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In vitro Susceptibility of Campylobacter jejuni and Campylobacter coli Isolated in Austria to Erythromycin and Ciprofloxacin
Zbl. Bakt. 272, 443-447 (1990) © Gustav Fischer Verlag, StuttgartlNew York
In vitro Susceptibility of Campylobacter jejuni and Campylobacter coli Isolated in Austria to Erythromycin and Ciprofloxacin * A. M. HIRSCHLl, D. WOLF 2 , ] . BERGER\ and M. L. ROTTER! 1 2
Hygiene-Institut der Universitiit, A-1095 Wien Institut rur Pathologie, Kaiser Franz Joseph Krankenhaus, A-lIOO Wien
Received May 10, 1989 . Accepted in revised form October 19, 1989
Summary More than 200 strains of Campylobaeter (C.) jejunilcoli isolated in 1985 and 1987/88 from human fecal specimens were tested for their susceptibility to erythromycin and ciprofloxacin. Their MIC 90 as assessed by agar dilution tests was 2.0 and 0.5 mg/l, respectively. Thus, all strains were regarded as susceptible to ciprofloxacin. With 2 out of 55 strains of C. coli the MIC of erythromycin was 8.0 mgll. Therefore, only 3.6% of the C. coli strains were resistant to erythromycin. All 209 strains of C. jejuni proved to be susceptible to erythromycin.
Zusammenfassung Uber 200 Campylobaeter (C.) jejunileoli-Stiimme aus menschlichen Stuhlproben der Jahre 1985 sowie 1987/88 wurden im Agardilutionstest auf ihre Empfindlichkeit gegeniiber Erythromycin und Ciprofloxacin untersucht. Die MHK90 rur Erythromycin und Ciprofloxacin betrug 2,0 bzw. 0,5 mgll. Alle gepriiften Stiimme waren gegeniiber Ciprofloxacin empfindlich. Bei lediglich 2 von insgesamt 55 C. eoli-Stiimmen betrug die MHK von Erythromycin 8,0 mgll. Somit war der Anteil Erythromycin-resistenter C. coli-Stiimme 3,6%. Die 209 Stiimme von C. jejuni waren durchwegs Erythromycin empfindlich.
Introduction Worldwide, Campylobaeter (C.) jejuni and C. coli range among the most frequent causes of bacterial diarrhoea. Although usually benign and self-limiting, this disease may also occur as a severe and long-lasting infection justifying antibiotic therapy.
* Dedicated to Professor Dr. med. Dr. med. h.c. Heinz Flamm on the occasion of his 60th birthday.
444
A. M. Hirschi, D. Wolf, J. Berger, and M. L. Rotter
Since long, erythromycin has been thought to be the drug of choice for treatment of
Campylobaeter-associated enteritis. The sensitivity of C. jejuni and C. coli to this drug, however, shows great regional differences and, especially for C. coli erythromycin-
resistant strains have often been reported (1, 2, 4, 5, 6, 7, 10, 11, 13, 14). For the Vienna area, the antibiotic sensitivity of these species was studied by comparing strains isolated in 1985 and in 1987/88. Besides erythromycin, also ciprofloxacin was tested which is also frequently used to treat Campylobaeter-associated enteritis.
Material and Methods
Strains. All strains were human fecal isolates which had been cultured and identified by standard methods (3). They were stored at-70°C in horse serum with 17% glycerol added. Most of the strains were isolates from sporadic cases. Only one strain per patient was included in the study. After thawing, the strains were grown on Mueller-Hinton (MH)-agar (Oxoid) with 5% defibrinated sheeep blood in a microaerophilic atmosphere (85% N2, 10%, CO 2, 5% O2) at 37°C for 48 h. From this, a suspension was prepared in isotonic saline to give approximately 109 cfulml, of which after a dilution by a factor of 10-2, volumes of 3 !Jl each were used to point-inoculate solid agar media. Assessment of MIC. The agar-dilution test was employed using MH-agar with 5% defibrinated sheep blood as a nutrient medium. For each of both antibiotics, final concentrations between 0.03 and 128mgll in a log2 series were tested. The plates were incubated under microaerophilic conditions for 48 h at 37°C. Definition of MIC. MIC was defined as the lowest antibiotic concentration reducing growth very distinctly as compared to the growth on the antibiotic-free control plate. Statistical methods. Differences in frequency were tested for statistical significance by chisquare tests, either in 2 x 2 or 2 x k contingency tables. As level of significance 2p = 0,05 was chosen. Results In Tables 1 and 2, the results of susceptibility testing have been summarized. Irrespective of the Campylobaeter species, i. e. C. jejuni or C. coli, there was a slight but significant (2p < 0.001) increase of sensitivity to erythromycin by one log2 step between strains isolated in 1985 and 1987/88 but not so to ciprofloxacin. Since for either drug, a break-point of > 4 mg/l indicating resistance had been defined (9, 12), none of the 209 strains of C. jejuni tested was resistant. Only 2 out of 55 C. coli strains were resistant to erythromycin, representing 3.6%, the difference to C. jejuni not being significant (2p > 0.05). The MIC so and MIC 90 values of all tested Campylobaeter strains were: for erythromycin 1.0 and 2.0 mgll, respectively, and for ciprofloxacin, 0.12 and 0.5 mgll.
Discussion As compared to results of other studies (2, 4, 10) the susceptibility pattern of enteric
Campylobaeter strains to erythromycin seems to have been rather favourable in this investigation. It is noticeable that - except for 2 resistant C. coli strains - there was no
1985
1985
C.jejuni
C. coli
>16,0
1987/88
15 12
12 23
4 6 2 9
4 3
'""
:t
2.
~
~:
§
ll>
r::r
'$.. o
ll>
a
1985
33 41 16
8,0
o ......
~.
g:
::t.
"0
~
C. coli
17 36
4,0
~
CJ) V>
70 109 18 36
2,0
2
>16,0
1985
1987/88
0,12
1,0
1
1
8,0
C.jejuni
::;0,06 0,50
9
20 9
4,0
Number of strains inhibited at concentration (mg/I)
42 57 12 6
2,0
No. of strains tested
8 69 4 18
1,0
Year of isolation
0,25
3
0,50
Number of strains inhibited at concentration (mg/I)
0,25
Species
3
0,12
"0
::;0,06
(j
70 139 18 37
No. of strains tested
Table 2. Susceptibility of C. jejuni and C. coli to Ciprofloxacin
1987/88
1987/88
Year of isolation
Species
Table 1. Susceptibility of C. jejuni and C. coli to Erythromycin
446
A. M. Hirschi, D. Wolf, J. Berger, and M. L. Rotter
difference in sensitivity to erythromycin in both species. This is in contrast to reports by Elharrif et a1. (2) who found markedly higher resistance rates in C. coli especially in isolates from pigs; the MICso values were> 128 mg/1. Also Fliegelman et al. (4) encountered erythromycin-resistant strains of C. coli more often (MIC 9o > 32 mgll), and in another study from Japan (10), the resistance rate was reported to be 40%, though in a very small sample of strains (n = 10). Erythromycin-resistant strains of C. jejuni seem to be less frequent; the proportion reported in literature ranges from 0 to 17% (1,2,4, 7, 10, 13,14). The excellent efficacy of ciprofloxacin found in this study was reported also by others (6, 8). From the results of this study, the following 3 practical consequences may be drawn: i) There is a case for distinction between C. jejuni and C. coli as the latter species is more often resistant. ii) Erythromycin sensitivity should always be tested if chemotherapy with this drug is considered. iii) There is also a case for - at least sporadically - testing the sensitivity of Campylobaeter to ciprofloxacin in order to detect occurrence of resistant strains in time. References
1. Andreasen, j. j.: In vitro susceptibility of Campylobaeter jejuni and Campylobaeter coli isolated in Denmark to 14 antimicrobial agents. Acta path. microbiol. immunol. scand. Sect. B 95 (1987) 189-192 2. Elharrif, Z., F. Megraud, and A. M. Marchand: Susceptibility of Campylobaeter jejuni and Campylobaeter coli to macrolides and related compounds. Antimicrob. Agents Chemother. 28 (1985) 695-697 3. Lior, H.: New extended biotyping scheme for Campylobaeter jejuni, Campylobaeter coli and "Campylobaeter laridis". J. Clin. Microbiol. 20 (1984) 636-640 4. Fliegelman, R. M., R. M. Petrak, L. j. Goodman, j. Segreti, G. N. Trenholme, and R. L. Kaplan: Comparative in vitro activities of 12 antimicrobial agents against Campylobaeter species. Antimicrob. Agents Chemother. 27 (1985) 429-430 5. Freydiere, A. M., G. Barbe, Y. Gille, and P. Vincent: Susceptibility of Campylobaeter fetus subsp. jejuni to paromomycin. Eur. J. Clin. Microbiol. 3 (1984) 322-323 6. Goodman, L. j., R. M. Fliegelman, G. M. Trenholme, and R. L. Kaplan: Comparative in vitro activity of ciprofloxacin against Campylobaeter spp. and other bacterial enteric pathogens. Antimicrob. Agents Chemother. 25 (1984) 505-506 7. Michel, j., M. Rogol, and D. Dickman: Susceptibility of clinical isolates of Campylobaeter jejuni to 16 antimicrobial agents. Antimicrob. Agents Chemother. 23 (1983) 796-797
8. Mikhail, I. A., A. L. Bourgeois, K. C. Hyams, j. K. Podgore, and C. R. Lissner: In vitro activity of ciprofloxacin compared to trimethoprim - sulfamethoxazole against Campy[obaeter spp., Shigella spp., and enterotoxigenic Escherichia coli causing travelers diarrhoea in Egypt. Scand. J. Infect. Dis. 19 (1987) 479-481 9. Report by Working Party of the British Society for Antimicrobial Chemotherapy: Breakpoints in in-vitro antibiotic sensitivity testing. J. Antimicrob. Chemother. 21 (1988) 701-710
to. Sagara, H., A. Mochizuki, N. Okamura, and R. Nakaya: Antimicrobial resistance of Campylobaeter jejuni and Campylobaeter coli with special reference to plasmid profiles
of Japanese clinical isolates. Antimicrob. Agents Chemother. 31 (1987) 713-719 11. Shanker, S. and T. C. Sorrell: Susceptibility of Campylobaeter jejuni to 23 antimicrobial agents. Pathology 15 (1983) 61-63
Susceptibility of Campylobacter jejuni
447
12. Thrupp, L. D.: Susceptibility testing of antibiotics in liquid media. In: Antibiotics in Laboratory Medicine, 2nd edition (V. Lorian, ed.), pp. 93-150. Williams and Wilkins Co., Baltimore (1986)
13. Van der Auwera, P. and B. Seorneaux: In vitro susceptibility of Campylobaeter jejuni to
27 antimicrobial agents and various combinations of betalactams with clavulanic acid or sulbactam. Antimicrob. Agents Chemother. 28 (1985) 37-40 14. Welkos, S. L.: A modified broth-disk antibiotic susceptibility test for Campylobaeter jejuni. Eur. J. Clin. Microbiol. 1 (1982) 354-360. Univ. Doz. Dr. A. M. Hirschi, Hygiene-Institute, University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria
In vitro Susceptibility of Campylobacter jejuni and Campylobacter coli Isolated in Austria to Erythromycin and Ciprofloxacin * A. M. HIRSCHLl, D. WOLF 2 , ] . BERGER\ and M. L. ROTTER! 1 2
Hygiene-Institut der Universitiit, A-1095 Wien Institut rur Pathologie, Kaiser Franz Joseph Krankenhaus, A-lIOO Wien
Received May 10, 1989 . Accepted in revised form October 19, 1989
Summary More than 200 strains of Campylobaeter (C.) jejunilcoli isolated in 1985 and 1987/88 from human fecal specimens were tested for their susceptibility to erythromycin and ciprofloxacin. Their MIC 90 as assessed by agar dilution tests was 2.0 and 0.5 mg/l, respectively. Thus, all strains were regarded as susceptible to ciprofloxacin. With 2 out of 55 strains of C. coli the MIC of erythromycin was 8.0 mgll. Therefore, only 3.6% of the C. coli strains were resistant to erythromycin. All 209 strains of C. jejuni proved to be susceptible to erythromycin.
Zusammenfassung Uber 200 Campylobaeter (C.) jejunileoli-Stiimme aus menschlichen Stuhlproben der Jahre 1985 sowie 1987/88 wurden im Agardilutionstest auf ihre Empfindlichkeit gegeniiber Erythromycin und Ciprofloxacin untersucht. Die MHK90 rur Erythromycin und Ciprofloxacin betrug 2,0 bzw. 0,5 mgll. Alle gepriiften Stiimme waren gegeniiber Ciprofloxacin empfindlich. Bei lediglich 2 von insgesamt 55 C. eoli-Stiimmen betrug die MHK von Erythromycin 8,0 mgll. Somit war der Anteil Erythromycin-resistenter C. coli-Stiimme 3,6%. Die 209 Stiimme von C. jejuni waren durchwegs Erythromycin empfindlich.
Introduction Worldwide, Campylobaeter (C.) jejuni and C. coli range among the most frequent causes of bacterial diarrhoea. Although usually benign and self-limiting, this disease may also occur as a severe and long-lasting infection justifying antibiotic therapy.
* Dedicated to Professor Dr. med. Dr. med. h.c. Heinz Flamm on the occasion of his 60th birthday.
444
A. M. Hirschi, D. Wolf, J. Berger, and M. L. Rotter
Since long, erythromycin has been thought to be the drug of choice for treatment of
Campylobaeter-associated enteritis. The sensitivity of C. jejuni and C. coli to this drug, however, shows great regional differences and, especially for C. coli erythromycin-
resistant strains have often been reported (1, 2, 4, 5, 6, 7, 10, 11, 13, 14). For the Vienna area, the antibiotic sensitivity of these species was studied by comparing strains isolated in 1985 and in 1987/88. Besides erythromycin, also ciprofloxacin was tested which is also frequently used to treat Campylobaeter-associated enteritis.
Material and Methods
Strains. All strains were human fecal isolates which had been cultured and identified by standard methods (3). They were stored at-70°C in horse serum with 17% glycerol added. Most of the strains were isolates from sporadic cases. Only one strain per patient was included in the study. After thawing, the strains were grown on Mueller-Hinton (MH)-agar (Oxoid) with 5% defibrinated sheeep blood in a microaerophilic atmosphere (85% N2, 10%, CO 2, 5% O2) at 37°C for 48 h. From this, a suspension was prepared in isotonic saline to give approximately 109 cfulml, of which after a dilution by a factor of 10-2, volumes of 3 !Jl each were used to point-inoculate solid agar media. Assessment of MIC. The agar-dilution test was employed using MH-agar with 5% defibrinated sheep blood as a nutrient medium. For each of both antibiotics, final concentrations between 0.03 and 128mgll in a log2 series were tested. The plates were incubated under microaerophilic conditions for 48 h at 37°C. Definition of MIC. MIC was defined as the lowest antibiotic concentration reducing growth very distinctly as compared to the growth on the antibiotic-free control plate. Statistical methods. Differences in frequency were tested for statistical significance by chisquare tests, either in 2 x 2 or 2 x k contingency tables. As level of significance 2p = 0,05 was chosen. Results In Tables 1 and 2, the results of susceptibility testing have been summarized. Irrespective of the Campylobaeter species, i. e. C. jejuni or C. coli, there was a slight but significant (2p < 0.001) increase of sensitivity to erythromycin by one log2 step between strains isolated in 1985 and 1987/88 but not so to ciprofloxacin. Since for either drug, a break-point of > 4 mg/l indicating resistance had been defined (9, 12), none of the 209 strains of C. jejuni tested was resistant. Only 2 out of 55 C. coli strains were resistant to erythromycin, representing 3.6%, the difference to C. jejuni not being significant (2p > 0.05). The MIC so and MIC 90 values of all tested Campylobaeter strains were: for erythromycin 1.0 and 2.0 mgll, respectively, and for ciprofloxacin, 0.12 and 0.5 mgll.
Discussion As compared to results of other studies (2, 4, 10) the susceptibility pattern of enteric
Campylobaeter strains to erythromycin seems to have been rather favourable in this investigation. It is noticeable that - except for 2 resistant C. coli strains - there was no
1985
1985
C.jejuni
C. coli
>16,0
1987/88
15 12
12 23
4 6 2 9
4 3
'""
:t
2.
~
~:
§
ll>
r::r
'$.. o
ll>
a
1985
33 41 16
8,0
o ......
~.
g:
::t.
"0
~
C. coli
17 36
4,0
~
CJ) V>
70 109 18 36
2,0
2
>16,0
1985
1987/88
0,12
1,0
1
1
8,0
C.jejuni
::;0,06 0,50
9
20 9
4,0
Number of strains inhibited at concentration (mg/I)
42 57 12 6
2,0
No. of strains tested
8 69 4 18
1,0
Year of isolation
0,25
3
0,50
Number of strains inhibited at concentration (mg/I)
0,25
Species
3
0,12
"0
::;0,06
(j
70 139 18 37
No. of strains tested
Table 2. Susceptibility of C. jejuni and C. coli to Ciprofloxacin
1987/88
1987/88
Year of isolation
Species
Table 1. Susceptibility of C. jejuni and C. coli to Erythromycin
446
A. M. Hirschi, D. Wolf, J. Berger, and M. L. Rotter
difference in sensitivity to erythromycin in both species. This is in contrast to reports by Elharrif et a1. (2) who found markedly higher resistance rates in C. coli especially in isolates from pigs; the MICso values were> 128 mg/1. Also Fliegelman et al. (4) encountered erythromycin-resistant strains of C. coli more often (MIC 9o > 32 mgll), and in another study from Japan (10), the resistance rate was reported to be 40%, though in a very small sample of strains (n = 10). Erythromycin-resistant strains of C. jejuni seem to be less frequent; the proportion reported in literature ranges from 0 to 17% (1,2,4, 7, 10, 13,14). The excellent efficacy of ciprofloxacin found in this study was reported also by others (6, 8). From the results of this study, the following 3 practical consequences may be drawn: i) There is a case for distinction between C. jejuni and C. coli as the latter species is more often resistant. ii) Erythromycin sensitivity should always be tested if chemotherapy with this drug is considered. iii) There is also a case for - at least sporadically - testing the sensitivity of Campylobaeter to ciprofloxacin in order to detect occurrence of resistant strains in time. References
1. Andreasen, j. j.: In vitro susceptibility of Campylobaeter jejuni and Campylobaeter coli isolated in Denmark to 14 antimicrobial agents. Acta path. microbiol. immunol. scand. Sect. B 95 (1987) 189-192 2. Elharrif, Z., F. Megraud, and A. M. Marchand: Susceptibility of Campylobaeter jejuni and Campylobaeter coli to macrolides and related compounds. Antimicrob. Agents Chemother. 28 (1985) 695-697 3. Lior, H.: New extended biotyping scheme for Campylobaeter jejuni, Campylobaeter coli and "Campylobaeter laridis". J. Clin. Microbiol. 20 (1984) 636-640 4. Fliegelman, R. M., R. M. Petrak, L. j. Goodman, j. Segreti, G. N. Trenholme, and R. L. Kaplan: Comparative in vitro activities of 12 antimicrobial agents against Campylobaeter species. Antimicrob. Agents Chemother. 27 (1985) 429-430 5. Freydiere, A. M., G. Barbe, Y. Gille, and P. Vincent: Susceptibility of Campylobaeter fetus subsp. jejuni to paromomycin. Eur. J. Clin. Microbiol. 3 (1984) 322-323 6. Goodman, L. j., R. M. Fliegelman, G. M. Trenholme, and R. L. Kaplan: Comparative in vitro activity of ciprofloxacin against Campylobaeter spp. and other bacterial enteric pathogens. Antimicrob. Agents Chemother. 25 (1984) 505-506 7. Michel, j., M. Rogol, and D. Dickman: Susceptibility of clinical isolates of Campylobaeter jejuni to 16 antimicrobial agents. Antimicrob. Agents Chemother. 23 (1983) 796-797
8. Mikhail, I. A., A. L. Bourgeois, K. C. Hyams, j. K. Podgore, and C. R. Lissner: In vitro activity of ciprofloxacin compared to trimethoprim - sulfamethoxazole against Campy[obaeter spp., Shigella spp., and enterotoxigenic Escherichia coli causing travelers diarrhoea in Egypt. Scand. J. Infect. Dis. 19 (1987) 479-481 9. Report by Working Party of the British Society for Antimicrobial Chemotherapy: Breakpoints in in-vitro antibiotic sensitivity testing. J. Antimicrob. Chemother. 21 (1988) 701-710
to. Sagara, H., A. Mochizuki, N. Okamura, and R. Nakaya: Antimicrobial resistance of Campylobaeter jejuni and Campylobaeter coli with special reference to plasmid profiles
of Japanese clinical isolates. Antimicrob. Agents Chemother. 31 (1987) 713-719 11. Shanker, S. and T. C. Sorrell: Susceptibility of Campylobaeter jejuni to 23 antimicrobial agents. Pathology 15 (1983) 61-63
Susceptibility of Campylobacter jejuni
447
12. Thrupp, L. D.: Susceptibility testing of antibiotics in liquid media. In: Antibiotics in Laboratory Medicine, 2nd edition (V. Lorian, ed.), pp. 93-150. Williams and Wilkins Co., Baltimore (1986)
13. Van der Auwera, P. and B. Seorneaux: In vitro susceptibility of Campylobaeter jejuni to
27 antimicrobial agents and various combinations of betalactams with clavulanic acid or sulbactam. Antimicrob. Agents Chemother. 28 (1985) 37-40 14. Welkos, S. L.: A modified broth-disk antibiotic susceptibility test for Campylobaeter jejuni. Eur. J. Clin. Microbiol. 1 (1982) 354-360. Univ. Doz. Dr. A. M. Hirschi, Hygiene-Institute, University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria