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Antibiotic Susceptibility and Serotype Distribution of Streptococcus pneumoniae Causing Meningitis in Italy, 1997–1999

  1. Annalisa Pantosti,
  2. Fabio D'Ambrosio,
  3. Agapito Tarasi,
  4. Simona Recchia,
  5. Graziella Orefici, and
  6. Paola Mastrantonio
  1. Laboratory of Bacteriology and Medical Mycology, Istituto Superiore di Sanità, Rome, Italy
  1. Reprints or correspondence: Dr. Annalisa Pantosti, Laboratory of Medical Bacteriology and Mycology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy (pantosti{at}iss.it).
 
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Abstract

Because few data are available in Italy regarding antimicrobial susceptibility and serotype distribution of invasive Streptococcus pneumoniae strains, meningeal isolates collected at Italian hospitals during the years 1997–1999 were studied. The 12 most common serogroups, representing >85% of the isolates, were 14, 23, 6, 4, 3, 9, 19, 8, 1, 12, 18, and 7 (in order of frequency). The serogroups identified in children <5 years old were more limited in number: 80% are included in the 7-valent conjugate vaccines. Penicillin resistance was observed in 14 (9.5%) of 148 strains and increased from 5% in the first part of the study to 13% in the last part. Only 2 strains were fully penicillin resistant, and these belonged to serotype 9V. Thirty percent of the strains, mostly belonging to serogroups 14 or 6 and carrying either the ermB or the mef genes, were resistant to erythromycin.

Infections due to antibiotic-resistant Streptococcus pneumoniae are becoming increasingly common all over the world [1]. Of special concern is resistance to penicillin, because this antibiotic has always been considered to be the first-line therapy for pneumococcal diseases, including serious infections such as meningitis and bacteremia. Although the clinical impact of penicillin-resistant S. pneumoniae (PRSP) strains has not been clearly defined [2], meningitis due to penicillin-resistant strains has been shown to have a less favorable outcome [3]. Moreover, PRSP strains are often multiresistant; therefore, infections due to these strains pose serious problems for therapy [4].

The magnitude of the problem can be estimated from the number of reports published in recent years, not only from Western countries but also from east Asia [5, 6]. In the United States, the prevalence of PRSP strains rose from <0.1% during the 1980s to 3.5% in 1994 [7], up to 25%–35% in some areas of the country in more recent surveys [8, 9]. In Europe, the prevalence of PRSP strains varies greatly from region to region. In Spain, PRSP strains had become very common at the end of the 1980s and, since then, their prevalence has remained stable at ∼40% [10, 11]. In France, penicillin resistance showed a sharp increase at the beginning of the 1990s, reaching a rate >40% in 1996 [12, 13]. In the United Kingdom, the prevalence of penicillin resistance increased gradually, although significantly, from 0.3% in 1989 to 3.9% in 1995 [14, 15]. Conversely, in Germany and the Scandinavian countries [16–18], resistance to penicillin is infrequent: <2% of the strains have been reported as being penicillin resistant, with very few strains being fully resistant (MIC ⩾2 µg/mL). Two factors are considered to be important in the development of penicillin resistance in a certain area: the restrictiveness of the country's policy regarding prescription of antibiotics [19] and the circulation of successful clones, such as the Spanish clone 23F, that are capable of geographically spreading throughout countries and continents [20].

The therapeutic problems connected with antibiotic-resistant pneumococcal infections have renewed the interest in the prevention of pneumococcal disease by active immunization. Use of the licensed polyvalent polysaccharide vaccine is encouraged [21], and new pneumococcal conjugate vaccines for infants and children are under development [22]. Consequently, an important aspect of surveillance is the identification of the most prevalent pneumococcal serotypes, especially those comprising invasive or antibiotic-resistant strains.

Although no systematic surveillance of PRSP strains has been carried out in Italy, published reports indicate that the prevalence of penicillin resistance is low to moderate, at 5%–8% [23]. This level of prevalence cannot be explained by a prudent antibiotic policy, because, in Italy, consumption of antibiotics is high and, until few years ago, consumption was not much restricted. The aim of the present study was to determine the prevalence of antibiotic resistance in invasive strains of S. pneumoniae isolated in Italy from patients with meningitis in 1997–1999 and to describe the most common invasive serotypes circulating in the country.

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Materials and Methods

Bacterial strains

S. pneumoniae strains isolated from the CSF of subjects with meningitis were sent to the Laboratory of Bacteriology and Medical Mycology of the Istituto Superiore di Sanità on a voluntary basis, as part of the National Surveillance System of Bacterial Meningitis. The identification of all strains was confirmed by colony morphology, Gram's stain, and the Optochin test. For strains resistant to Optochin by the standard criteria [24], a polymerase chain reaction (PCR) assay for the pneumolysin gene was performed with primers designed by Dagan et al. [25]. The strains were stored in Microbank vials (Pro-Lab Diagnostics, Neston, UK) at −80°C until used. Columbia agar (Oxoid, Basingstoke, UK) supplemented with 5% sheep's blood was used for all passages.

Serotyping

The strains were serotyped by quellung reaction with the Pneumotest antisera panel (Statenserum Institut, Copenhagen, Denmark), which is designed to identify the serogroups included in the current 23-valent antipneumococcal polysaccharide vaccine. Factorial serum samples were used to identify the most frequent serotypes inside serogroups, when needed.

Antibiotic susceptibility testing

Susceptibilities to penicillin, ceftriaxone, erythromycin, clindamycin, chloramphenicol, tetracycline and trimethoprim-sulfamethoxazole (TMP-SMZ) were determined by the E test (AB Biodisk, Solne, Sweden) with Mueller-Hinton II agar (Becton-Dickinson Italia, Milan, Italy) supplemented with 5% sheep's blood, according to the procedures recommended by the manufacturer. Incubation was performed in CO2 at 35°C for 24 h. The E test was shown elsewhere to compare well with the reference agar dilution method [26]. MICs obtained with the E test were rounded to the next higher log2 value if the MIC fell between the standard 2-fold increments. The breakpoints recommended by the National Committee for Clinical Laboratory Standards [27] were used. Two strains with predetermined MIC values, 1 intermediate (ATCC 49619), and 1 resistant to penicillin (IC3, kindly provided by A. Tomasz, Rockefeller University, NY), were tested with any new batch of agar medium.

Molecular fingerprinting by pulsed-field gel electrophoresis (PFGE)

The method used for PFGE has been described elsewhere [28]. In brief, S. pneumoniae strains were grown for 6–8 h in Todd-Hewitt broth (Difco Laboratories, Becton-Dickinson Italia). The bacterial cells were washed with PIV buffer (10 mM Tris and 1 M NaCl, pH 8.0), were placed into 100-µL agarose plugs, and were lysed following the standard procedure [29]. For restriction analysis, each plug was treated with 20 U of SmaI or ApaI (Roche Molecular Biochemicals, Milan, Italy) in 150 µL of buffer, according to the manufacturer's recommendations. Electrophoresis was performed with a CHEF-Mapper apparatus (Bio-Rad Laboratories, Milan, Italy), at 6 V/cm for 21 h at 14°, with an included angle of 120° and a linear ramp of 1–20 s.

PCR amplification for erythromycin resistance genes

To detect the erythromycin resistance of genetic determinants ermB and mef, PCR amplifications were performed with the oligonucleotide primers suggested by Sutcliff et al. [30]. Pneumococcal cells at a density of 1 OD600 were boiled for 10 min and centrifuged briefly; the supernatants were used as templates. PCR reactions were performed in a Gene Amp PCR System 9600 apparatus (Applied Biosystems, PE Italia, Monta, Italy), in 100-µL volumes containing 5 µL of template, 100 µ M of each deoxynucleotide, 50 pmol of each primer, and 2 U of DynaZyme II Taq polymerase (Finnzyme, Oy, Finland). Each amplification was preceded by 5 min at 94°C and consisted of 35 cycles of 60 s at 94°C, 60 s at 54°C, and 60 s at 72°C, followed by a final 5-min period at 72°C.

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Results

From December 1996 to March 1999, 148 S. pneumoniae strains isolated from the CSF of patients with meningitis were sent to the Laboratory of Bacteriology and Medical Mycology of the Istituto Superiore di Sanità. Pneumococci were isolated at 47 hospitals in different areas of Italy. The strains represented approximately one-third of all pneumococcal meningeal isolates in the country, as estimated from the reported cases of pneumococcal meningitis in the same period [31]. The distributions of pneumococcal meningitis cases and of the strains obtained for this study in the different areas of Italy are shown in table 1. If 3 macroregions are considered, the distribution of strains appears to be similar to the distribution of reported cases of meningitis, although a slightly higher number of strains (compared with cases) was obtained from south Italy. This was mainly due to a higher number of strains obtained from the city of Naples.

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Table 1

Distribution of cases of pneumococcal meningitis and of strains among the different areas of Italy.

Ages were known for 141 patients; ages ranged from 2 months to 87 years, with a median of 47 years. Twenty-five patients were ⩽5 years old, and 50 were ⩾60 years old. Although the percentage of strains from young children was low (15%), compared with other studies [32], this reflected the percentage of cases in this age group in Italy [31]. There were 83 male patients and 65 female patients.

Distribution of serotypes

In total, 147 strains were serotyped, and 142 were assigned to a serotype or serogroup with the available antisera panel. Five strains were recognized only by the antisera pools, which indicates that they do not belong to serogroups included in the 23-valent vaccine. Nineteen different serogroups were identified; however, 12 serogroups accounted for 87% of the isolates (figure 1). The top-ranking 6 serogroups were 14 (11.6% of the strains), 23 (11.6%), 6 (11%), 4 (9.5%), 3 (8.8%), and 9 (7.5%). Overall, 96% of the strains examined belonged to serogroups included in the current 23-valent polysaccharide vaccine. The most frequent serotypes inside serogroups were also identified and are indicated in figure 1. A substantial portion of the strains (14%) belonged to serotypes that are not directly included in the polysaccharide vaccine (e.g., 6A, 23 other than F) but that are considered to be cross-reactive with the vaccine serotypes [33]. Serogroups 2 and 5 were not found among the isolates examined.

Figure 1
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Figure 1

Percentage distribution of serogroups, in order of frequency, among meningeal Streptococcus pneumoniae isolated in Italy. The most common serotypes inside serogroups are indicated inside the bars. Serotypes included in the current 23-valent polysaccharide antipneumococcal vaccine are shown (shaded bars). Serotypes not present in the vaccine are indicated (hatched bars).

In children ⩽5 years old, only 9 serotypes were found, the most frequent being 14 (28% of the strains), 6B (20%), and 23F (16%). Because of the low number of strains isolated from this age group, the relative frequency of the other serotypes identified (1, 4, 18C, 19A, 6A, and 7F), could not be reliably assessed. In elderly adults (⩾60 years old), the most frequent serotypes were 3 (16% of the strains), 8 (10%), 23 (10%), 4 (8%), and 19 (8%). Six other serogroups (6, 7, 10, 11, 12, and 18) were found at approximately the same frequency, representing 6%–4% of the isolates.

Antibiotic susceptibilities

Table 2 shows the distribution of the MICs for 7 antibiotics in invasive S. pneumoniae strains. Fourteen strains (9.5%) showed decreased susceptibility to penicillin (MIC ⩾0.12 µg/mL): 12 were intermediate, and only 2 strains (1.3%), with MICs of 2 µg/mL, were resistant according to the currently accepted criteria [27] (table 2). If the data are stratified per study periods, the penicillin resistance rate increased from 5% (3 of 59) in strains isolated from December 1996 to December 1997 to 13% (11 of 89) in strains isolated from January 1998 to March 1999. All but 1 of the strains were fully susceptible to ceftriaxone, the only exception being a penicillin-resistant strain, which showed intermediate susceptibility to ceftriaxone (MIC, 1 µg/mL). PRSP strains were isolated in different regions of Italy; however, half were from a single city (Naples) in south Italy (table 3).

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Table 2

Distribution of MICs in 148 strains of invasive Streptococcus pneumoniae from Italy.

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Table 3

Characteristics of 14 penicillin-intermediate and -resistant Streptococcus pneumoniae isolated in Italy from CSF samples during 1997–1999.

Forty-two strains (28.3%) were resistant to erythromycin. There was an apparent trend of increase in the resistance rate for this antibiotic as well, from 23.7% in 1996–1997 to 31.4% in 1998–1999. The distribution of MICs to erythromycin was not modal: the susceptible strains showed MICs ⩽0.25 µg/mL, whereas the resistant strains showed 2 clusters. One group, comprising 27% of the resistant strains, included strains with moderate-level resistance (MIC, 4–32 µg/mL); the other group included strains with high-level resistance (MIC ⩾256 µg/mL). The strains with high-level resistance were all resistant to clindamycin, and, by PCR, they were found to harbor the ermB gene. The strains with low-level resistance were susceptible to clindamycin and yielded an amplification product in the PCR assay for the gene mef, but not in the assay for the gene ermB (data not shown).

Thirty strains (20%) were resistant to clindamycin, with high MICs (⩾256 µg/mL); they corresponded to the strains showing high resistance to erythromycin and bearing the ermB gene, in accordance with the resistance phenotype MLSB. Seven strains (4.7% of the isolates) were resistant to chloramphenicol, and 28 (19%) were resistant to tetracycline. Two strains were intermediate to tetracycline. Of 138 strains tested for susceptibility to TMP-SMZ, 16 (11.6%) were resistant, and 26 (19%) were intermediate.

Of 14 PRSP isolates, 10 were multiresistant, being resistant to ⩾2 other classes of antibiotics (table 3). The most frequent pattern, present in 8 strains, was resistance to penicillin, erythromycin, clindamycin, and tetracycline. No one of the PRSP strains was resistant to chloramphenicol. All 7 chloramphenicol-resistant strains were multiresistant, being resistant also to erythromycin and tetracycline (data not shown).

Relationship between antibiotic resistance and serotypes

The PRSP strains belonged to 7 different serotypes (table 3). Four strains, including the 2 fully penicillin-resistant strains, belonged to serotype 9V. A penicillin-resistant 9V clone was responsible for the geographic spreading seen in several European countries, including Spain, France, and the United Kingdom [34, 35]. A strain from this international clone, originally isolated in the United Kingdom (provided by A. Efstriatou, Central Public Health Laboratory, London, UK), was examined concurrently with the Italian isolates by determining the SmaI and ApaI macrorestriction profiles. By use of either enzyme, the profiles of the Italian isolates were similar but not identical, showing a 1–3 band difference from each other (figure 2). According to currently accepted criteria [36], these strains can be considered to be clonally related. When compared to the 9V resistant strain from the United Kingdom, none of them appeared to be identical to it, as their profiles showed a 2–5-band difference with SmaI and a 4–7-band difference with ApaI (figure 2). Therefore, the Italian strains can be considered to be possibly related to the European clone [36].

Figure 2
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Figure 2

Pulsed-field gel electrophoresis of penicillin-resistant Streptococcus pneumoniae (PRSP) serotype 9V and serotype 24F. PRSP serotype 9V was digested with SmaI (lanes 1–5) or ApaI (lanes 6–10) before PFGE. Lanes 1 and 6, 9V from the United Kingdom. Lanes 2 and 7, PN60. Lanes 3 and 8, PN157. Lanes 4 and 9, PN144. Lanes 5 and 10, PN141. The 2 serotype 24F strains (lanes 11 and 12) were digested with SmaI. M, molecular mass marker.

Two PRSP strains, not typeable by the antisera used, appeared to be identical by PFGE (figure 2, lanes 11 and 12). They were referred to the Statens Seruminstitut of Copenhagen, where they both were identified as belonging to serotype 24F. The 2 strains were isolated from patients in the same city, several months apart.

The erythromycin-resistant strains belonged to various serogroups, the most common being 14 and 6: 88% and 62%, respectively, of the strains belonging to these serogroups were erythromycin resistant. The erythromycin-resistant type 14 strains showed very similar SmaI macrorestriction profiles, whereas a randomly chosen susceptible type 14 gave a distinct profile (figure 3). Of interest, strains with high MICs to erythromycin (⩾256 µg/mL) shared an identical pattern, slightly different from the patterns shown by strains with a low level of resistance (strains with MICs 4–32 µg/mL). The similar macrorestriction profiles imply that erythromycin-resistant type 14 strains belong to the same clone.

Figure 3
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Figure 3

Pulsed-field gel electrophoresis of erythromycin-resistant serotype 14 Streptococcus pneumoniae. Lane 1, erythromycin-susceptible strain. Lanes 2, 4, 6, 7, and 9, moderate-level erythromycin-resistant strains (MIC 4–32 µg/mL). Lanes 3, 5, and 8, high-level erythromycin-resistant strains (MIC ⩾256 µg/mL). M, molecular mass marker.

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Discussion

In S. pneumoniae, 90 different serological types, linked to different structures of the polysaccharide capsule, have been recognized. These types can be grouped into 46 serogroups, because of some degree of cross-reaction among type-specific rabbit antisera [37]. However, only a limited number of types are capable of producing serious or invasive diseases. These types are approximately the same worldwide, although their relative frequency can vary according to geographic area, age of patients, and type of disease and can fluctuate from year to year.

The distribution of serogroups found in our study is similar to that reported in other industrialized countries [32]. The top-ranking types are 14 and 23. Type 14 was reported to be on the rise in several European countries in recent years [11, 17] and also was the most prevalent in a meta-analysis of invasive strains from different areas of the globe [32]. Serogroup 23 is composed of different serotypes, among which 23F is the most common. If considered alone, serotype 23F ranks fifth in our study, after serogroups 14, 6, 4, and 3. The third most common serogroup was 6, comprising serotypes 6A and 6B, the latter being more common than the former in our country, as in other studies. Serogroup 1 was found to be the most common among invasive isolates in Germany and Denmark [16, 18] and among the most frequent serogroups in Spain and the United Kingdom [11, 38], but ranks only ninth in Italy. We never isolated strains belonging to serogroups 2 or 5, whose polysaccharides are included in the current 23-valent vaccine. Both serogroups are reported as very rare or absent in other recent surveys conducted in Western countries [32]. Overall, 96% of the strains belonged to types included or cross-reactive with the current pneumococcal polysaccharide vaccine.

The serotypes isolated from children <5 years old are more limited in number, similar to those seen in other Western countries [32]. Eighty percent belong to serotypes included in the 7-valent conjugate vaccines presently being tested in clinical trials [22]. Although the strains examined in our study are few, this rate is similar to the expected coverage for pneumococcal meningitis in infants in the United States [39].

The frequency of the serotypes isolated from adults ⩾60 years old is somehow different from that seen in the general population. The most common serotypes were 3 (16% of isolates), 8, 23, and 4. Serotype 14, which is one most commonly seen in the general population, was never isolated from patients in this age group. Overall, 94% of pneumococcal meningitis cases among patients ⩾60 years old were potentially preventable by the current 23-valent polysaccharide vaccine.

In our study, we confirmed that, in Italy, there is a moderate prevalence of PRSP strains, including among invasive isolates. The resistance rate to penicillin (9.5%) is much lower than that reported in neighboring countries, such as Spain and France [11,12–13], and resistance to third-generation cephalosporins is practically absent. In Italy, we also observed a low resistance rate to β-lactams in invasive strains of Haemophilus influenzae type b [28].

However, it is apparent that PRSP strains are on the rise in Italy, too. There were no PRSP strains among invasive strains isolated in 1980 [40]; in 1993 PRSP strains represented 5.4% of the isolates from different laboratories [23]; in 1996 in Genoa (north Italy), combined penicillin-intermediate and resistant strains accounted for 8.8% of the respiratory tract isolates [41]. In our study, we saw an increase in the prevalence of PRSP strains among meningeal isolates, from 5% in the first part of the study to 13% in 1998–1999. It is possible that we are witnessing a rapid evolution in penicillin resistance, similar to what has already happened in other southern European countries [19]. Moreover, it is possible that resistance is higher in certain areas of the country, as several PRSP strains were isolated in the city of Naples. This aspect deserves further investigation.

The low prevalence of PRSP strains in Italy has been ascribed to the different use of antibiotics, compared with that in other European countries. In 1992, Italy accounted for 20% of the total European antibiotic market, but it accounted for ∼50% of the market for injectable cephalosporins [42]. Parenteral cephalosporins were the first-line antibiotics used for the treatment of community-acquired lower respiratory tract infections in a survey conducted in 1993–1994 [43]. Because the use of injectable cephalosporins assures higher compliance and higher serum antibiotic levels, as opposed to oral broad-spectrum β-lactam drugs, it has been speculated that these conditions might limit the selection of strains with reduced susceptibility to penicillin and the development of fully resistant strains [23, 42]. Narrow-spectrum β-lactams are still often used in Germany and the United Kingdom, where the prevalence of PSRP strains appears to stay low [42], but not in Italy.

In our study, most PRSP strains showed an intermediate level of penicillin resistance; this suggests that resistance has developed independently in different strains. Accordingly, we did not find a prevalent widespread resistant clone. Four strains, including the 2 strains with high-level penicillin resistance, belong to serotype 9V. By molecular fingerprinting, they appeared different, but all showed some relatedness with the resistant 9V clone, which has geographically spread to several European countries [34, 35]. It is possible that the Italian strains are variant members of this same clone. The presence of the European 9V resistant clone in Italy has been reported elsewhere [44].

We observed 2 cases of meningitis in Naples due to a penicillin-resistant 24F clone. Serotype 24F, which is not included in the 23-valent vaccine, has been reported to be relatively common in meningitis strains in Denmark [18], but, to our knowledge, it has not been reported to be penicillin resistant to date. With regard to resistance to other antibiotics, the resistance rates to chloramphenicol and tetracycline appear to be intermediate between the low rates reported in Germany, the United Kingdom, and the United States (5%–11% for tetracycline and <2% for chloramphenicol) [7, 14, 16] and the very high rates reported in Spain and France (>30% for tetracycline and >25% for chloramphenicol) [11, 41]. The percentage of strains found to be nonsusceptible to TMP-SMZ was very high. This is in accordance with an increase in resistance to this drug, already reported in the United States and several European countries [7]. Data about susceptibility to TMP-SMZ obtained by E test should be regarded with caution, however, because we and others have demonstrated that they do not correlate entirely with susceptibility obtained by reference methods [26, 45].

The most remarkable finding in our study was the very high rate of resistance to erythromycin (30%). This resistance developed in just a few years: in the study by Marchese et al. [23], conducted in 1993, only 3.8% of the isolates were erythromycin resistant. In our study, we found a moderate increase in the resistance rate, from 24% during 1996–1997 to 31% during 1998–1999. In Italy, rapid development of erythromycin resistance in S. pneumoniae has paralleled a similar dramatic increase in Streptococcus pyogenes [46, 47]. An increase in macrolide resistance has been noted in various countries, although to a lower extent than in Italy, and especially in noninvasive isolates [11, 48]. A recent report, which compares the use of different kind of macrolides in several European countries, has linked the development of erythromycin resistance to the common use of new, long-acting macrolide drugs [49]. These drugs have been increasingly used in Italy in the last few years, especially for the treatment of upper respiratory tract infections [47, 50].

In our study, resistance to erythromycin was associated with serotype 6 and, mostly, with serotype 14, as already noted in the United Kingdom [34]. The predominant phenotype was that of high-level erythromycin resistance (MLSB), due to the presence of the ermB determinant, but almost one third of the resistant strains carried the efflux gene mef, which produces moderate-level resistance. The MLSB phenotype was often associated with resistance to tetracycline, which suggests that these resistant traits are carried by common genetic elements, such as transposons [51]. A further association with chloramphenicol resistance was present in strains belonging to serotype 6. This same pattern of multiresistance (erythromycin, tetracycline, and chloramphenicol, with or without resistance to TMP-SMZ, but with susceptibility to penicillin) was found to be characteristic of serotype 6 strains isolated from the nasopharynx of healthy children in central Italy [52]. Therefore, the diffusion of erythromycin resistance in Italy appears to be due to a combination of the spreading of a particular clone, as in the case of serotype 14 or multiresistant serotype 6, and the diffusion of different antibiotic resistance determinants among the isolates.

In conclusion, the situation of antibiotic resistance in S. pneumoniae in Italy appears to be in evolution: penicillin resistance is moderate, although on the rise, whereas erythromycin resistance has already reached one of the highest rates in Europe. This pattern of antibiotic resistance is different from that of the other European and Western countries and probably correlates with a different pattern of use of antibiotics in Italy.

In spite of the obvious limitations of our study, which includes only meningeal isolates, we have gained some insight on the serotypes and susceptibility of invasive S. pneumoniae strains in Italy. As meningitis is one of the most dreadful and therapeutically problematic pneumococcal diseases and is potentially preventable by vaccination, it is extremely important to provide up-to-date information that can be used by countries' medical personnel as they consider treatment and prevention.

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Acknowledgments

We thank all the participants in the Italian National Surveillance System of Bacterial Meningitis, for providing strains and information, and J. Henrichsen (Staten Seruminstitut, Copenhagen), for serotyping the 24F strains.

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Footnotes

  • Financial support: This study was supported in part by a grant from Ministero della Sanità, Programma per la Ricerca Finalizzata 1998.

  • Received December 13, 1999.
  • Revision received May 4, 2000.
  • Accepted November 29, 2000.
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  1. Clin Infect Dis. (2000) 31 (6): 1373-1379. doi: 10.1086/317502
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