Journal of Antimicrobial Chemotherapy (1992) 30, 463-474
Characterization of antibiotic-resistant Corynebacterium striatum strains Marilyn C. Roberts", Rebecca B. Leonard*, Ann Briselden*, Fritz D. Scfaoenknecht^ and Marie B. Coyk**
Antibiotic-resistant Corynebacterium striatum strains were isolated from 14 Harborview and one Veterans Administration Hospital patients in Seattle during the period 1987-90. These clindamycin-erythromycin resistant strains were shown to hybridize with the ermCd gene, which was cloned from a Corynebacterium diphtheriae plasmid and encodes for a rRNA methylase. Thirteen of these strains also hybridized with the tetM gene probes, and were tetracycline resistant The ermCd gene could be transferred, by conjugation, while the tetM gene was not transferable.
Introduction
Non-diphtheria bacteria of the genus Corynebacterium have been increasingly recognized as important opportunistic pathogens (Coyle & Lipsky, 1990). Most Corynebacterium jeikeium isolates (CDC group JK) are resistant to the majority of clinically useful antibiotics such as clindamycin, erythromycin, gentamicin, penicillins, and tetracycline (Larson et al., 1986). Similarly, most of the CDC Corynebacterium group D2 are highly resistant to a number of antibiotics (Soriano et al., 1987). However, the mechanisms of resistance to these antibiotics have not been characterized. The first description of an erythromycin and lincomycin resistant strain of Corynebacterium diphlheriae was in 1973 (Jellard & Lipinski, 1973). This was followed by a report from Seattle of an erythromycin (EmO and clindamycin resistant (CY) strain of C. diphtheriae and two resistant skin coryneforms isolated from a single outbreak in 1978 (Coyle et al., 1979). All six of the C. diphtheriae strains carried a single plasmid of 9-6 MDa (Schiller, Groman & Coyle, 1980). A 1606 bp £coRI fragment, containing the ermCd gene, was identified and shown to encode an inducible rRNA methylase. This gene shares 18-30% amino acid identity with other Gram-positive erm resistance genes (Serwold-Davies & Groman, 1988). We have characterized antibiotic resistant isolates of Corynebacterium striatum collected from patients between 1987 and 1990 and shown that they also carry the ermCd gene. In addition, most isolates were resistant to tetracycline and carried the tetM tetracycline resistance gene. •Corresponding author. 0305-7453/92/100463 + 12 J08.00/0
463 © 1992 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at Mount Royal University on June 10, 2015
Departments of'Pathobiology, bLaboratory Medicine, and'Microbiology, University of Washington, Seattle, Washington 98195, USA
464
M. C. Roberts et aL Methods
Bacterial strains
Susceptibility testing Antimicrobial susceptibility testing was performed on Mueller-Hinton agar (Difco, Detroit, MI, USA) supplemented with 5% defibrinated sheep blood. Standard disc and agar incorporation MIC methods were used and interpreted according to the guidelines of the National Committee for Clinical Laboratory Standards (1990o,£). Culture plates were examined at 24 and 48 h. Induction experiments Inducibility of erythromycin resistance was demonstrated when the inocula were pre-grown in the presence of clindamycin or erythromycin (0K)5 mg/L) for 24 h before testing by the disc diffusion method. The majority of the isolates were also tested after 4 h of pre-growth. No difference was seen between pre-growth at 4 h as compared with 24 h. Media The strains of C. diphtheriae, C. striatum, C. xerosis and Enterococcus faecalis were grown on supplemented GC-medium-base (Difco) at 36-5°C in 5% CO2 (Roberts & Lansciardi, 1990). For mating experiments, the media were supplemented with 8 mg/L of tetracycline and 12-5 mg/L fusidic acid, or 25 mg/L of erythromycin and 12-5 mg/L of fusidic acid, for the selection of transconjugants. Donors and recipients were selected on media supplemented with, 8 mg/L of tetracycline, or either 12-5 mg/L of fusidic acid or 10 mg/L of rifampicin, respectively. Plasmid isolation Plasmid DNA extractions from the C. striatum strains were done as previously described for coryneforms using the C. diphtheriae strain containing plasmid pNG2 as
Downloaded from http://jac.oxfordjournals.org/ at Mount Royal University on June 10, 2015
Antibiotic-resistant C. striatum isolates were cultured from 14 patients in Harborview Medical Center (HMQ between 1987 and 1990. Ten of the 14 resistant isolates were from sputum and produced a characteristic brown pigment. A similar brown pigmented C. striatum isolate (R198), obtained from a blood culture at the Seattle Veterans Affairs Medical Center (SVAM), was included in the study. Susceptible strains, R160 and R161, isolated from two HMC patients during this period were included as controls (Table I). The clinical isolates were identified according to the criteria of Hollis & Weaver (1981). A representative isolate was confirmed as C. striatum by the Division of Bacterial Diseases at the Centers for Disease Control. C. striatum type strain ATCC 6940, and the related Corynebacterium xerosis strains ATCC 373, ATCC 7711, ATCC 9016, and NCTC 9755 were included in the study as references. The clindamycin-erythromycin resistant C. diphtheriae S601, which carries the original pNG2 plasmid, was also included in the study (Coyle et al., 1979) (Table I).
25 21 17 27 6
C. xerosis ATCC 373 ATCC 7711 ATCC 9016 ATCC 9755
C. diphtheria* S601 6
44 19 40 38
24 12 11 35 35 44
19-23 21 20-24 20-24 18
37
32 27 36 36
6-13 34 32 30 34 38
22-29 33 (28-29)* 27-29 27-29 16
>128
0-25 0-25 1 0-12
>128 >128 > 128 1 ND 1
>128 >128 ND' ND >128
a
>128
). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically. 2nd edn; Approved Standard M7-A2. NCCLS, VUlanova, PA. Roberts, M. C. (1989). Plasmid-mediated Tet M in Haemophilia ducreyi. Antimicrobial Agents and Chemotherapy 33, 1611-3. Roberts, M. C. & Hillier, S. L. (1990). Genetic basis of tetracycline resistance in urogenital bacteria. Antimicrobial Agents and Chemotherapy 34, 261-4. Roberts, M. C. & Lansciardi, J. (1990). Transferable Tet M in Fusobacterium nucleatum. Antimicrobial Agents and Chemotherapy 34, 1836-8. Sanchez-Pescador, R., Brown, J. T., Roberts, M. & Urdea, M. S. (1988). The nucleotide sequence of the tetracycline resistance determinant TetM from Ureaplasma urealyticum. Nucleic Acids Research 16, 1216-7. Schiller, J., Groman, N. & Coyle, M. (1980). Plasmids in Corynebacterium diphtheriae and diphtheroids mediating erythromycin resistance. Antimicrobial Agents and Chemotherapy 18, 814-21. Schiller, J., Strom, M., Groman, N. & Coyle, M. (1983). Relationship between pNG2, an Em' plasmid in Corynebacterium diphtheriae, and plasmids in aerobic skin coryneforms. Antimicrobial Agents and Chemotherapy 24, 892-901. Serwold-Davies, T. M. & Groman, N. B. (1986). Mapping and cloning of Corynebacterium diphtheriae plasmid pNG2 and characterization of its relatedness to plasmids from skin coryneforms. Antimicrobial Agents and Chemotherapy 30, 69-72. Serwold-Davies, T. M. & Groman, N. B. (1988). Identification of a methylase gene for erythromycin resistance within the sequence of a spontaneously deleting fragment of Corynebacterium diphtheriae plasmid pNG2. FEMS Microbiology Letters 56, 7-14. Soriano, F., Ponte, C , Santamaria, M., Torres, A. & Fernandez-Roblas, R. (1987). Susceptibility of urinary isolates of Corynebacterium group D2 to fifteen antimicrobials and acetohydroxamic acid. Journal of Antimicrobial Chemotherapy 20, 349-55. Taylor, D. E., Hiratsuka, K., Ray, H. & Manavathu, E. K. (1987). Characterization and expression of a cloned tetracycline resistance determinant from Campylobacter jejuni plasmid pUA466. Journal of Bacteriology 169, 2984-9. Zilhao, R., Papadopoulou, B. & Courvalin, P. (1988). Occurrence of the Campylobacter resistance gene tetO in Enterococcus and Streptococcus spp. Antimicrobial Agents and Chemotherapy 32, 1793-6.
Characterization of antibiotic-resistant Corynebacterium striatum strains Marilyn C. Roberts", Rebecca B. Leonard*, Ann Briselden*, Fritz D. Scfaoenknecht^ and Marie B. Coyk**
Antibiotic-resistant Corynebacterium striatum strains were isolated from 14 Harborview and one Veterans Administration Hospital patients in Seattle during the period 1987-90. These clindamycin-erythromycin resistant strains were shown to hybridize with the ermCd gene, which was cloned from a Corynebacterium diphtheriae plasmid and encodes for a rRNA methylase. Thirteen of these strains also hybridized with the tetM gene probes, and were tetracycline resistant The ermCd gene could be transferred, by conjugation, while the tetM gene was not transferable.
Introduction
Non-diphtheria bacteria of the genus Corynebacterium have been increasingly recognized as important opportunistic pathogens (Coyle & Lipsky, 1990). Most Corynebacterium jeikeium isolates (CDC group JK) are resistant to the majority of clinically useful antibiotics such as clindamycin, erythromycin, gentamicin, penicillins, and tetracycline (Larson et al., 1986). Similarly, most of the CDC Corynebacterium group D2 are highly resistant to a number of antibiotics (Soriano et al., 1987). However, the mechanisms of resistance to these antibiotics have not been characterized. The first description of an erythromycin and lincomycin resistant strain of Corynebacterium diphlheriae was in 1973 (Jellard & Lipinski, 1973). This was followed by a report from Seattle of an erythromycin (EmO and clindamycin resistant (CY) strain of C. diphtheriae and two resistant skin coryneforms isolated from a single outbreak in 1978 (Coyle et al., 1979). All six of the C. diphtheriae strains carried a single plasmid of 9-6 MDa (Schiller, Groman & Coyle, 1980). A 1606 bp £coRI fragment, containing the ermCd gene, was identified and shown to encode an inducible rRNA methylase. This gene shares 18-30% amino acid identity with other Gram-positive erm resistance genes (Serwold-Davies & Groman, 1988). We have characterized antibiotic resistant isolates of Corynebacterium striatum collected from patients between 1987 and 1990 and shown that they also carry the ermCd gene. In addition, most isolates were resistant to tetracycline and carried the tetM tetracycline resistance gene. •Corresponding author. 0305-7453/92/100463 + 12 J08.00/0
463 © 1992 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at Mount Royal University on June 10, 2015
Departments of'Pathobiology, bLaboratory Medicine, and'Microbiology, University of Washington, Seattle, Washington 98195, USA
464
M. C. Roberts et aL Methods
Bacterial strains
Susceptibility testing Antimicrobial susceptibility testing was performed on Mueller-Hinton agar (Difco, Detroit, MI, USA) supplemented with 5% defibrinated sheep blood. Standard disc and agar incorporation MIC methods were used and interpreted according to the guidelines of the National Committee for Clinical Laboratory Standards (1990o,£). Culture plates were examined at 24 and 48 h. Induction experiments Inducibility of erythromycin resistance was demonstrated when the inocula were pre-grown in the presence of clindamycin or erythromycin (0K)5 mg/L) for 24 h before testing by the disc diffusion method. The majority of the isolates were also tested after 4 h of pre-growth. No difference was seen between pre-growth at 4 h as compared with 24 h. Media The strains of C. diphtheriae, C. striatum, C. xerosis and Enterococcus faecalis were grown on supplemented GC-medium-base (Difco) at 36-5°C in 5% CO2 (Roberts & Lansciardi, 1990). For mating experiments, the media were supplemented with 8 mg/L of tetracycline and 12-5 mg/L fusidic acid, or 25 mg/L of erythromycin and 12-5 mg/L of fusidic acid, for the selection of transconjugants. Donors and recipients were selected on media supplemented with, 8 mg/L of tetracycline, or either 12-5 mg/L of fusidic acid or 10 mg/L of rifampicin, respectively. Plasmid isolation Plasmid DNA extractions from the C. striatum strains were done as previously described for coryneforms using the C. diphtheriae strain containing plasmid pNG2 as
Downloaded from http://jac.oxfordjournals.org/ at Mount Royal University on June 10, 2015
Antibiotic-resistant C. striatum isolates were cultured from 14 patients in Harborview Medical Center (HMQ between 1987 and 1990. Ten of the 14 resistant isolates were from sputum and produced a characteristic brown pigment. A similar brown pigmented C. striatum isolate (R198), obtained from a blood culture at the Seattle Veterans Affairs Medical Center (SVAM), was included in the study. Susceptible strains, R160 and R161, isolated from two HMC patients during this period were included as controls (Table I). The clinical isolates were identified according to the criteria of Hollis & Weaver (1981). A representative isolate was confirmed as C. striatum by the Division of Bacterial Diseases at the Centers for Disease Control. C. striatum type strain ATCC 6940, and the related Corynebacterium xerosis strains ATCC 373, ATCC 7711, ATCC 9016, and NCTC 9755 were included in the study as references. The clindamycin-erythromycin resistant C. diphtheriae S601, which carries the original pNG2 plasmid, was also included in the study (Coyle et al., 1979) (Table I).
25 21 17 27 6
C. xerosis ATCC 373 ATCC 7711 ATCC 9016 ATCC 9755
C. diphtheria* S601 6
44 19 40 38
24 12 11 35 35 44
19-23 21 20-24 20-24 18
37
32 27 36 36
6-13 34 32 30 34 38
22-29 33 (28-29)* 27-29 27-29 16
>128
0-25 0-25 1 0-12
>128 >128 > 128 1 ND 1
>128 >128 ND' ND >128
a
>128
). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically. 2nd edn; Approved Standard M7-A2. NCCLS, VUlanova, PA. Roberts, M. C. (1989). Plasmid-mediated Tet M in Haemophilia ducreyi. Antimicrobial Agents and Chemotherapy 33, 1611-3. Roberts, M. C. & Hillier, S. L. (1990). Genetic basis of tetracycline resistance in urogenital bacteria. Antimicrobial Agents and Chemotherapy 34, 261-4. Roberts, M. C. & Lansciardi, J. (1990). Transferable Tet M in Fusobacterium nucleatum. Antimicrobial Agents and Chemotherapy 34, 1836-8. Sanchez-Pescador, R., Brown, J. T., Roberts, M. & Urdea, M. S. (1988). The nucleotide sequence of the tetracycline resistance determinant TetM from Ureaplasma urealyticum. Nucleic Acids Research 16, 1216-7. Schiller, J., Groman, N. & Coyle, M. (1980). Plasmids in Corynebacterium diphtheriae and diphtheroids mediating erythromycin resistance. Antimicrobial Agents and Chemotherapy 18, 814-21. Schiller, J., Strom, M., Groman, N. & Coyle, M. (1983). Relationship between pNG2, an Em' plasmid in Corynebacterium diphtheriae, and plasmids in aerobic skin coryneforms. Antimicrobial Agents and Chemotherapy 24, 892-901. Serwold-Davies, T. M. & Groman, N. B. (1986). Mapping and cloning of Corynebacterium diphtheriae plasmid pNG2 and characterization of its relatedness to plasmids from skin coryneforms. Antimicrobial Agents and Chemotherapy 30, 69-72. Serwold-Davies, T. M. & Groman, N. B. (1988). Identification of a methylase gene for erythromycin resistance within the sequence of a spontaneously deleting fragment of Corynebacterium diphtheriae plasmid pNG2. FEMS Microbiology Letters 56, 7-14. Soriano, F., Ponte, C , Santamaria, M., Torres, A. & Fernandez-Roblas, R. (1987). Susceptibility of urinary isolates of Corynebacterium group D2 to fifteen antimicrobials and acetohydroxamic acid. Journal of Antimicrobial Chemotherapy 20, 349-55. Taylor, D. E., Hiratsuka, K., Ray, H. & Manavathu, E. K. (1987). Characterization and expression of a cloned tetracycline resistance determinant from Campylobacter jejuni plasmid pUA466. Journal of Bacteriology 169, 2984-9. Zilhao, R., Papadopoulou, B. & Courvalin, P. (1988). Occurrence of the Campylobacter resistance gene tetO in Enterococcus and Streptococcus spp. Antimicrobial Agents and Chemotherapy 32, 1793-6.
