J Vet Diagn Invest 4:170-174 (1992)
Antimicrobial resistance patterns and plasmid profiles of Streptococcus suis isolates Mario Cantin, Josée Harel, Robert Higgins, Marcelo Gottschalk Abstract. Streptococcus suis isolates recovered from diseased animals in Quebec and western Canada and from human cases in Europe were tested for their susceptibility to different antimicrobial agents and screened for their plasmid content. Most isolates from Quebec were clindamycin, erythromycin, and tetracycline resistant; animal isolates from western Canada were notably less resistant to clindamycin and erythromycin, whereas human isolates were considerably more susceptible to most antimicrobials tested. More than 60% of isolates had plasmids that ranged from 1.5 to 35 kilobases (kb). Of the 7 plasmid profiles found, 2 were particularly frequent in isolates from Quebec and western Canada, suggesting the presence of epidemic strains in the swine population. A particular plasmid band of about 5 kb was present in most Canadian isolates. When this band was used as a probe in colony and Southern blot hybridization, most isolates harboring the 5-kb plasmid hybridized, even though their plasmid profiles were different. Human isolates from Europe differed in their plasmid content from Canadian isolates of animal origin. Although a high degree of antimicrobial in most isolates, it was not possible to establish a resistance was associated with the presence of causative relationship.
Streptococcus suis infections in swine have been diagnosed in all countries where the swine industry is developed. This microorganism was originally implicated in cases of meningitis and septicemia, but it is now recognized to be responsible for a variety of other infections in pigs. 11 It has also been described as a 12 6 1 pathogen for ruminants, horses and humans. O f 7,19 the 23 capsular types characterized, type 2 is the type most frequently isolated from diseased pigs in North America, followed by types 3, 1/2, and 8. 11 Among recently described capsular types 9-22, type 22 also appears to be an important pathogen for pigs.8 Streptococcus suis infections in swine are usually treated with penicillin. However, penicillin-resistant beta-lactamase-negative isolates have recently been detected. 9 Resistance to other antimicrobial agents normally effective against gram-positive organisms, such as erythromycin, streptomycin, and tetracycline, has also been reported.20,25 are associated with the ability to confer antimicrobial resistance among bacteria. The majority of the genes mediating resistance to antibiotics in Streptococcus are located on or on transposons and are readily disseminated.5,13,17,18 Plasmid analysis also allows the differentiation of bacterial isolates belonging to the same serotype. Thus, are an important epidemiological tool for determining the orFrom the Groupe de Recherche sur les Maladies Infectieuses du Port, Faculté de médecine vétérinaire, Université de Montreal, C.P. 5000, Saint-Hyacinthe, Québec J2S 7C6, Canada. Received for publication August 28, 1991.
igin of a disease outbreak or for investigating the dissemination of an infectious agent in a population.24 Data concerning the prevalence of resistant S. suis strains and information about the presence of in this species are rare.22 The objective of the present study was to compare S. suis isolates belonging to different capsular types, recovered from diseased animals in Quebec and western Canada and from human cases in Europe, on the basis of their susceptibility to different antimicrobial agents and their plasmid content.
Materials and methods Bacterial isolates. A total of 92 S. suis isolates were examined. Sixty capsular type 2 isolates from animals originated from Quebec (45 isolates) and from western Canada (15 isolates, supplied by S. A. Rawluk, Alberta). All of these isolates were from pigs with different types of infections, except 1 isolate from a case of bovine abortion.12 Twelve isolates from human cases of meningitis were obtained from J. P. Arends, The Netherlands. Four groups of 5 isolates belonging to capsular types l/2, 3, 8, and 22, respectively, and from diseased pigs in Quebec were also included in the study. All of these isolates were biochemically identified and serotyped in our laboratory using previously described procedures 10 and were stored as lyophilized cultures. Antimicrobial susceptibility testing. Antimicrobial susceptibility patterns for S. suis isolates were determined by the Kirby-Bauer disk diffusion method.2 Antimicrobial agents tested were clindamycin, ampicillin, cephalothin, chloramphenicol, erythromycin, gentamicin, neomycin, oxacillin, penicillin, streptomycin, trimethoprim-sulfamethoxazole, sulfonamides, and tetracycline. Readings were performed according to the NCCLS guidelines (approved standard M2A4, 1990). The presence of beta-lactamase was tested in pen-
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Antimicrobial resistance and plasmids of S. suis Table 1. Percent resistance of 92 Streptococcus suis isolates against various antimicrobial agents.
icillin-resistant isolates using Nitrocefin,a a chromogenic cephalosporin. Plasmid preparation. The plasmid preparation procedure was carried out in triplicate. Plasmid DNA was extracted by using a rapid alkaline lysis procedure3 with some modifications. Bacteria were grown in Todd-Hewitt broth in a shaker at 37 C at midlog phase, as determined by growth curves. Cells were harvested, washed, and resuspended in a solution containing 50 mM glucose, 25 mM Tris-HC1 (pH 8.0) 10 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0), and lysozyme (10 mg/ml)b After incubation at room temperature with frequent mixing, a solution of 0.2 N NaOH and 1% sodium dodecyl sulfate (SDS) was added to the mixture and incubated for 5 min on ice. An ice-cold solution of potassium acetate (pH 4.8) was added to the lysate. Following 3 phenol and chloroform extractions, the DNA was precipitated with 100% ethanol. Gel electrophoresis. Plasmid DNA was separated by electrophoresis for 2 hr in a 0.8% agarose gel containing 0.5 µg/ ml of ethidium bromide, in Tris-acetate buffer (0.04 M Trisacetate, 0.001 M EDTA), at 50 V. Plasmids were photographed with UV-light illumination (Polaroid #667). The Escherichia coli plasmids V517 (54, 7, 5.6, 5.1, 3.9, 3.0, 2.7 and 2.1 kilobases [kb]14 and pSAS 1393 (94, 54.5, 35, and 6.3 kb) were used as molecular weight markers. Preparation of the DNA probe. The S. suis probe consisted of a 5.1-kb band from a capsular type 2 isolate (#89-4303) and was purified from a low melting point agarose gel. 15 The probe was labeled with alpha 32P-dCTP using the random primer kit.c Hybridization. For colony hybridization, isolates of S. suis and positive (S. suis #89-4303) and negative (E. coli HB101) controls were spotted onto blood agar and incubated at 37 C. Colonies were replicated by placing filter paperd on the surface of the blood agar for 8 hr and were processed for hybridization as described previously.4 The filters were hybridized with S. suis 5.1-kb probe. For gel hybridization, the DNA preparations were depurinated by washing in 0.25 N HC1 for 15 min, denatured with 0.2 M NaOH-1.5 M NaCl for 45 min, and neutralized with 1 M Tris-HC1 (pH 8.0)
Table 2.
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Distribution of plasmids among 92 Streptococcus suis
isolates.
containing 1.5 M NaCl for 45 min. The fragments were transferred overnight to a nylon membrane by the Southern method 23 in 10 x standard saline citrate (SSC) solution (1 x SSC is 0.15 M NaCl containing 0.015 M sodium citrate, pH 7.0). The nylon membrane was then prehybridized for 4 hr at 65 C in the hybridization solution, which consisted of 5 x SSPE (pH 7.4) (1 x SSPE is 180 mM NaCl, 10 mM NaH2PO 4, and 20 mM EDTA), 5 x Denhardt solution (1 x Denhardt solution is 0.02% Ficoll [molecular weight 400 000c], 0.02% polyvinylpyrolidone [molecular weight 360 000], and 0.02% bovine serum albumin), and 100 µ1/ml of heat-denatured salmon sperm DNA.15 The membrane was hybridized overnight at 65 C in a fresh hybridization solution with the denatured probe. Finally, the membrane was washed twice for 5 min with 2 x SSC at room temperature, twice for 30 min at 65 C with 2 x SSC containing 0.5% SDS, and twice for 5 min with 0.1 x SSC. The filter was air dried, and autoradiographs were made by exposing the filter to X-Ray-Filme for few hours at -70 C. The films were developed according to the manufacturer’s recommendations.
Results
Antimicrobial resistance. The antimicrobial resistance patterns of S. suis isolates belonging to different capsular types are shown in Table 1. All isolates were susceptible to ampicillin, cephalothin, chloramphenicol, and trimethoprim-sulfamethoxazole. Capsular type 2 isolates from Quebec were consistently more resistant to antimicrobial agents than were those from westem Canada and those from Europe (human cases). Nonetheless, the human isolates were more resistant to neomycin, sulfonamides, and, to a lesser extent, gentamicin than were capsular type 2 isolates of animal origin. One capsular type 2 isolate and 1 capsular type 22 isolate were resistant to penicillin but were betalactamase negative. Capsular type 22 was more resistant to oxacillin than were other capsular types. There was no correlation between the type of specimen or
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Figure 1. Plasmid patterns of Streptococcus suis. A. Electrophoresis in 0.8% agarose gel of plasmid preparations. Lanes A-F, capsular type 2 isolates from Quebec; lane G capsular type 2 isolate from western Canada; lane M, supercoiled DNA standard from E. coli V157. B. Southern blot hybridization of plasmid preparations used in panel A hybridized with the 5.1-kb probe. Lanes A-G correspond to those shown in panel A. Arrow shows the 5.1-kb related plasmids.
their tissue of origin and the resistance patterns to antimicrobial agents. Plasmid content and plasmid profiles. More than 60% of isolates had plasmids that ranged from 1.5 to 35 kb. All but 3 capsular type 2 isolates from Quebec (93%), 5 western Canada isolates (33%), and only 2 European isolates of human origin (17%) harbored plasmids (Table 2). Isolates belonging to other capsular types also contained plasmids. A particular plasmid band of about 5 kb was present in most capsular type 2 isolates from Quebec and from western Canada and in Quebec isolates belonging to capsular types 1/2, 3, and 8 (Table 2). Seven different plasmid profiles were found. Two profiles, 1 with 9-kb and 5-kb bands and another with 9-kb, 7-kb, 5-kb, and 3-kb bands, were the most prevalent, especially among capsular type 2 isolates (Fig. 1). The 2 European capsular type 2 isolates of human origin that harbored plasmids showed profiles different from those of animal origin. The bovine capsular type 2 isolate had a profile with 9-kb and 5-kb bands, similar to that seen in most porcine isolates. Isolates belonging to other capsular types showed various profiles, but most of them contained the 5-kb band. Most isolates that were resistant to ≥ 1 antimicrobial
lates contained a 5-kb plasmid in their plasmid profile, a specific plasmid band of 5.1 kb from isolate #894303 was used as a probe in colony and Southern blot hybridization (Table 2, Fig. 1). All but 1 capsular type 2 isolates from Quebec, which harbored 5-kb plasmids, hybridized with the 5.1-kb probe. Most isolates belonging to capsular types 1/2, 3, and 8 also hybridized by colony hybridization with the 5.1-kb probe, even though their plasmid profiles were different from predominant profiles. When each of the positive isolates were tested using the Southern hybridization technique, the 5.1-kb probe hybridized with bands of about 5 and 9 kb; the 9-kb band was probably the open circular form of the 5.1-kb plasmid. Table 3. Plasmid DNA content of Streptococcus suis isolates in relation to their resistance to different antimicrobial agents.
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Discussion The amount of information about S. suis antimicrobial susceptibility is limited.9,20,25 Most porcine S. suis isolates from Quebec were clindamycin, erythromycin, and tetracycline resistant, which is in accordance with data from Ontario.20 Isolates from western Canada were less resistant to clindamycin and erythromycin than were those from Quebec. Differences in the antimicrobial susceptibility patterns could be explained by a more intensive use of antimicrobial therapy in industrial swine operations during the recent years. Human isolates were considerably more susceptible to most antimicrobials, with the exception of neomycin, gentamicin, and sulfonamides. Therapeutic use of penicillin has been reported in human patients infected with S. suis capsular type 2.1 In some cases the elimination of the infection could not be achieved with usual dosages of this antibiotic.26 In our study, 2 S. suis isolates were penicillin resistant, 1 belonging to capsular type 2 and the other to capsular type 22. Streptococcus suis strains belonging to other capsular types and that are resistant to penicillin and are beta-lactamase negative have previously been reported.9 As has been previously suggested,9 antimicrobial susceptibility testing should be carried out on S. suis isolates before the establishment of a treatment. Two plasmid patterns predominated among capsular type 2 isolates of animal origin, suggesting the presence of “epidemic” strains in the swine population that could eventually be transmitted to other species. The 12 European human isolates differed from Canadian capsular type 2 isolates of animal origin in their plasmid content. In this instance, plasmid profile determination offers precision beyond serotyping. The presence of the 5.1-kb plasmid in the different capsular types among isolates of S. suis from widely separated geographic regions, such as Quebec and western Canada, suggests that molecular diagnoses can be made by using this 5.1-kb as an epidemiologic marker. In addition to serotyping, antimicrobial susceptibil16 ity determination, and DNA fingerprinting, fingerprinting appears to be a useful tool for determining the presence of an epidemic strain of S. suis and for tracing the movement of that strain within or among herds. The procedure is easy to perform and can distinguish epidemic from sporadic isolates.24 Plasmids have been used as strain markers to investigate community-based outbreaks in infectious diseases.24 Streptococci have a variety of means for exchanging genetic information.5 The presence of plasmids in S. suis suggests a potential for the acquisition or the transfer of existing or additional genetic material; some material could be cryptic or could encode for additional drug resistance. In the present study, although a high
degree of antimicrobial resistance in association with the presence of plasmids was observed in most isolates, it was not possible to establish a causative relationship. Investigations on the transferability of resistance to different antimicrobials would indicate if the resistance is correlated with the presence of in S. suis. Moreover, preliminary experiments using the streptococcal erythromycin-resistant transposon Tn91721 as a probe in a hybridization procedure revealed that some erythromycin-resistant S. suis isolates carried the Tn917 homologous sequence (data not shown). However, the 5.1 -kb did not hybridize with Tn917. In swine populations fed with subtherapeutic levels of antimicrobial agents, the opportunity for plasmid-associated antibiotic resistance development in S. suis could be enhanced. Acknowledgements This study was supported in part by a grant from Fonds de la Recherche en Santé du Québec (2243) and by the Minister-e de 1’Enseignement Supérieur et de la Science du Québec. We thank D. B. Clewell (University of Michigan) for his gift of strain pAM225 and A. Sasarman (University of Montreal) for his gift of strain pSAS 1393.
Sources and manufacturers a. Cefinase disk, BBL Microbiology system, Cockeysville, MD. b. Sigma Chemical Co., St. Louis, MO. c. Pharmacia LKB Biotechnology, Baie d’Urfe, Quebec, Canada. d. Whatman, Clifton, NJ. e. Eastman Kodak Co., Rochester, NY.
References 1. Arends JP, Zanen HC: 1988, Meningitis caused by Streptococcus suis in humans. Rev Infect Dis 10: 131-136. 2. Bauer AW, Kirby WMM, Sherris JC: 1966, Antibiotic susceptibility testing by standardized single disk method. Am J Clin Pathol 45:493-496. 3. Bimboim HC, Doly J: 1979, A rapid alkaline extraction proDNA. Nucleic Acids cedure for screening recombinant Res 7:1513-1523. 4. Broes A, Fairbrother JM, Mainil J, et al.: 1988, Phenotypic and genotypic characterization of enterotoxigenic Escherichia coli serotype 08:K.X 105 and 08:K“2829” strains isolated from piglets with diarrhea. J Clin Microbiol 26:2402-2409. 5. Clewell DB: 1981, Plasmid, drug resistance, and gene transfer in genus Streptococcus. Microbiol Rev 45:409-436. 6. Devriese LA, Sustronk B, Mahenhout J, Haesebrouck F: 1990, Streptococcus suis meningitis in a horse. Vet Rec 127:168. 7. Gottschalk M, Higgins R, Jacques M, et al.: 1989, Description of 14 new capsular types of Streptococcus suis. J Clin Microbiol 27:2633-2636. 8. Gottschalk M, Higgins R, Jacques M, et al.: 1991, Isolation and characterization of Streptococcus suis capsular types 9-22. J Vet Diagn Invest 3:60-65. 9 . Gottschalk M, Turgeon P, Higgins R, et al.: 1991, Susceptibility of Streptococcus suis to penicillin. J Vet Diagn Invest 3: 170172. 10. Higgins R, Gottschalk M: 1990, An update on Streptococcus suis identification. J Vet Diagn Invest 2:249-252.
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11. Higgins R, Gottschalk M, Beaudoin M: 1990, Streptococcus suis infections in swine. A 16-month study. Can J Vet Res 54: 170-173. 12. Higgins R, Gottschalk M, Fecteau G, et al.: 1990, Isolation of Streptococcus suis from cattle. Can Vet J 31:529. 13. Leblanc DJ, Lee N, Banai M, et al.: 1987, Dissemination of streptococcal antibiotic resistance determinants in the natural environment. In: Streptococcal genetics, ed. Ferretti JF, Curtiss R III, pp. 43-53. American Society for Microbiology, Washington, DC. 14. Macrina FL, Kopecko DJ, Jones RR, et al.: 1978, A multiple plasmid containing Escherichia coli strain: convenient source of size reference plasmid molecules. Plasmid 1:417-420. 15. Maniatis T, Fritsch EF, Sambrook J: 1982, Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 16. Mogollon JD, Pijoan C, Murtaugh MP, et al.: 1991, Identification of epidemic strains of Streptococcus suis by genomic fingerprinting. J Clin Microbiol 29:782-787. 17. Murphy E: 1989, Transposable elements in gram-positive bacteria. In: Mobile DNA, ed. Berg DJ, Howe MM, pp. 269-288. American Society for Microbiology, Washington, DC. 18. Pepper K, de Cespedes G, Horaud T: 1988, Heterogeneity of
chromosomal gene encoding chloramphenicol resistance in streptococi. Plasmid 19:71-74. 19. Perch B, Pedersen KB, Henrichsen J: 1983, Serology of capsulated streptococci pathogenic for pigs: six new serotypes of Streptococcus suis. J Clin Microbiol 17:993-996. 20. Sanford SE, Tilker AME: 1989, Streptococcus suis antimicrobial susceptibility. Can Vet J 30:673-680. 21. Shaw JH, Clewell DB: 1985, Complete nucleotide sequence of macrolide-lincosamide-streptogramin P-resistance transposon Tn917 in Streptococcus faecalis. J Bacterial 164:782-796. 22. Shryloch TR, Mortensen JE, Himes SL: 1990, Characterization of Streptococcus suis isolates. Proc Annu Meet Am Soc Microbiol, p. D-18. 23. Southern EM: 1975, Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Bio1 98: 503-517. 24. Tenover FC: 1985, Plasmid fingerprinting: a tool for bacterial strain identification and surveillance of nosocomial and community-acquired infections. Clin Lab Med 5:413-436. 25. Touil F, Higgins R, Nadeau M: 1988, Isolation of Streptococcus suis from pigs in Canada. Vet Microbiol 17: 171-177. 26. Woo J, Li EK: 1987, Streptococcus suis meningitis requires prolonged treatment with penicillin. Infection 15: 129-130.
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Antimicrobial resistance patterns and plasmid profiles of Streptococcus suis isolates Mario Cantin, Josée Harel, Robert Higgins, Marcelo Gottschalk Abstract. Streptococcus suis isolates recovered from diseased animals in Quebec and western Canada and from human cases in Europe were tested for their susceptibility to different antimicrobial agents and screened for their plasmid content. Most isolates from Quebec were clindamycin, erythromycin, and tetracycline resistant; animal isolates from western Canada were notably less resistant to clindamycin and erythromycin, whereas human isolates were considerably more susceptible to most antimicrobials tested. More than 60% of isolates had plasmids that ranged from 1.5 to 35 kilobases (kb). Of the 7 plasmid profiles found, 2 were particularly frequent in isolates from Quebec and western Canada, suggesting the presence of epidemic strains in the swine population. A particular plasmid band of about 5 kb was present in most Canadian isolates. When this band was used as a probe in colony and Southern blot hybridization, most isolates harboring the 5-kb plasmid hybridized, even though their plasmid profiles were different. Human isolates from Europe differed in their plasmid content from Canadian isolates of animal origin. Although a high degree of antimicrobial in most isolates, it was not possible to establish a resistance was associated with the presence of causative relationship.
Streptococcus suis infections in swine have been diagnosed in all countries where the swine industry is developed. This microorganism was originally implicated in cases of meningitis and septicemia, but it is now recognized to be responsible for a variety of other infections in pigs. 11 It has also been described as a 12 6 1 pathogen for ruminants, horses and humans. O f 7,19 the 23 capsular types characterized, type 2 is the type most frequently isolated from diseased pigs in North America, followed by types 3, 1/2, and 8. 11 Among recently described capsular types 9-22, type 22 also appears to be an important pathogen for pigs.8 Streptococcus suis infections in swine are usually treated with penicillin. However, penicillin-resistant beta-lactamase-negative isolates have recently been detected. 9 Resistance to other antimicrobial agents normally effective against gram-positive organisms, such as erythromycin, streptomycin, and tetracycline, has also been reported.20,25 are associated with the ability to confer antimicrobial resistance among bacteria. The majority of the genes mediating resistance to antibiotics in Streptococcus are located on or on transposons and are readily disseminated.5,13,17,18 Plasmid analysis also allows the differentiation of bacterial isolates belonging to the same serotype. Thus, are an important epidemiological tool for determining the orFrom the Groupe de Recherche sur les Maladies Infectieuses du Port, Faculté de médecine vétérinaire, Université de Montreal, C.P. 5000, Saint-Hyacinthe, Québec J2S 7C6, Canada. Received for publication August 28, 1991.
igin of a disease outbreak or for investigating the dissemination of an infectious agent in a population.24 Data concerning the prevalence of resistant S. suis strains and information about the presence of in this species are rare.22 The objective of the present study was to compare S. suis isolates belonging to different capsular types, recovered from diseased animals in Quebec and western Canada and from human cases in Europe, on the basis of their susceptibility to different antimicrobial agents and their plasmid content.
Materials and methods Bacterial isolates. A total of 92 S. suis isolates were examined. Sixty capsular type 2 isolates from animals originated from Quebec (45 isolates) and from western Canada (15 isolates, supplied by S. A. Rawluk, Alberta). All of these isolates were from pigs with different types of infections, except 1 isolate from a case of bovine abortion.12 Twelve isolates from human cases of meningitis were obtained from J. P. Arends, The Netherlands. Four groups of 5 isolates belonging to capsular types l/2, 3, 8, and 22, respectively, and from diseased pigs in Quebec were also included in the study. All of these isolates were biochemically identified and serotyped in our laboratory using previously described procedures 10 and were stored as lyophilized cultures. Antimicrobial susceptibility testing. Antimicrobial susceptibility patterns for S. suis isolates were determined by the Kirby-Bauer disk diffusion method.2 Antimicrobial agents tested were clindamycin, ampicillin, cephalothin, chloramphenicol, erythromycin, gentamicin, neomycin, oxacillin, penicillin, streptomycin, trimethoprim-sulfamethoxazole, sulfonamides, and tetracycline. Readings were performed according to the NCCLS guidelines (approved standard M2A4, 1990). The presence of beta-lactamase was tested in pen-
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Antimicrobial resistance and plasmids of S. suis Table 1. Percent resistance of 92 Streptococcus suis isolates against various antimicrobial agents.
icillin-resistant isolates using Nitrocefin,a a chromogenic cephalosporin. Plasmid preparation. The plasmid preparation procedure was carried out in triplicate. Plasmid DNA was extracted by using a rapid alkaline lysis procedure3 with some modifications. Bacteria were grown in Todd-Hewitt broth in a shaker at 37 C at midlog phase, as determined by growth curves. Cells were harvested, washed, and resuspended in a solution containing 50 mM glucose, 25 mM Tris-HC1 (pH 8.0) 10 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0), and lysozyme (10 mg/ml)b After incubation at room temperature with frequent mixing, a solution of 0.2 N NaOH and 1% sodium dodecyl sulfate (SDS) was added to the mixture and incubated for 5 min on ice. An ice-cold solution of potassium acetate (pH 4.8) was added to the lysate. Following 3 phenol and chloroform extractions, the DNA was precipitated with 100% ethanol. Gel electrophoresis. Plasmid DNA was separated by electrophoresis for 2 hr in a 0.8% agarose gel containing 0.5 µg/ ml of ethidium bromide, in Tris-acetate buffer (0.04 M Trisacetate, 0.001 M EDTA), at 50 V. Plasmids were photographed with UV-light illumination (Polaroid #667). The Escherichia coli plasmids V517 (54, 7, 5.6, 5.1, 3.9, 3.0, 2.7 and 2.1 kilobases [kb]14 and pSAS 1393 (94, 54.5, 35, and 6.3 kb) were used as molecular weight markers. Preparation of the DNA probe. The S. suis probe consisted of a 5.1-kb band from a capsular type 2 isolate (#89-4303) and was purified from a low melting point agarose gel. 15 The probe was labeled with alpha 32P-dCTP using the random primer kit.c Hybridization. For colony hybridization, isolates of S. suis and positive (S. suis #89-4303) and negative (E. coli HB101) controls were spotted onto blood agar and incubated at 37 C. Colonies were replicated by placing filter paperd on the surface of the blood agar for 8 hr and were processed for hybridization as described previously.4 The filters were hybridized with S. suis 5.1-kb probe. For gel hybridization, the DNA preparations were depurinated by washing in 0.25 N HC1 for 15 min, denatured with 0.2 M NaOH-1.5 M NaCl for 45 min, and neutralized with 1 M Tris-HC1 (pH 8.0)
Table 2.
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Distribution of plasmids among 92 Streptococcus suis
isolates.
containing 1.5 M NaCl for 45 min. The fragments were transferred overnight to a nylon membrane by the Southern method 23 in 10 x standard saline citrate (SSC) solution (1 x SSC is 0.15 M NaCl containing 0.015 M sodium citrate, pH 7.0). The nylon membrane was then prehybridized for 4 hr at 65 C in the hybridization solution, which consisted of 5 x SSPE (pH 7.4) (1 x SSPE is 180 mM NaCl, 10 mM NaH2PO 4, and 20 mM EDTA), 5 x Denhardt solution (1 x Denhardt solution is 0.02% Ficoll [molecular weight 400 000c], 0.02% polyvinylpyrolidone [molecular weight 360 000], and 0.02% bovine serum albumin), and 100 µ1/ml of heat-denatured salmon sperm DNA.15 The membrane was hybridized overnight at 65 C in a fresh hybridization solution with the denatured probe. Finally, the membrane was washed twice for 5 min with 2 x SSC at room temperature, twice for 30 min at 65 C with 2 x SSC containing 0.5% SDS, and twice for 5 min with 0.1 x SSC. The filter was air dried, and autoradiographs were made by exposing the filter to X-Ray-Filme for few hours at -70 C. The films were developed according to the manufacturer’s recommendations.
Results
Antimicrobial resistance. The antimicrobial resistance patterns of S. suis isolates belonging to different capsular types are shown in Table 1. All isolates were susceptible to ampicillin, cephalothin, chloramphenicol, and trimethoprim-sulfamethoxazole. Capsular type 2 isolates from Quebec were consistently more resistant to antimicrobial agents than were those from westem Canada and those from Europe (human cases). Nonetheless, the human isolates were more resistant to neomycin, sulfonamides, and, to a lesser extent, gentamicin than were capsular type 2 isolates of animal origin. One capsular type 2 isolate and 1 capsular type 22 isolate were resistant to penicillin but were betalactamase negative. Capsular type 22 was more resistant to oxacillin than were other capsular types. There was no correlation between the type of specimen or
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Figure 1. Plasmid patterns of Streptococcus suis. A. Electrophoresis in 0.8% agarose gel of plasmid preparations. Lanes A-F, capsular type 2 isolates from Quebec; lane G capsular type 2 isolate from western Canada; lane M, supercoiled DNA standard from E. coli V157. B. Southern blot hybridization of plasmid preparations used in panel A hybridized with the 5.1-kb probe. Lanes A-G correspond to those shown in panel A. Arrow shows the 5.1-kb related plasmids.
their tissue of origin and the resistance patterns to antimicrobial agents. Plasmid content and plasmid profiles. More than 60% of isolates had plasmids that ranged from 1.5 to 35 kb. All but 3 capsular type 2 isolates from Quebec (93%), 5 western Canada isolates (33%), and only 2 European isolates of human origin (17%) harbored plasmids (Table 2). Isolates belonging to other capsular types also contained plasmids. A particular plasmid band of about 5 kb was present in most capsular type 2 isolates from Quebec and from western Canada and in Quebec isolates belonging to capsular types 1/2, 3, and 8 (Table 2). Seven different plasmid profiles were found. Two profiles, 1 with 9-kb and 5-kb bands and another with 9-kb, 7-kb, 5-kb, and 3-kb bands, were the most prevalent, especially among capsular type 2 isolates (Fig. 1). The 2 European capsular type 2 isolates of human origin that harbored plasmids showed profiles different from those of animal origin. The bovine capsular type 2 isolate had a profile with 9-kb and 5-kb bands, similar to that seen in most porcine isolates. Isolates belonging to other capsular types showed various profiles, but most of them contained the 5-kb band. Most isolates that were resistant to ≥ 1 antimicrobial
lates contained a 5-kb plasmid in their plasmid profile, a specific plasmid band of 5.1 kb from isolate #894303 was used as a probe in colony and Southern blot hybridization (Table 2, Fig. 1). All but 1 capsular type 2 isolates from Quebec, which harbored 5-kb plasmids, hybridized with the 5.1-kb probe. Most isolates belonging to capsular types 1/2, 3, and 8 also hybridized by colony hybridization with the 5.1-kb probe, even though their plasmid profiles were different from predominant profiles. When each of the positive isolates were tested using the Southern hybridization technique, the 5.1-kb probe hybridized with bands of about 5 and 9 kb; the 9-kb band was probably the open circular form of the 5.1-kb plasmid. Table 3. Plasmid DNA content of Streptococcus suis isolates in relation to their resistance to different antimicrobial agents.
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Antimicrobial resistance and plasmids of S. suis
Discussion The amount of information about S. suis antimicrobial susceptibility is limited.9,20,25 Most porcine S. suis isolates from Quebec were clindamycin, erythromycin, and tetracycline resistant, which is in accordance with data from Ontario.20 Isolates from western Canada were less resistant to clindamycin and erythromycin than were those from Quebec. Differences in the antimicrobial susceptibility patterns could be explained by a more intensive use of antimicrobial therapy in industrial swine operations during the recent years. Human isolates were considerably more susceptible to most antimicrobials, with the exception of neomycin, gentamicin, and sulfonamides. Therapeutic use of penicillin has been reported in human patients infected with S. suis capsular type 2.1 In some cases the elimination of the infection could not be achieved with usual dosages of this antibiotic.26 In our study, 2 S. suis isolates were penicillin resistant, 1 belonging to capsular type 2 and the other to capsular type 22. Streptococcus suis strains belonging to other capsular types and that are resistant to penicillin and are beta-lactamase negative have previously been reported.9 As has been previously suggested,9 antimicrobial susceptibility testing should be carried out on S. suis isolates before the establishment of a treatment. Two plasmid patterns predominated among capsular type 2 isolates of animal origin, suggesting the presence of “epidemic” strains in the swine population that could eventually be transmitted to other species. The 12 European human isolates differed from Canadian capsular type 2 isolates of animal origin in their plasmid content. In this instance, plasmid profile determination offers precision beyond serotyping. The presence of the 5.1-kb plasmid in the different capsular types among isolates of S. suis from widely separated geographic regions, such as Quebec and western Canada, suggests that molecular diagnoses can be made by using this 5.1-kb as an epidemiologic marker. In addition to serotyping, antimicrobial susceptibil16 ity determination, and DNA fingerprinting, fingerprinting appears to be a useful tool for determining the presence of an epidemic strain of S. suis and for tracing the movement of that strain within or among herds. The procedure is easy to perform and can distinguish epidemic from sporadic isolates.24 Plasmids have been used as strain markers to investigate community-based outbreaks in infectious diseases.24 Streptococci have a variety of means for exchanging genetic information.5 The presence of plasmids in S. suis suggests a potential for the acquisition or the transfer of existing or additional genetic material; some material could be cryptic or could encode for additional drug resistance. In the present study, although a high
degree of antimicrobial resistance in association with the presence of plasmids was observed in most isolates, it was not possible to establish a causative relationship. Investigations on the transferability of resistance to different antimicrobials would indicate if the resistance is correlated with the presence of in S. suis. Moreover, preliminary experiments using the streptococcal erythromycin-resistant transposon Tn91721 as a probe in a hybridization procedure revealed that some erythromycin-resistant S. suis isolates carried the Tn917 homologous sequence (data not shown). However, the 5.1 -kb did not hybridize with Tn917. In swine populations fed with subtherapeutic levels of antimicrobial agents, the opportunity for plasmid-associated antibiotic resistance development in S. suis could be enhanced. Acknowledgements This study was supported in part by a grant from Fonds de la Recherche en Santé du Québec (2243) and by the Minister-e de 1’Enseignement Supérieur et de la Science du Québec. We thank D. B. Clewell (University of Michigan) for his gift of strain pAM225 and A. Sasarman (University of Montreal) for his gift of strain pSAS 1393.
Sources and manufacturers a. Cefinase disk, BBL Microbiology system, Cockeysville, MD. b. Sigma Chemical Co., St. Louis, MO. c. Pharmacia LKB Biotechnology, Baie d’Urfe, Quebec, Canada. d. Whatman, Clifton, NJ. e. Eastman Kodak Co., Rochester, NY.
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