Journal of Antimicrobial Chemotherapy (2002) 50, 201 209 DOI: 10.1093/jac/dkf099 Antimicrobial susceptibility of Streptococcus suis isolated from swine in France and from humans in different countries between 1996 and 2000 J. Marie 1, H. Morvan 2, F. Berthelot-Hérault 1, P. Sanders 3, I. Kempf 1, A. V. Gautier-Bouchardon 1, E. Jouy 1 and M. Kobisch 1 * 1 Agence Française de Sécurité Sanitaire des Aliments, Laboratoire d Etudes et de Recherches Avicoles et Porcines, Unité Mycoplasmologie-Bactériologie, BP 53, F-22440 Ploufragan; 2 Laboratoire de Développement et d Analyses, Zoopôle, 7 rue du Sabot, BP 54, F-22440 Ploufragan; 3 Agence Française de Sécurité Sanitaire des Aliments, Laboratoire d Etudes et de Recherches sur les Médicaments Vétérinaires et les Désinfectants, La Haute Marche, Javené, F-35133 Fougères, France Received 23 August 2001; returned 26 November 2001; revised 18 April 2002; accepted 29 April 2002 The susceptibility of 135 Streptococcus suis strains isolated from pigs (n = 110) and from humans (n = 25) to 13 antimicrobial agents was studied by microdilution and disc diffusion methods using Mueller Hinton Agar II (MH) supplemented with either defibrinated sheep blood (MHSB) or horse serum (MHHS). Results were similar for both methods used except for penicillin G whose zone diameters were reduced with MHSB compared with MHHS. When MH was supplemented with sheep blood, 39% of S. suis strains classified as penicillin susceptible by MHHS microdilution showed intermediate susceptibility. Nearly all strains were susceptible to penicillin G (except by disc diffusion in MHSB), amoxicillin, ceftiofur, florfenicol, gentamicin and bacitracin. The least active antimicrobial agents were doxycycline and macrolides/lincosamides. High-level resistance (MIC > 500 mg/l or zone diameters < 10 mm) to streptomycin and kanamycin was detected in only a few strains. The virulence of strains did not seem to be related to antimicrobial resistance because no statistical difference was reported between the proportion of resistant strains of S. suis isolated from pigs with meningitis, septicaemia and arthritis, and those from tonsils and nasal cavities. However, significant differences were found in the proportions of macrolide- or doxycycline-resistant strains between S. suis serotype 2 and other serotypes. The results of antibiotic susceptibility testing presented in this study indicate that β-lactams can be used in empirical treatment of human and pig S. suis infections in France. Introduction Streptococcus suis is an important swine pathogen causing meningitis, arthritis, septicaemia and endocarditis, but these bacteria can also be isolated from nasal cavities or tonsils of healthy pigs. 1,2 Moreover, S. suis is a zoonotic agent and may cause meningitis in humans. 3 S. suis is divided into 35 serotypes, according to its polysaccharide capsular antigens. Virulence differs within the serotypes, serotype 2 being the serotype most often associated with disease. 1 Autogenous vaccines are used to protect swine against S. suis infections but since protection is incomplete, different types of vaccine are under investigation. 2 Among antimicrobial agents for treatment and prevention, the most active are the β-lactams, aminoglycosides, phenicols and fluoroquinolones. 4 Previous studies have investigated the antimicrobial susceptibility of clinical isolates but few have reported the susceptibility of S. suis strains isolated from healthy pigs. 5 Most reports of resistance concentrate on streptococci, notably Streptococcus pneumoniae. 6 9 Shneerson et al. 10 described a case of human meningitis with a penicillin-resistant S. suis. β-lactam-resistant S. suis strains have also been isolated from swine in Spain. 11 According to Gottschalk et al., 12 resistance has not been associated with the production of β-lactamase. More recently, resistance of S. suis to different antibiotics has been described. 1,2... *Corresponding author. Tel: +33-2-96-01-62-80; Fax: +33-2-96-01-62-73; E-mail: m.kobisch@ploufragan.afssa.fr... 201 2002 The British Society for Antimicrobial Chemotherapy
J. Marie et al. We have undertaken a study to determine the antimicrobial susceptibility of 110 S. suis strains isolated in France from 1996 to 2000, from diseased as well as from healthy pigs, testing 13 antimicrobial agents by agar diffusion and microdilution methods. Twenty-five strains isolated from humans were also analysed. Materials and methods Antimicrobial susceptibility testing was carried out on all strains by disc diffusion and microdilution tests according to recommendations reported by the Comité de l Antibiogramme de la Société Française de Microbiologie (CA- SFM). 13,14 Bacterial strains Eighty-three S. suis strains collected from diseased pigs presenting with septicaemia, meningitis or arthritis, and 27 strains isolated from tonsils or nasal cavities of healthy pigs were tested in the study. Twenty-five S. suis strains isolated from humans from Canada (four strains), England (one strain), France (seven strains), the Netherlands (11 strains) and Mexico (two strains) were also included. Among these human strains, 24 were associated with pyrexia, meningitis, endocarditis or spondylitis, and one was isolated from a healthy human. 15 Identification and serotyping of S. suis were performed as described by Gottschalk et al. 16 The following reference strains were used for each batch of antimicrobial testing by both methods: Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853. Antimicrobial susceptibility testing Disc diffusion method (DDM). The culture medium was Mueller Hinton (MH) agar supplemented with 5% defibrinated sheep blood [MHSB (AES, Combourg, France) as recommended by the CA-SFM for streptococci 14 but also by the NCCLS 17 ] or with 5% horse serum [MHHS (Life- Technologies, Cergy-Pontoise, France)]. The antimicrobial agents used in this study were: β-lactams: penicillin G (6 µg), amoxicillin (25 µg), ceftiofur (30 µg); aminoglycosides: streptomycin (500 µg), kanamycin (1000 µg), gentamicin (500 µg); polypeptides: bacitracin (10 IU); tetracyclines: doxycycline (30 IU); macrolides/ lincosamides: erythromycin (15 IU), spiramycin (100 µg), lincomycin (15 µg) (Bio-Rad, Marnes la Coquette, France), tylosin (30 µg) (Lilly-France, Saint-Cloud, France); phenicols: florfenicol (30 µg) (Schering-Plough, Segré, France). The method used was in accordance with CA-SFM recommendations. 13,14 Briefly, three or four colonies from an overnight culture on Columbia agar supplemented with 5% sheep blood (AES) were suspended in MH broth (Becton Dickinson, Pont de Claix, France). The suspension was adjusted to a 0.5 McFarland standard and diluted to obtain an inoculum of 10 6 cfu/ml S. suis. For each strain two plates were inoculated by flooding MH agar (4 mm depth) supplemented with either 5% defibrinated sheep blood or 5% horse serum. The antibiotic discs were placed with a disc dispenser (Bio- Rad) and plates were incubated at 37 C in 5% CO 2 for 18 h. The zone diameters of S. suis strains and the reference strains were measured on the same day using a sliding caliper. Microdilution method (MDM). Susceptibility to antimicrobial agents was also determined by the MDM with MH broth supplemented with 5% horse serum using the following antimicrobial agents: penicillin G (0.12 32 mg/l), amoxicillin (0.25 32 mg/l), ceftiofur (1 64 mg/l), streptomycin (32 2048 mg/l), kanamycin (16 2048 mg/l), gentamicin (32 2048 mg/l), bacitracin (1 16 mg/l), doxycycline (0.25 16 mg/l), erythromycin (0.25 16 mg/l), spiramycin (0.25 16 mg/l), tylosin (0.25 16 mg/l), lincomycin (0.5 32 mg/l) and florfenicol (0.25 32 mg/l). All antimicrobial agents were purchased from Sigma (Saint Quentin Fallavier, France) except for ceftiofur (Pharmacia & Upjohn, Saint Quentin en Yvelines, France) and florfenicol (Schering- Plough). The antimicrobial solutions were prepared as described in the 2000 2001 report of the CA-SFM, 14 and each dilution was distributed in 96-well microtitration plates (Merck Eurolab, Strasbourg, France). Bacterial suspensions were prepared as described for the DDM and adjusted to 5 10 5 cfu/ml. The microtitration plates were incubated at 37 C in 5% CO 2 for 18 h. The MIC was defined as the lowest concentration of antibiotic that inhibited development of visible growth. Interpretation of susceptibility testing results A strain was considered susceptible if the zone diameter was greater than or equal to the diameter breakpoint for the DDM or if the MIC was smaller than or equal to the MIC breakpoint for the MDM. The antibiotic breakpoints were taken from the CA-SFM 13,14 and the NCCLS 17 reports except for florfenicol, for which the breakpoints used were those given by the manufacturer. The distributions of resistant (R), intermediate (I) or susceptible (S) strains were compared according to the three methods used and the agreement of these classifications was analysed for each strain. If a discord was detected, it was defined as major if a strain was classified S to an antimicrobial agent with one method and R with the other method. The discord was defined as minor if a strain was classified I to an antimicrobial agent with one method and S or R with the other method. 202
Antimicrobial susceptibility of Streptococcus suis Statistical analysis For each antimicrobial agent, the effects of capsular type and origin of S. suis on the antimicrobial susceptibility were analysed with Fisher s exact test. This test was also used for the evaluation of the medium effect (MHSB or MHHS) on the antimicrobial susceptibility determined by the DDM. In all cases, differences were considered significant when P was <0.05. Results The distribution of S. suis isolates according to serotype is presented in Tables 1 and 2. Sixty of the 110 strains (54.5%) isolated from pigs belonged to serotype 2. Most of them were associated with clinical signs and pathology: 55% with septicaemia, 50% with meningitis and 100% with arthritis. However, 44% of strains from healthy pigs were serotype 2. In humans, 96% of serotype 2 strains were associated with meningitis. The four reference strains presented zone diameters and MICs similar to those reported by the CA-SFM (Tables 3 and 4). Good agreement was obtained for results by the DDM using MHSB and MHHS media for all antimicrobial agents except for penicillin G. For this molecule, the population of S. suis strains was divided by the larger diameter breakpoint into two sub-populations with MHSB, whereas all but one strain were classified as susceptible with MHHS (Figure 1). Moreover, for the two reference strains susceptible to penicillin G (S. aureus ATCC 25923 and E. faecalis 29212), zone diameters were slightly smaller with MHHS than with MHSB. The other two reference strains were R to penicillin G and their zone diameters were identical with both media. According to S, I or R classification of strains, significant differences (P < 0.05) were noted between the DDM and the MDM for streptomycin and bacitracin. For streptomycin, only four strains were classified as R by DDM, and seven were classified as R and five as I by the MDM. For bacitracin, only one strain was classified as R by the DDM, whereas nine strains were R by the MDM (Tables 1 and 2). Minor discords were reported mainly for penicillin G. Fiftythree strains were classified as I by the DDM with MHSB (Figure 1a) and as S by the DDM with MHHS (Figure 1b) and by the MDM (Table 2). One strain was classified as I by the DDM (Figure 1b) and S by the MDM (Table 2). Minor differences were also observed for macrolides/lincosamides, streptomycin, kanamycin and doxycycline. For these last molecules two to 10 strains were classified differently by the DDM or MDM, and one to four strains were classified differently by diffusion in MHSB or MHHS. The numbers of susceptible S. suis strains according to the DDM and MDM are reported in Tables 1 and 2, respectively. A large proportion of S. suis strains was classified as R to doxycycline (79% with the DDM and MDM). Resistance to macrolides and lincosamides was also widespread: for erythromycin, spiramycin, tylosin and lincomycin, only about 30% of the strains were classified as S (Tables 1 and 2). Most of the strains were classified as S to streptomycin and kanamycin. All strains were classified as S to penicillin G (except by the DDM with MHSB), amoxicillin, ceftiofur, gentamicin and florfenicol. Most strains were also S to bacitracin (99% with the DDM and 93% with the MDM). No correlation (P 0.14) could be found between the proportions of S S. suis isolated from diseased pigs (n = 83), healthy pigs (n = 27) or humans (n = 25). However, S. suis strains isolated from humans were significantly more frequently S (P < 0.02) to erythromycin, spiramycin, tylosin, lincomycin and doxycycline than strains isolated from diseased pigs (meningitis, arthritis or septicaemia) or healthy pigs. The S. suis capsular type 2 strains (n = 85) were also significantly more frequently S than other serotypes (n = 50) to erythromycin, spiramycin, tylosin and lincomycin (45 46% versus 8%, P < 0.0001) and doxycycline (31% versus 4%, P < 0.0001). Discussion This study was undertaken in order to propose a standardized method for testing the antimicrobial sensitivity of S. suis. As the DDM is very commonly used in France, it was decided to evaluate this technique, using either MHSB or MHHS, and to compare it with the broth dilution test, which seems more usual in many other countries. The use of horse serum instead of sheep blood in the DDM seemed justified because the opacity of the medium due to supplementation with blood hinders precise measuring of zone diameters without opening the plates, a manipulation that might be dangerous with a zoonotic agent such as S. suis. Moreover, a large quantity of a single batch of horse serum can be stored at 20 C for periods up to 1 year, whereas sheep blood must be ordered regularly (several times a month), a feature that results in the use of many different batches of this component, which may influence the reproducibility of the test over time. It is important to underline that this study showed that the presence in MH of horse serum, compared with sheep blood, resulted in increased inhibitory zone diameters of S. suis when testing penicillin G. The difference reported might be explained by a modification of penicillin G diffusion in the medium according to the supplement used. This explanation is in agreement with the slightly different results obtained with S. aureus ATCC 25923 and E. faecalis ATCC 29212 strains. Andrews et al. 18 have also reported a difference in zone diameters for Gram-positive pathogens to dalfopristin/ 203
Table 1. Distribution of S. suis serotypes and number of susceptible S. suis isolates determined by disc diffusion Capsular types isolated from diseased pigs (n = 83) Septicaemia (n = 54) Meningitis (n = 22) Arthritis (n = 7) Capsular types isolated from healthy pigs (nasal cavity, tonsils) (n = 27) Capsular type isolated from humans (n = 25) ATB a BP b (mm) 1/2 c (n = 7) 2 3 7 9 (n = 30) (n = 4) (n = 5) (n = 6) Aut d Total (n = 2) (n = 54) 1/2 (n = 1) 2 (n = 11) 7 (n = 3) 9 (n = 5) Aut Total 2 (n = 2) (n = 22) (n = 7) Total 1/2 (n = 7) (n = 2) 2 (n = 12) 3 7 Aut Total 2 (n = 2) (n = 6) (n = 5) (n = 27) (n = 25) Total (n = 25) 204 P e 29 7 30 4 5 6 2 54 1 11 3 4 2 21 7 7 2 12 2 6 5 27 25 25 2 17 2 4 3 2 30 1 5 1 3 0 10 6 6 2 7 1 4 3 17 18 18 AMX 21 7 30 4 5 6 2 54 1 11 3 5 2 22 7 7 2 12 2 6 5 27 25 25 XNL 21 7 30 4 5 6 2 54 1 11 3 5 2 22 7 7 2 12 2 6 5 27 25 25 FFC 19 7 30 4 5 6 2 54 1 11 3 5 2 22 7 7 2 12 2 6 5 27 25 25 B 15 7 30 4 5 6 2 54 1 11 3 5 2 22 7 7 2 12 2 6 4 26 25 25 GEN 17 7 30 4 5 6 2 54 1 11 3 5 2 22 7 7 2 12 2 6 5 27 25 25 STR 14 7 30 4 5 5 2 53 1 10 3 5 2 21 7 7 2 12 2 6 4 26 24 24 KAN 14 5 27 4 5 5 2 48 1 10 3 4 2 20 6 6 2 11 2 6 4 25 25 25 E 22 2 11 0 0 0 0 13 0 6 0 1 0 7 1 1 0 3 0 0 0 3 18 18 SP 24 2 8 0 0 0 0 10 0 4 0 1 0 5 1 1 0 3 0 0 0 3 17 17 TYL 18 2 11 0 0 0 0 13 0 6 0 1 0 7 1 1 0 3 0 0 0 3 18 18 L 21 2 11 0 0 0 0 13 0 6 0 1 0 7 1 1 0 3 0 0 0 3 17 17 DO 19 1 6 0 0 0 0 7 0 4 0 0 0 4 0 0 0 0 1 1 0 2 15 15 J. Marie et al. a ATB, antibiotic: P, penicillin G; AMX, amoxicillin; XNL, ceftiofur; FFC, florfenicol; B, bacitracin; GEN, gentamicin; STR, streptomycin; KAN, kanamycin; E, erythromycin; L, lincomycin; SP, spiramycin; TYL, tylosin; DO, doxycycline. b Diameter breakpoint for susceptibility (the strains were classified as S when the zone diameters were superior or equal to the diameter breakpoint) according to guidelines 13,14 or manufacturers. c Serotype. d Autoagglutinable isolates. e For penicillin G, the first line reports the number of S strains by DDM with MHHS (horse serum), and the second line reports the number of S strains by DDM with MHSB (sheep blood). For all other antibiotics the number of S strains by DDM with MHHS is only reported (no statistical differences were observed between the two media).
Table 2. Distribution of S. suis serotypes and number of susceptible S. suis isolates determined by microdilution 205 ATB a BP b (mg/l) Capsular types isolated from diseased pigs (n = 83) Septicaemia (n = 54) Meningitis (n = 22) Arthritis (n = 7) 1/2 c 2 3 7 9 Aut d Total (n = 7) (n = 30) (n = 4) (n = 5) (n = 6) (n = 2) (n = 54) 1/2 (n = 1) 2 (n = 11) 7 (n = 3) 9 (n = 5) Aut Total (n = 2) (n = 22) 2 (n = 7) Capsular types isolated from healthy pigs (nasal cavity, tonsils) (n = 27) Total 1/2 2 3 7 Aut Total (n = 7) (n = 2) (n = 12) (n = 2) (n = 6) (n = 5) (n = 27) Capsular type isolated from humans (n = 25) P 0.25 7 30 4 5 6 2 54 1 11 3 5 2 22 7 7 2 12 2 6 5 27 25 25 AMX 4 7 30 4 5 6 2 54 1 11 3 5 2 22 7 7 2 12 2 6 5 27 25 25 XNL 2 7 30 4 5 6 2 54 1 11 3 5 2 22 7 7 2 12 2 6 5 27 25 25 FFC 4 7 30 4 5 6 2 54 1 11 3 5 2 22 7 7 2 12 2 6 5 27 25 25 B 2 6 30 4 5 3 2 50 1 11 3 2 1 18 7 7 2 12 2 6 4 26 25 25 GEN 250 7 30 4 5 6 2 54 1 11 3 5 2 22 7 7 2 12 2 6 5 27 25 25 STR 250 4 27 4 5 5 2 47 0 10 3 5 2 20 7 7 2 11 2 6 4 25 24 24 KAN 250 5 27 4 5 5 1 47 0 10 3 4 2 19 6 6 2 11 2 6 4 25 24 24 E 1 2 11 0 0 0 0 13 0 6 0 1 0 7 1 1 0 3 0 0 0 3 18 18 SP 1 2 11 0 0 0 0 13 0 5 0 1 0 6 1 1 0 3 0 0 0 3 18 18 TYL 1 2 11 0 0 0 0 13 0 6 0 1 0 7 1 1 0 3 0 0 0 3 18 18 L 2 2 11 0 0 0 0 13 0 6 0 1 0 7 1 1 0 3 0 0 0 3 18 18 DO 4 1 6 0 0 0 0 7 0 4 0 0 0 4 0 0 0 1 1 0 0 2 15 15 2 (n = 25) Total (n = 25) Antimicrobial susceptibility of Streptococcus suis a ATB, antibiotic: P, penicillin G; AMX, amoxicillin; XNL, ceftiofur; FFC, florfenicol; B, bacitracin; GEN, gentamicin; STR, streptomycin; KAN, kanamycin; E, erythromycin; L, lincomycin; SP, spiramycin; TYL, tylosin; DO, doxycycline. b MIC breakpoint (the strains were classified as S when the MICs were inferior or equal to the MIC breakpoint) according to guidelines 13,14 or manufacturers. c Serotype. d Autoagglutinable isolates.
J. Marie et al. Table 3. Zone diameters (mm) obtained with the four reference strains, compared with CA-SFM 13,14 and NCCLS 17 breakpoints E. coli ATCC 25922 S. aureus ATCC 25923 P. aeruginosa ATCC 27853 E. faecalis ATCC 29212 ATB a R b CA-SFM NCCLS R CA-SFM NCCLS R CA-SFM NCCLS R CA-SFM NCCLS P 6 35 31.0 38.5 26 37 6 26 20.0 28.0 AMX 23 22.0 26.5 31 6 29 28.5 36.0 XLN 29 26 31 31 27 31 17 14 18 16 FFC B 6 18 6 19 GEN 22 22.0 26.5 19 26 26 24.0 28.5 19 27 19 15.5 22.5 16 21 STR KAN 25 22.3 26.0 17 25 26 25.0 30.0 19 26 6 E 9 25 26.5 31.5 22 30 6 22 19.0 26.5 SP TYL 6 20 6 22 L 6 23 24.5 29.5 6 9 9.0 13.0 DO 23 19.5 25.0 30 28.0 32.0 9 18 11.5 20.0 a ATB, antibiotic: P, penicillin G; AMX, amoxicillin; XNL, ceftiofur; FFC, florfenicol; B, bacitracin; GEN, gentamicin; STR, streptomycin; KAN, kanamycin; E, erythromycin; L, lincomycin; SP, spiramycin; TYL, tylosin; DO, doxycycline. b Results obtained in this study. Table 4. MICs (mg/l) obtained with the four reference strains, compared with CA-SFM 13,14 and NCCLS 17 breakpoints E. coli ATCC 25922 S. aureus ATCC 25923 P. aeruginosa ATCC 27853 E. faecalis ATCC 29212 ATB a R b CA-SFM NCCLS R CA-SFM NCCLS R CA-SFM NCCLS R CA-SFM NCCLS P 32 <0.12 0.03 0.12 >32 2 1 4 1 4 AMX XLN <1 0.25 1 <1 16 16 64 32 FFC 2 8 >16 2 8 B GEN 1 0.25 1 0.25 1 <0.25 0.12 0.25 < 0.25 0.12 0.5 0.5 2 4 0.5 2 4 16 STR 8 4 4 >16 KAN 4 1 4 1 4 2 1 4 >64 16 16 64 16 64 E >16 <0.25 0.12 0.5 >16 1 0.5 4 1 4 SP >16 16 >16 1 TYL >16 >32 1 >16 >32 1 0.5 4 L >32 1 0.5 2 >32 16 16 64 DO 0.5 <0.5 >16 8 a ATB, antibiotic: P, penicillin G; AMX, amoxicillin; XNL, ceftiofur; FFC, florfenicol; B, bacitracin; GEN, gentamicin; STR, streptomycin; KAN, kanamycin; E, erythromycin; L, lincomycin; SP, spiramycin; TYL, tylosin; DO, doxycycline. b Results obtained in this study. 206
Antimicrobial susceptibility of Streptococcus suis Figure 1. Distribution of zone diameters for penicillin G in MHSB (a) or MHHS (b) (n = 135 strains of S. suis). The vertical lines represent the zone diameter breakpoints defined by the CA-SFM 13,14 for streptococci except S. pneumoniae. S suis isolates were classified as: R (zone diameter < 8 mm), I (zone diameter 8 mm and < 29 mm) or S (zone diameter 29 mm). quinupristin: the presence of blood in the base medium (Iso- Sensitest agar) also resulted in reduced zone diameters for Staphylococcus cohni, Staphylococcus capitis, Staphylococcus warneri, Enterococcus gallinarum and for the reference strains used in their study (S. aureus NCTC 6571, S. aureus ATCC 25923 and E. faecalis ATCC 29212). Although the distribution of the strains for penicillin G susceptibility appeared monomodal whatever the medium used, when using MHSB the CA-SFM-recommended breakpoint (29 mm) cut the population into two categories (60% susceptible and 40% intermediate), whereas all but one strain appeared susceptible when the test was performed with MHHS. As reported by Acar & Goldstein, 19 from an epidemiological point of view, the breakpoint should fit within the limits of clusters of microbial organisms with comparable susceptibility. Thus, in our opinion, it would be judicious to advise laboratories to use horse serum in preferance to sheep blood as a supplement in MH for S. suis susceptibility testing by the DDM. All S. suis strains were found to be S to penicillin G (except by the DDM: 40% of strains were I with MHSB and only one strain was I with MHHS), amoxicillin, ceftiofur, florfenicol and gentamicin, and nearly all strains were S to bacitracin. These results are in accordance with many other studies that have described S. suis as mainly susceptible to these antimicrobial agents. 20 24 The efficacy of β-lactams and gentamicin indicates that these antibiotics can still be chosen empirically when S. suis pig infection is detected in France. Moreover, in human meningitis the usual antimicrobial treatment (β-lactams) should be effective. However, S. suis resistance to β-lactams has been reported, 11,12,25 and streptococcal resistance to β-lactams has been described largely for S. pneumoniae. 6 8,26,27 Resistance to macrolides/lincosamides and tetracyclines described in this study has also been described in the literature to varying degrees in different countries. In France, Morvan 28 reported that 19% of 400 S. suis strains were susceptible to spiramycin, 38% to lincomycin and 18% to tetracycline. In Spain, Reams et al. 25 reported that 33%, 32% and 19% of S. suis strains were susceptible to erythromycin, clindamycin and tetracycline, respectively. In Denmark, 20% of S. suis strains, isolated from 1995 to 1997, were resistant to erythromycin, spiramycin, tylosin and lincomycin and 44% to tetracycline. These high rates of resistance to macrolides/ lincosamides and to tetracyclines might be explained by intensive use of tylosin (growth promoter) and tetracycline (therapeutic) in Danish pig production between 1992 and 1997. 29 Our study showed a high level of resistance to streptomycin and to kanamycin and confirmed the results published by other authors who also described this kind of resistance in varying degrees. 21,24,29,30 The differences observed between countries might be explained by different usage of antimicrobial agents but could also be due to methodological differences between studies. Standardization of susceptibility testing methods at an international level would facilitate comparisons. In our study, serotype 2 was found to be resistant to macrolides/lincosamides less frequently than other serotypes, 207
J. Marie et al. in agreement with results reported by Aarestrup et al. 23 However, these results are inconsistent with those reported previously by Cantin et al. 31 No explanation can be found for this difference. The difference between serotype susceptibility shown in our study could be explained by the fact that serotype 2 is considered to be the serotype most commonly associated with disease, 1 and most commonly isolated from healthy animals. Austin et al. 32 demonstrated that levels of colonization by antibiotic-resistant bacteria are related to antibiotic exposure and prevalence of bacteria. For a proportion of subjects treated, the proportion of resistance decreased when the prevalence of bacteria increased. This difference in resistance in relation to serotype may explain the difference in macrolide/ lincosamide and doxycycline susceptibility observed between human and pig strains, because all human strains tested belonged to serotype 2. In conclusion, this study showed the efficacy of β-lactams against French isolates of S. suis. However, reduced susceptibility or resistance to penicillin G in S. suis has been reported in other countries. 11,12,21,24 Therefore, it seems very important to monitor susceptibility of S. suis to penicillins, still the treatment of choice for human or pig S. suis infections. In France, the evolution of antibiotic susceptibility of S. suis will be surveyed using a novel epidemiological network, RESAP- ATH, which has been running since the beginning of the year 2001. The aim of this network is to collect results of susceptibility testing carried out by regional veterinary laboratories and thus to follow the evolution of resistance in common pathogenic bacteria from cattle, pigs and poultry. These laboratories routinely use the disc diffusion method described in this article and about 70% of them use sheep blood. Mechanisms of resistance detected by this network will be studied in our laboratory or in collaboration with other French research centres. Acknowledgements This research was supported by the Direction Générale de l Alimentation (DGAl), Ministère de l Agriculture et de la Pêche. References 1. Staats, J. J., Feder, I., Okwumabua, O. & Chengappa, M. M. (1997). Streptococcus suis: past and present. Veterinary Research Communications 21, 381 407. 2. Higgins, R. & Gottschalk, M. (1999). Streptococcal diseases. In Diseases of Swine, 8th edn (Straw, B. E., D Allaire, S., Mengeling, W. L. & Taylor, D. J., Eds), pp. 563 78. Iowa State University Press, Ames, IA, USA. 3. Arends, J. P. & Zanen, H. C. (1988). Meningitis caused by Streptococcus suis in humans. Reviews of Infectious Diseases 10, 131 7. 4. Euzeby, J. P. (1999). Streptococcus suis: a short review. Revue de Médecine Vétérinaire 150, 981 8. 5. Dee, S. A., Carlson, A. R., Winkelman, N. L. & Corey, M. M. (1993). Effect of management practices on the Streptococcus suis carrier rate in nursery swine. Journal of the American Veterinary Medical Association 203, 295 9. 6. Tomasz, A. (1997). Antibiotic resistance in Streptococcus pneumoniae. Clinical Infectious Diseases 24, Suppl. 1, S85 8. 7. Manzor, O., Pawlak, J. & Saravolatz, L. (1999). In-vitro activity of 29 antimicrobial agents against penicillin-resistant and -intermediate isolates of Streptococcus pneumoniae. Journal of Antimicrobial Chemotherapy 43, 31 6. 8. Granizo, J. J., Aguilar, L., Casal, J., Garcia-Rey, C., Dal-Re, R. & Baquero, F. (2000). Streptococcus pneumoniae resistance to erythromycin and penicillin in relation to macrolide and β-lactam consumption in Spain (1979 1997). Journal of Antimicrobial Chemotherapy 46, 767 73. 9. Sahm, D. F., Karlowsky, J. A., Kelly, L. J., Critchley, I. A., Jones, M. E., Thornsberry, C. et al. (2001). Need for annual surveillance of antimicrobial resistance in Streptococcus pneumoniae in the United States: 2-year longitudinal analysis. Antimicrobial Agents and Chemotherapy 45, 1037 42. 10. Shneerson, J. M., Chattopadhyay, B., Murphy, M. F. & Fawcett, I. W. (1980). Permanent perceptive deafness due to Streptococcus suis type II infection. Journal of Laryngology and Otology 94, 425 7. 11. Prieto, C., Garcia, F. J., Suarez, P., Imaz, M. & Castro, J. M. (1994). Biochemical traits and antimicrobial susceptibility of Streptococcus suis isolated from slaughtered pigs. Zentralblatt für Veterinarmedizin, Reihe B 41, 608 17. 12. Gottschalk, M., Turgeon, P., Higgins, R., Beaudoin, M. & Bourgault, A. M. (1991). Susceptibility of Streptococcus suis to penicillin. Journal of Veterinary Diagnostic Investigations 3, 170 2. 13. Courvalin, P. & Soussy C. J., Eds. (1996). Comité de l Antibiogramme de la Société Française de Microbiologie guidelines. Clinical Microbiology and Infection 2, Suppl. 1. 14. Soussy, C., Carret, G., Cavallo, J. D., Chardon, H., Chidiac, C., Choutet, P. et al. (2000). Comité de l Antibiogramme de la Société Française de Microbiologie. Communiqué 2000 2001. Pathologie Biologie 48, 832 71. 15. Berthelot-Herault, F., Morvan, H., Keribin, A. M., Gottschalk, M. & Kobisch, M. (2000). Production of muraminidase-released protein (MRP), extracellular factor (EF) and suilysin by field isolates of Streptococcus suis capsular types 2, 1/2, 9, 7 and 3 isolated from swine in France. Veterinary Research 31, 473 9. 16. Gottschalk, M., Higgins, R., Jacques, M., Mittal, K. R. & Henrischen, J. (1989). Description of 14 new capsular types of Streptococcus suis. Journal of Clinical Microbiology 27, 2633 6. 17. National Committee for Clinical Laboratory Standards. (1999). Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals: Approved Standard M31-A. NCCLS, Wayne, PA. 18. Andrews, J. M., Ashby, J. P. & Wise, R. (1999). Study to assess the reliability of a disc diffusion method for determining the sensitivity of Gram-positive pathogens to dalfopristin/quinupristin. Journal of Antimicrobial Chemotherapy 43, 141 3. 208
Antimicrobial susceptibility of Streptococcus suis 19. Acar, J. F. & Goldstein, F. W. (1996). In Antibiotics in Laboratory Medicine, 4th edn (Lorian, V., Ed.), pp. 1 51. Lippincott, Williams and Wilkins, Baltimore, MD, USA. 20. Shryock, T. R., Mortensen, J. E. & Rhoads, L. R. (1992). Antibiotic susceptibility in Streptococcus suis. Current Therapeutic Research 52, 419 24. 21. Stuart, J. G., Zimmerer, E. J. & Maddux, R. L. (1992). Conjugation of antibiotic resistance in Streptococcus suis. Veterinary Microbiology 30, 213 22. 22. Tarradas, M. C., Arenas, A., Maldonado, A., Vicente, S., Miranda, A. & Perea, A. (1994). Susceptibility of Streptococcus suis to various antimicrobial agents. Zentralblatt für Veterinarmedizin, Reihe B 41, 685 8. 23. Aarestrup, F. M., Jorsal, S. E. & Jensen, N. E. (1998). Serological characterization and antimicrobial susceptibility of Streptococcus suis isolates from diagnostic samples in Denmark during 1995 and 1996. Veterinary Microbiology 60, 59 66. 24. Kataoka, Y., Yoshida, T. & Sawada, T. (2000). A 10-year survey of antimicrobial susceptibility of Streptococcus suis isolates from swine in Japan. Journal of Veterinary Medical Science 62, 1053 7. 25. Reams, R. Y., Glickman, L. T., Harrington, D. D., Bowersock, T. L. & Thacker, H. L. (1993). Streptococcus suis infection in swine: a retrospective study of 256 cases. Part I. Epidemiologic factors and antibiotic susceptibility patterns. Journal of Veterinary Diagnostic Investigations 5, 363 7. 26. Hofmann, J., Cetron, M. S., Farley, M. M., Baughman, W. S., Facklam, R. R., Elliott, J. A. et al. (1995). The prevalence of drugresistant Streptococcus pneumoniae in Atlanta. New England Journal of Medicine 333, 481 6. 27. Baquero, F., Garcia-Rodriguez, J. A., Garcia de Lomas, J. & Aguilar, L. (1999). Antimicrobial resistance of 1,113 Streptococcus pneumoniae isolates from patients with respiratory tract infections in Spain: results of a 1-year (1996 1997) multicenter surveillance study. The Spanish Surveillance Group for Respiratory Pathogens. Antimicrobial Agents and Chemotherapy 43, 357 9. 28. Morvan, H. (1994). Sensibilité aux antibiotiques en élevage industriel. In Antimicrobials in Animal Intensive Production, pp. 209 22. European Symposium Proceedings. Institut Supérieur des Productions animales et des Industries Agro-alimentaires, Ploufragan, France. 29. Aarestrup, F. M., Rasmussen, S. R., Artursson, K. & Jensen, N. E. (1998). Trends in the resistance to antimicrobial agents of Streptococcus suis isolates from Denmark and Sweden. Veterinary Microbiology 63, 71 80. 30. Turgeon, P. L., Higgins, R., Gottschalk, M. & Beaudoin, M. (1994). Antimicrobial susceptibility of Streptococcus suis isolates. British Veterinary Journal 150, 263 9. 31. Cantin, M., Harel, J., Higgins, R. & Gottschalk, M. (1992). Antimicrobial resistance patterns and plasmid profiles of Streptococcus suis isolates. Journal of Veterinary Diagnostic Investigations 4, 170 4. 32. Austin, D. J., Kakehashi, M. & Anderson, R. M. (1997). The transmisssion dynamics of antibiotic-resistant bacteria: the relationship between resistance in commensal organisms and antibiotic consumption. Proceedings of the Royal Society of London. Series B. Biological Sciences 264, 1629 38. 209