Abstract
A study of 257 French invasive pneumococci isolated between 2000 and 2002 showed high rates of nonsusceptibility to penicillin and macrolides (50%), contrasting with a low frequency of resistance to amoxicillin or levofloxacin (<1%) and tolerance to vancomycin (0%). The genetic homogeneity of some serogroups, including serogroup 1, enhanced the risk of epidemiological changes.
Streptococcus pneumoniae is one major causative agent of severe infectious diseases. In France the incidence of invasive pneumococcal infections was 9 per 100,000 inhabitants in 2000 (12). The worldwide emergence of antibiotic resistance in this species has required alternative strategies such as vancomycin therapy in meningitis, fluoroquinolone therapy in lower respiratory tract infections, and large vaccination programs (14). The need for national surveillance programs to evaluate the impact of these strategies on the epidemiology of S. pneumoniae is well established (27, 29). The ColBVH Study Group, a network of 105 nonteaching sentinel hospitals representative of general hospitals in France, conducts a yearly survey of pneumococcal bacteremia. We report the antibiotic resistance prevalence, serotype distribution, and molecular epidemiology of 257 invasive pneumococci recently isolated in France.
Nonduplicate pneumococci isolated from blood cultures performed on hospitalized patients were consecutively collected during each November in 2000, 2001, and 2002 (112, 69, and 76 isolates, respectively) by the ColBVH Study Group and sent to a central laboratory (Service d'Hygiène, Centre Hospitalier de Versailles). The MICs of penicillin G, amoxicillin, cefuroxime, cefotaxime, ceftriaxone, erythromycin, quinupristin-dalfopristin (70/30 ratio), ciprofloxacin, levofloxacin, gatifloxacin, and moxifloxacin were determined by agar dilution method in ambient air according to NCCLS guidelines (22, 23). S. pneumoniae ATCC 49619 was used as a control strain. Erythromycin-resistant strains were screened by PCR for the presence of erm(B) and mef(A) genes, as described previously (5, 21). Tolerance to vancomycin was evaluated as previously reported, with use of R6 and the tolerant autolysin-defective laboratory mutant Lyt 4-4 as control strains (5, 11). The criterion for vancomycin tolerance was a decrease of viability of less than 2 logs (5, 11). Serotype determination was performed by the Quellung reaction (15). Pulsed-field gel electrophoresis (PFGE) of total DNA was performed using the GenePath kit (Bio-Rad, Marnes-la-Coquette, France) according to the recommendations of the manufacturer and with the SmaI enzyme as previously reported (5). Visual comparison of PFGE patterns with a panel of international clones recognized by the Pneumococcal Molecular Epidemiology Network (19) was performed according to current consensus protocols (30).
Among the 257 pneumococci tested, 41 (16%) were penicillin resistant and 79 (30.7%) were penicillin intermediate. Erythromycin resistance occurred in 144 isolates (56%). Very few isolates or none were resistant to amoxicillin, cefotaxime-ceftriaxone, quinupristin-dalfopristin, or fluoroquinolones with enhanced antipneumococcal activity. Susceptibility data are reported according to the age of the patients in Tables 1 and 2. All macrolide-resistant isolates carried erm(B) exclusively. Vancomycin tolerance was not detected in any of the strains studied, and no significant difference in lysis was observed for any clinical strains. Serogroup distribution of the 241 typeable strains is reported according to the age of the patients in Tables 3 and 4. Serogroups of penicillin-susceptible isolates varied widely, but serogroups 1 (15.4%), 3 (12.2%), and 4 (10.6%) were predominant. Serogroups of penicillin-nonsusceptible S. pneumoniae (PNSSP) were limited, including serogroups 14 (35.9%), 23 (16.2%), 19 (15.4%), 9 (12.8%), and 6 (8.5%). No significant differences in antibiotic susceptibility or serogroup distribution were observed during the three periods of the study (data not shown). The repartition of pulsotypes among the main serogroups is reported in Table 5 and Fig. 1. The PFGE patterns of isolates from other serogroups showed various pulsotypes (data not shown).
TABLE 1.
Distribution of MICs of antibiotics for 222 invasive pneumococci recovered from adults in France
| Antimicrobial agent | MIC (μg/ml)a
|
Susceptibilityb (%)
|
||||
|---|---|---|---|---|---|---|
| 50% | 90% | Range | S | I | R | |
| Penicillin G | 0.064 | 2 | 0.008-4 | 52.0 | 31.5 | 16.0 |
| Amoxicillin | 0.032 | 1 | 0.008-4 | 98.2 | 1.8 | 0.0 |
| Cefuroxime | 0.125 | 4 | 0.016-16 | 62 | 2.7 | 35.3 |
| Cefotaxime | 0.032 | 1 | 0.016-4 | 95.6 | 4.0 | 0.4 |
| Ceftriaxone | 0.016 | 0.5 | 0.008-4 | 99.6 | 0.0 | 0.4 |
| Erythromycin | 1 | 1,024 | 0.064-1,024 | 43.2 | 1.4 | 55.4 |
| Ciprofloxacin | 2 | 2 | 0.5-64 | NDc | ND | ND |
| Quinupristin-dalfopristin | 0.5 | 1 | 0.125-1 | 100.0 | 0.0 | 0.0 |
| Levofloxacin | 1 | 2 | 0.25-32 | 99.6 | 0.0 | 0.4 |
| Gatifloxacin | 0.25 | 0.5 | 0.064-8 | 99.6 | 0.0 | 0.4 |
| Moxifloxacin | 0.125 | 0.25 | 0.032-4 | 99.6 | 0.0 | 0.4 |
50% and 90%, MICs at which 50 and 90%, respectively, of the isolates tested are inhibited.
S, susceptible; I, intermediate; R, resistant, all according to the NCCLS criteria.
ND, not determined (no available interpretative breakpoint for S. pneumoniae). The proportion of strains for which the, MIC was ≥4 μg/ml was 5.8% (13 of 222).
TABLE 2.
Distributions of MICs of antibiotics for 35 invasive pneumococci isolated from children in France
| Antimicrobial agent | MIC (μg/ml)a
|
Susceptibilityb (%)
|
||||
|---|---|---|---|---|---|---|
| 50% | 90% | Range | S | I | R | |
| Penicillin G | 0.032 | 2 | 0.016-2 | 54.2 | 31.5 | 14.3 |
| Amoxicillin | 0.016 | 1 | 0.016-8 | 94.2 | 2.9 | 2.9 |
| Cefuroxime | 0.064 | 4 | 0.016-16 | 65.7 | 0.0 | 34.3 |
| Cefotaxime | 0.032 | 1 | 0.016-2 | 97.1 | 2.9 | 0.0 |
| Ceftriaxone | 0.016 | 0.1 | 0.016-1 | 100.0 | 0.0 | 0.0 |
| Erythromycin | 1 | 1,024 | 0.064-1,024 | 51.4 | 0.0 | 48.6 |
| Quinupristin-dalfopristin | 0.5 | 1 | 0.25-1 | 100.0 | 0.0 | 0.0 |
| Ciprofloxacin | 2 | 2 | 0.5-4 | NDc | ND | ND |
| Levofloxacin | 1 | 2 | 0.5-2 | 0.0 | 0.0 | 0.0 |
| Gatifloxacin | 0.25 | 0.5 | 0.125-1 | 0.0 | 0.0 | 0.0 |
| Moxifloxacin | 0.125 | 0.25 | 0.125-0.5 | 0.0 | 0.0 | 0.0 |
50% and 90%, MICs at which 50 and 90%, respectively, of the isolates tested are inhibited.
S, susceptible; I, intermediate; R, resistant, all according to the NCCLS criteria.
ND, not determined (no available interpretative breakpoint for S. pneumoniae). The proportion of strains for which the MIC was ≥4 μg/ml was 2.9% (1 of 35).
TABLE 3.
Serogroup distribution of the 209 typeable pneumococci isolated from adults, according to their penicillin susceptibility
| Serogroup | No. susceptible (n = 106) | No. with intermediate resistance (n = 70) | No. resistant (n = 33) |
|---|---|---|---|
| 1 | 16 | 0 | 0 |
| 3 | 13 | 0 | 0 |
| 4 | 12 | 2 | 0 |
| 6 | 7 | 6 | 2 |
| 7 | 4 | 0 | 1 |
| 8 | 8 | 0 | 0 |
| 9 | 5 | 11 | 4 |
| 10 | 6 | 1 | 0 |
| 11 | 4 | 0 | 0 |
| 12 | 2 | 1 | 1 |
| 14 | 0 | 20 | 16 |
| 15 | 1 | 2 | 0 |
| 17 | 1 | 0 | 0 |
| 18 | 5 | 0 | 0 |
| 19 | 5 | 14 | 2 |
| 20 | 2 | 0 | 0 |
| 21 | 1 | 0 | 0 |
| 22 | 1 | 0 | 0 |
| 23 | 5 | 8 | 8 |
| 24 | 0 | 5 | 0 |
| 31 | 1 | 0 | 0 |
| 32 | 1 | 0 | 0 |
| 33 | 5 | 0 | 0 |
TABLE 4.
Serogroup distribution of the 32 typeable pneumococci isolated from children according to their penicillin susceptibility
| Serogroup | No. susceptible (n = 17) | No. with intermediate resistance (n = 10) | No. resistant (n = 5) |
|---|---|---|---|
| 1 | 3 | 1 | 0 |
| 2 | 1 | 0 | 0 |
| 3 | 2 | 0 | 0 |
| 4 | 1 | 0 | 0 |
| 6 | 1 | 2 | 0 |
| 7 | 2 | 0 | 0 |
| 14 | 0 | 4 | 2 |
| 17 | 1 | 0 | 0 |
| 18 | 2 | 0 | 0 |
| 19 | 2 | 1 | 2 |
| 23 | 0 | 2 | 1 |
| 24 | 1 | 0 | 0 |
| 33 | 1 | 0 | 0 |
TABLE 5.
Repartition of pulsotypes among the main serogroup in terms of numbers of strains
| Serotype | Pulsotype(s) | No. of strains/ pulsotype | Relation to international clone |
|---|---|---|---|
| 1 | A | 15 | |
| B to D | 1 | ||
| 9 or 14 | A | 41 | Spain9V-1 |
| B to G | 1 or 2 | ||
| 19 | A | 15 | |
| B to K | 1 or 2 | ||
| 23 | A | 12 | Spain23F-1 |
| B to 1 | 1 or 2 |
FIG. 1.
PFGE patterns of SmaI-digested DNA of the main pneumococcal serotypes isolated from blood cultures.
The prevalence of antibiotic resistance in S. pneumoniae is higher in France than in other countries, in particular that to penicillin G and macrolides (1). In this work the rate of PNSSP among invasive pneumococci seemed stable (46.7%) after more than a decade of dramatic increase (7% in 1991 [3], 27% in 1997 [17], and 48.3% in 2000 [4]), and the frequency of strains fully resistant to amoxicillin or cefotaxime-ceftriaxone remained very low (0.4%) (3, 4, 17). However, the rate of erythromycin-resistant strains increased further, reaching 45.4% in 2000 (4) and 54.9% in this study. As previously reported, erythromycin resistance is due to the erm(B) methylase gene (5, 9). Our results also confirm the low occurrence of fluoroquinolone resistance among French pneumococci (4, 5, 28), but the method used could not detect any first-stage mutants (16, 25). Vancomycin tolerance in S. pneumoniae has been involved in clinical failure (11, 18). In a previous study, 3% of clinical isolates of pneumococci originating mainly from Sweden exhibited a phenotype of tolerance to vancomycin (11). No strain showing vancomycin tolerance was detected in our work. In a recent regional survey (5), we did not detect any vancomycin-tolerant strains among invasive isolates from French children, and a similar observation was established by two different authors in Spain (2, 24). Despite the relatively limited number of strains studied, the distribution of serogroups among PNSSP seemed similar to those in other countries (15) whereas the predominance of serotypes 1 and 3 among penicillin-susceptible strains was of concern. Serotype 1 was responsible for 26.5% of all invasive pneumococcal infections in Denmark in 1996 and for a greater-than-fourfold increase in the rate of pneumococcal bacteremia in Sweden between 1987 and 1997 (10, 15), and serotype 3 dominated in fatal cases in an international study (13). Unfortunately, a recent French study did not include typing of these serotypes (17). A larger prospective surveillance program is necessary to detect potential significant variation in serogroup distribution. In France no recent national data about the spread of clones were available, in particular for susceptible strains. As in other countries, PNSSP belonged to a small number of clones, most of them international resistant epidemic clones, whereas penicillin-susceptible isolates belonged to sporadic clones (6, 7, 8, 20, 26). However, the serotype 1 isolates demonstrated a high degree of genetic homogeneity. A similar finding was observed in Sweden: a single novel penicillin-susceptible clone of type 1 was responsible for the increase of invasive pneumococcal diseases over several years (10). These findings should be confirmed by a large-scale analysis of the different pulsotypes with multilocus sequence typing methodology.
In summary, this work provides nationwide data on the epidemiology of invasive S. pneumoniae in France and underlines the need for a large national surveillance program.
Acknowledgments
We thank E. Varon from the French Pneumococci National Reference Centre for proving R6 and Lyt 4-4 strains; R. R. Reinert from the National Reference Center for Streptococci, Aachen, Germany; for providing the reference strains of international clones recognized by the Pneumococcal Molecular Epidemiology Network; and the microbiologists who participated in the ColBHV Study Group.
Participating members of the ColBHV Study Group in various cities in France include the following: J. Akli (Blois), C. Alba-Sauviat (Chaumont), G. Aubert (Saint Etienne), P. Amirault (Vierzon), J. Assens (St. Afrique), J. P. Aubry (Quimperle), P. Aucher (Saint Jean D'Angely), C. Auvray (Charleville Mezieres), A. Bailly (Albi), A. Barrans (Sete), D. Barraud (Gonesse), C. Benoit (Fontainebleau), E. Bichier (Saumur), H. Biessy (La Rochelle), M. Bietrix (Martigues), P. Bineau (Saint Dizier), V. Blanc (Antibes), S. Bland (Annecy), A. Boisivon (St. Germain en Laye), Y. Boucaud-Maitre (Lyon), C. Bouguigny-Saison (Soissons), P. Brisou (Toulon Naval), S. Brovedani (Rambouillet), M. Caillaux (Tourcoing), B. Cancet (Villeneuve sur Lot), J. Carre-Cavelier (Bayeux), G. L. Cartolano (St. Germain en Laye), J. Cartron (Dreux), G. Chambreuil (La Roche sur Yon), P. Chantelat (Vesoul), A. Chapelle (Aubenas), C. Chaplain (Saint Denis), H. Chardon (Aix en Provence), B. Chaurang (Neuilly sur Seine), A. Clarac (Foix), P. Clergeau (Sallanches), E. Collot (Bar le Duc), P. Courrier (Metz Armees), M. F. Danjoux (Tarbes), J. P. Darchis (Compiegne), H. de Montclos (Bourg en Bresse), A. Decoster (Lomme les Lille), C. Delamare (Thionville), J. M. Delarbre (Mulhouse), P. Deligne (Remiremont), F. Delubac (Annonay), M. C. Demachy (Meaux), J. Deregnaucourt (Paris), M. A. Desailly-Chanson (La Roche sur Yon), J. Didion (Metz), F. Doucet-Populaire (Versailles), A. Dublanchet (Villeneuve St. Georges), B. Dubourdieu (Rodez), S. Dubourdieu (Gisors), D. R. Dupond (Laon), C. Durand (Provins), C. Eloy (Troyes), P. Emerique (Remiremont), F. Evreux (Le Havre), D. Fevre (Vienne), J. Flipo (Wissembourg), N. Fonsale (St. Etienne), A. Fremaux (Creteil), C. Fuhrmann (Lyon), S. Gabriel (Monaco), M. Galanti (Coulommiers), G. Gallou (Falaise), F. Gandhilhon-Crepet (Monbrison), I. Ganivala (Montauban), E. Gardien (Morlaix), P. Garnotel (Marseille-Armees), M. Gavignet (Bourges), F. Geffroy (Quimper), C. Grasmick (Cahors), B. Gravagna (Lyon), G. Grise (Elbeuf), C. Guier (St. Valler), P. Guiet (Nemours), A. Heidt (Hagueneau), M. Helfre (Firminy), J. Heurte (Beauvais), E. Heusse (Bayeux), M. C. Jaffar Bandjee (Saint Denis Reunion), D. Jan (Laval), E. Jaouen (Sable sur Sarthe), G. Khatib (Bagnols sur Ceze), J. P. Lafargue (Dax), R. Lamarca (Narbonne), V. Larroque (Carcassonne), E. Laurens (Cholet), A. Le Coustumier (Cahors), F. Le Turdu (Argenteuil), J. Y. Leberre (Saint Nazaire), E. Lecaillon Thibon (Perpignan), H. Lefrand-Crepin (Avignon), P. Lemaitre (Creil), C. Lemble (Selestat), M. Leneveu (Meulan), B. Lepilleur (St. Dizier), A. Mandjee (Romans), A. Mangeol (Montfermeil), M. F. Marchal (Annemasse), M. Marcolin (Arras), A. Marmonier (Le Mans), T. Masseron (Lyon), R. Meley (Saint Etienne), O. Menouni (Montceau les Mines), M. Menouar (Rang du Flier), A. Michel (Marseille), M. Mora (Frejus), B. Moreau (Cayenne), A. Morel (Le Havre), O. Morvan (Saint Brieuc), D. Neri Schiavini (Cannes), G. Otterbein (Bry sur Marne), X. Palette (Plaisir), B. Pangon (Versailles), J. Paul (Boulogne sur Mer), C. Payen (Brignoles), M. Perrin ((Thionville), D. Pierrejean (Auch), P. Pouedras (Vannes), D. Pressac (Tulle), G. Rast (Poissy), D. Reisz (Montceau les Mines), F. Richardin (Mantes La Jolie), Y. Rio (Metz), P. Roos (Thionville), P. Roussellier (Salon de Provence), D. R. Rousset (Beaune), M. Rouviere (Mende), O. Sabot (Belley), D. R. Saly (St. Denis de la Réunion), S. Samaille (Saint Omer), R. Sanchez (Perigueux), A. Scanvic (Argenteuil), Y. Scat (Paris), A. Secher (Chartres), H. Sep-Hieng (Avranches), D. P. Simeon (Langres), V. Simha (Hyeres), C. Sire-Bidault (Chalon sur Saone), S. Smati (Aubenas), A. Sommabere (Brive), P. Stoessel (Neufchateau), P. Stolidi (Aubagne), F. Templier (Armentieres), J. P. Thellier (Chateau Thierry), D. R. Thore (Beaune), J. Tous (Chambery), A. Trevoux (Mulhousse), A. Vachee (Roubaix), E. Vallee (Eaubonne), J. Vaucel (St. Brieuc), A. Verhaeghe (Dunkerque), M. Villemain (Aurillac), M. Viot (Nice), I. Vray (Voiron), and J. F. Ygout (Lorient).
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