/ml or larger were required to inhibit growth of the test strains. Clindamycin, doxycycline, and gentamicin were clearly the most active of the 20 drugs, with erythromycin and tetracycline slightly less effective. Trimethoprim and sulfamethoxazole were inactive. Metronidazole and tinidazole had marginal activities. Only three drugs, doxycycline, tetracycline, and gentamicin, were active against all test strains at concentrations attainable in serum with standard dosage. Chloramphenicol had an MIC9o of 12.5 yg/ml. This concentration is at the upper range of levels that can be attained in serum with oral dosage.
DISCUSSION The results of this study on the antimicrobial susceptibilities of 100 strains of C. fetus subsp. jejuni are in general agreement with those obtained by Butzler et al. (2), on 7 strains of C. fetus and 114 of related vibrios, and by Chow et al. (3) on 11 strains of C. fetus. The variations in susceptibility recorded in this study were greater than were reported previously. Erythromycin has been reported as the drug of choice for treatment of Campylobacter infections (1, 11, 13). The present investigation indicates a high degree of susceptibility to erythromycin for many of the clinical isolates of Campylobacter, with median MIC values of 1.6 ,ug/
ml. It is, however, of great interest that approximately 8% of the isolates were resistant to erythromycin, lincomycin, and clindamycin. Butzler et al. (2) reported one strain resistant to erythromycin in 1974. Because of this resistance, the use of these macrolide antibiotics in lifethreatening infections caused by Campylobacter is contra-indicated. This position is further supported by the fact that the resistant strains were from four endemic outbursts without any evident connection. C. fetus subsp. jejuni is fairly resistant to all f?-lactam antibiotics tested. With median MICs of 5 ,ug/ml or greater, penicillins are not the drugs of choice for treatment of infections caused by Campylobacter. The MICso values for mezlocillin and all cephalosporins are higher than serum concentrations that can be attained with standard dosage. The activity of metronidazole and tinidazole against Campylobacter is of some, although limited, interest. Until recently, these and other nitroimidazole derivatives were considered to be active only against obligate anaerobes. In this study, metronidazole and tinidazole exhibited activity at concentrations that could be attained in serum with dosages of 2 g/day (6). ADDENDUM After this study was completed and submitted for publication, Vanhoof et al. published a study on 95 strains of C. fetus subsp.jejuni against 29 antimicrobial agents (R. Vanhoof, M. P. Vanderlinden, R. Dierickx, S. Lauwers, E. Yourassowsky, and J. P. Butzler, Antimicrob. Agents Chemother. 14:553-556, 1978. Their results were in accordance with those reported above. LITERATURE CITED 1. Anonymous. 1978. Campylobacter infections in Britain 1977. Br. Med. J. i:1357. 2. Butzler, J. P., P. Dekeyser, and T. Lafontaine. 1974. Susceptibility of related vibrios and Vibrio fetus to twelve antibiotics. Antimicrob. Agents Chemother. 5: 86-89. 3. Chow, A. W., V. Patten, and D. Bednorz. 1978. Susceptibility of Campylobacter fetus to twenty-two antimicrobial agents. Antimicrob. Agents Chemother. 13: 416-418. 4. Dekeyser, P., M. Gossuin-Detrain, J. P. Butzler, and J. Sternon. 1972. Acute enteritis due to related vibrio: first positive stool cultures. J. Infect. Dis. 125:390-392. 5. De Mol, P., and E. Bosmans. 1978. Cantpylobacter enteritis in central Africa. Lancet i:604. 6. Forsgren, A., and J. Wallin. 1974. Tinidazole-a new preparation for Trichomonas vaginalis infections. I. Laboratory evaluation. Br. J. Vener. Dis. 50:146-147. 7. Garrod, L, H. Lambert, and F. O'Grady. 1973. Antibiotic and chemotherapy, 4th ed. Churchill, Livingstone, London. 8. Holdeman, L E., and W. E. C. Moore (ed.). 1977. Anaerobe Laboratory Manual, 4th ed., p. 114-115. Anaerobe Laboratory, Virginia Polytechnic Institute,
Blacksburg.
9. King, E. 0. 1962. The laboratory recognition of Vibrio
VOL. 16, 1979
10. 11. 12.
13. 14.
C. FETUS SUBSP. JEJUNI SUSCEPTIBILITY
fetus and a closely related Vibrio isolated from cases of human vibriosis. Ann. N.Y. Acad. Sci. 98:700-711. Krutchik, A. N., and W. Velasquez. 1977. Campylobacter fetus infection in a patient with Hodgkin's disease. J. Am. Med. Assoc. 238:1810-1911. Lancet. 1978. Campylobacter enteritis. Lancet ii:135. Lauwers, S., M. De Boeck, and J. P. Butzler. 1978. Camnpylobacter enteritis in Brussels. Lancet i:604-605. Skirrow, M. B. 1977. Campylobacter enteritis: a "new" disease. Br. Med. J. ii:9-11. Smibert, R. M. 1974. Genus II. Campylobacter Sebald and Veron 1963, 907, p. 207-212. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey's manual of determinative
39
bacteriology, 8th ed. The Williams & Wilkins Co., Baltimore. 15. Steers, E., E. L. Foltz, B. S. Graves, and J. Riden. 1959. Inocula-replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9:307-311. 16. Tiehan, W., and R L Voght. 1978. Waterbome Campylobacter gastroenteritis. Vermont. Morbid. Mortal. Weekly Rep. 27:207. 17. Wyatt, R. A., K, Younoszai, S. Anuras, and M. G. Myers. 1977. Campylobacter fetus septicaemia and hepatitis in a child with agammaglobulinemia. J. Pediatr. 91:441 442.
0066-4804/79/07-0037/03$02.00/0
Vol. 16, No. 1
Susceptibility of Campylobacter fetus subsp. jejuni to Twenty Antimicrobial Agents MATS WALDER
Department of Clinical Bacteriology, University of Lund, The General Hospital, Malm6, Sweden Received for publication 24, April 1979
One hundred recent clinical isolates of Campylobacter fetus subsp. jejuni were tested by an agar dilution technique for susceptibility to each of 20 antimicrobial agents. Doxycycline and gentamicin were the most active of the drugs examined, inhibiting all strains at concentrations achievable in serum. Although the median minimal inhibitory concentration of erythromycin was low, 8% of the isolates were highly resistant. All isolates of Campylobacter fetus subsp. jejuni were relatively resistant to the f,-lactam antibiotics. Some strains were highly resistant to metronidazole and tinidazole. Culture media and antibiotics. The medium used was Mueller-Hinton agar (Difco) enriched with 5% human blood. Test compounds and sources were: benzylpenicillin, lot 19351, Kabi, Sweden; ampicillin, batch 184, Astra, Sweden; mecillinam, batch G A 36375, Leo, Denmark; mezlocillin, Pt 3813C, Bayer, West Germany; carbenicillin, Al 21, Astra, Sweden; cephalothin, YV 0162, Lilly; cephalexin, lot 454-226-20, Lilly; cefuroxime, 1894-A4, Glaxo; cefotaxime, Ch-B-24, Hoechst AG, West Germany; trimethoprim, A 327 119, Roche, Switzerland; sulfamethoxazole, batch 390 060, Roche, Switzerland; clindamycin, A 7800 153, Upjohn; lincomycin, R 7700028, Upjohn; erythromycin, lot 4805CD, Abbot; tetracycline, lot 706-2364, Pfizer, doxycycline, lot GS 3065/A/3, Pfizer, gentamicin, 225 99009100, Schering Corp.; chloramphenicol, lot 668125 A, Parke Davis; metronidazole, 104300, Leo, Sweden; and tinidazole, Pfizer. Standard solutions of these drugs were prepared (with correction for compound impurities) and subjected to twofold serial dilution prior to incorporation of the latter in Mueller-Hinton agar. Susceptibility testing. The inoculum was derived
Campylobacter fetus subsp. intestinalis (14) has occasionally been recovered from blood, joint fluid, cerebrospinal fluid, and placenta of patients, especially those with compromised host defences (9, 10, 17). Using a special technique, Dekeyser et al. (4) isolated a related vibrio (9) from patients with diarrhea. With the development and application of a selective culture medium (13), isolation of C. fetus subsp. jejuni from the feces of enteritis patients (14) has been a frequent occurrence in many areas of the world (5, 12). Campylobacter enteritis may result from aninal contacts or ingestion of contaminated food and water (14). The illness is usually characterized by an acute diarrhea, with watery or even blood-stained stools, of several days duration, with or without fever (11). Apart from diarrhea, the outstanding symptom is abdominal pain, which often persists after diarrhea has ceased. Isolation of C. fetus subsp. jejuni from stools is not in itself an indication for antibiotic treatment, unless the patient is in acute distress. However, the organisms sometimes enter the bloodstream and then must be eradicated with antimicrobial agents. At the moment, there is comparatively little published informnation to guide the choice of drug for therapy (2, 3). The aim of the present investigation was to determine the susceptibility of C. fetus subsp. jejuni to commonly available antibiotics.
from a 48-h culture of C fetus subsp. jejuni grown on peptone blood agar plates and harvested in a medium containing, per liter of distilled water: Trypticase, 170 g; Phytone, 30 g; sodium chloride, 40 g; potassium nitrate, 10 g; sodium formate, 10 g; and hemin, 30 mg. The suspension was then diluted in this medium so that there were 106 to 107 organisms per ml. With a multipoint inoculator (15), 2 to 3 pd of the diluted bacterial suspension was applied to the surface of the antibiotic agar plates. These were incubated for 48 h at 37°C in gas jars containing at most 5% 02 in addition to a gas mixture of N2, H2, and CO2. Minimal inhibitory concentrations (MICs) were recorded as the lowest antibiotic concentrations that gave no visible growth.
MATERIALS AND METHODS Bacterial strains. Ninety-eight isolates were obtained in Malmo from patients with acute diarrhea; two reference strains were kindly provided by M. B. Skirrow, Worchester, England. After identification by standard procedures (8, 9), these strains were stored in Stuart medium at -20°C until tested.
RESULTS The range of MIC values for each of the 20 antimicrobial agents tested and the concentrations of these drugs required to inhibit 90 (MIC90) and 50% of the test strains are presented 37
38
WALDER
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Susceptibility of C. fetus subsp. jejuni to various antimicrobial drugs Inhibitory concn (pg/ml of medium) For % of
Drug
strain
Range
50
Benzylpenicillin Ampicillin Mecillinam Mezlocillin Carbenicillin Cephalothin Cephalexin Cefurosime Cefotaxime Trimethoprim Sulfamethoxazole Clindamycin Lincomycin Erythromycin
Tetracycline Doxycycline Gentamicin Chloramphenicol Metronidazole Tinidazole
2.5 2.5 2.5 20 5
40 40 40 160 80 >160 40 - >160 40 - >160 5 - 40 >160 5 - 80 0.1 - >50 1.6 - >50 0.2 - >50 0.4 6.2 3.1 0.05 3.1 0.4 3.1 - 12.5 0.4 - >25 0.4 - >25 -
5 5 5 40 20 >160 160 80 10 >160 20 0.4 6.2 1.6 0.8 0.2 0.8 6.2 3.1 1.6
90 20 20 20 80 40 >160 >160 >160 40 >160 40 0.8 12.5 3.1 3.1 1.6 1.6 12.5 >25 >25
in Table 1. These data show that, as a group, the f8-lactam antibiotics had low activity; MICso concentrations of 20 ,Ag/ml or larger were required to inhibit growth of the test strains. Clindamycin, doxycycline, and gentamicin were clearly the most active of the 20 drugs, with erythromycin and tetracycline slightly less effective. Trimethoprim and sulfamethoxazole were inactive. Metronidazole and tinidazole had marginal activities. Only three drugs, doxycycline, tetracycline, and gentamicin, were active against all test strains at concentrations attainable in serum with standard dosage. Chloramphenicol had an MIC9o of 12.5 yg/ml. This concentration is at the upper range of levels that can be attained in serum with oral dosage.
DISCUSSION The results of this study on the antimicrobial susceptibilities of 100 strains of C. fetus subsp. jejuni are in general agreement with those obtained by Butzler et al. (2), on 7 strains of C. fetus and 114 of related vibrios, and by Chow et al. (3) on 11 strains of C. fetus. The variations in susceptibility recorded in this study were greater than were reported previously. Erythromycin has been reported as the drug of choice for treatment of Campylobacter infections (1, 11, 13). The present investigation indicates a high degree of susceptibility to erythromycin for many of the clinical isolates of Campylobacter, with median MIC values of 1.6 ,ug/
ml. It is, however, of great interest that approximately 8% of the isolates were resistant to erythromycin, lincomycin, and clindamycin. Butzler et al. (2) reported one strain resistant to erythromycin in 1974. Because of this resistance, the use of these macrolide antibiotics in lifethreatening infections caused by Campylobacter is contra-indicated. This position is further supported by the fact that the resistant strains were from four endemic outbursts without any evident connection. C. fetus subsp. jejuni is fairly resistant to all f?-lactam antibiotics tested. With median MICs of 5 ,ug/ml or greater, penicillins are not the drugs of choice for treatment of infections caused by Campylobacter. The MICso values for mezlocillin and all cephalosporins are higher than serum concentrations that can be attained with standard dosage. The activity of metronidazole and tinidazole against Campylobacter is of some, although limited, interest. Until recently, these and other nitroimidazole derivatives were considered to be active only against obligate anaerobes. In this study, metronidazole and tinidazole exhibited activity at concentrations that could be attained in serum with dosages of 2 g/day (6). ADDENDUM After this study was completed and submitted for publication, Vanhoof et al. published a study on 95 strains of C. fetus subsp.jejuni against 29 antimicrobial agents (R. Vanhoof, M. P. Vanderlinden, R. Dierickx, S. Lauwers, E. Yourassowsky, and J. P. Butzler, Antimicrob. Agents Chemother. 14:553-556, 1978. Their results were in accordance with those reported above. LITERATURE CITED 1. Anonymous. 1978. Campylobacter infections in Britain 1977. Br. Med. J. i:1357. 2. Butzler, J. P., P. Dekeyser, and T. Lafontaine. 1974. Susceptibility of related vibrios and Vibrio fetus to twelve antibiotics. Antimicrob. Agents Chemother. 5: 86-89. 3. Chow, A. W., V. Patten, and D. Bednorz. 1978. Susceptibility of Campylobacter fetus to twenty-two antimicrobial agents. Antimicrob. Agents Chemother. 13: 416-418. 4. Dekeyser, P., M. Gossuin-Detrain, J. P. Butzler, and J. Sternon. 1972. Acute enteritis due to related vibrio: first positive stool cultures. J. Infect. Dis. 125:390-392. 5. De Mol, P., and E. Bosmans. 1978. Cantpylobacter enteritis in central Africa. Lancet i:604. 6. Forsgren, A., and J. Wallin. 1974. Tinidazole-a new preparation for Trichomonas vaginalis infections. I. Laboratory evaluation. Br. J. Vener. Dis. 50:146-147. 7. Garrod, L, H. Lambert, and F. O'Grady. 1973. Antibiotic and chemotherapy, 4th ed. Churchill, Livingstone, London. 8. Holdeman, L E., and W. E. C. Moore (ed.). 1977. Anaerobe Laboratory Manual, 4th ed., p. 114-115. Anaerobe Laboratory, Virginia Polytechnic Institute,
Blacksburg.
9. King, E. 0. 1962. The laboratory recognition of Vibrio
VOL. 16, 1979
10. 11. 12.
13. 14.
C. FETUS SUBSP. JEJUNI SUSCEPTIBILITY
fetus and a closely related Vibrio isolated from cases of human vibriosis. Ann. N.Y. Acad. Sci. 98:700-711. Krutchik, A. N., and W. Velasquez. 1977. Campylobacter fetus infection in a patient with Hodgkin's disease. J. Am. Med. Assoc. 238:1810-1911. Lancet. 1978. Campylobacter enteritis. Lancet ii:135. Lauwers, S., M. De Boeck, and J. P. Butzler. 1978. Camnpylobacter enteritis in Brussels. Lancet i:604-605. Skirrow, M. B. 1977. Campylobacter enteritis: a "new" disease. Br. Med. J. ii:9-11. Smibert, R. M. 1974. Genus II. Campylobacter Sebald and Veron 1963, 907, p. 207-212. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey's manual of determinative
39
bacteriology, 8th ed. The Williams & Wilkins Co., Baltimore. 15. Steers, E., E. L. Foltz, B. S. Graves, and J. Riden. 1959. Inocula-replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9:307-311. 16. Tiehan, W., and R L Voght. 1978. Waterbome Campylobacter gastroenteritis. Vermont. Morbid. Mortal. Weekly Rep. 27:207. 17. Wyatt, R. A., K, Younoszai, S. Anuras, and M. G. Myers. 1977. Campylobacter fetus septicaemia and hepatitis in a child with agammaglobulinemia. J. Pediatr. 91:441 442.
