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Antimicrobial Agents and Chemotherapy, June 2000, p. 1575-1577, Vol. 44, No. 6
Institut National de la Santé et de la
Recherche Médicale, EMI-U 9933, Hôpital Bichat-Claude
Bernard, 75877 Paris Cedex 18, France
Received 26 July 1999/Returned for modification 26 November
1999/Accepted 17 March 2000
We examined the relationship between penicillin susceptibility,
peritoneal virulence in Swiss mice, and capsular type in a selection of
122 clinical Streptococcus pneumoniae isolates belonging to
24 serotypes. Regardless of the serotype, all 32 virulent strains were susceptible to penicillin, and all 41 strains with diminished susceptibility or resistance to penicillin were avirulent. The remaining 49 strains were both susceptible to penicillin
and avirulent, irrespective of the serotype. On the basis of their
capsular type and pathogenic behavior, strains fell into one of four
groups. In the group consisting of serotypes 1, 3, and 4 (n = 16), strains were predominantly
virulent (81.3%), and all were penicillin susceptible. In the serotype
6 group (n = 32), the frequency of virulence was significantly lower (34.4 versus 81.3%, P = 0.002),
and strains were predominantly penicillin susceptible (71.9%). In the
group composed of serotypes 9, 14, 19, and 23 (n = 50), all strains were avirulent, and 56% had decreased susceptibility
(n = 12) or resistance to (n = 16)
penicillin. The fourth group was heterogenous, as it pooled 24 strains
of 15 different serotypes; in this group the frequency of virulence was
33.3%, and strains were predominantly penicillin susceptible (83.3%).
These data point to a complex relationship between penicillin
susceptibility and virulence in mice but do not entirely separate these
characteristics from the role of the capsular type. The possibility
that the mechanisms conferring penicillin resistance are related to
those leading to a loss of virulence is supported by these findings.
Streptococcus pneumoniae
remains a leading cause of human morbidity and mortality
worldwide (14, 19, 23). Antimicrobial agents have been used
successfully to treat infections by this pathogen for over six decades,
but genes expressing resistance to these agents have emerged and
disseminated, interfering seriously with antimicrobial therapy. In the
last two decades, and more rapidly in the last 7 years, the global
incidence of strains displaying resistance to penicillin and other
Epidemiological, clinical, and preliminary laboratory investigations of
pneumococcal infection have suggested a relationship between penicillin
susceptibility and capsular type (6, 10, 13, 17, 20, 27) and
between serotype and virulence (6, 9). However, penicillin
resistance is less common in blood isolates than in isolates recovered
from other sites (16), and pneumonia caused in adults by
penicillin-nonsusceptible pneumococci has been linked to milder
clinical manifestations (12). Experimental pneumococcal
pneumonia cannot be induced in immunocompetent mice by inoculation with
wild resistant strains, independent of serotype (4). These
data, and others suggesting that invasive strains are largely
penicillin susceptible while less invasive strains are largely
penicillin nonsusceptible (7, 20, 21, 22, 26), point to a
correlation between penicillin susceptibility and virulence.
By studying a collection of 122 well-characterized clinical isolates of
S. pneumoniae belonging to 24 serotypes, we investigated the
relationship between penicillin susceptibility, virulence in
mice, and capsular type.
S. pneumoniae.
The 122 S. pneumoniae study
strains were isolated in France between 1991 and 1993 from patients 15 to 90 years old with infections thought to be caused by pneumococci.
Most strains (105 of 122) came from the Microbiological Laboratory of
Bichat-Claude Bernard Hospital, a 1,200-bed tertiary university
hospital in Paris, and the rest were kindly provided by P. Geslin,
Centre de Référence du Pneumocoque, Créteil, France.
Isolates cultured from blood (n = 45), cerebrospinal
fluid (n = 8), and the lower respiratory tract
(n = 33) were clinically defined as invasive; the
others, originated from the upper respiratory tract (n = 14) and middle ear (n = 19), were defined as
noninvasive; and the last three, originated from lymph node, vagina,
and knee infections, were also defined as noninvasive. None of the
strains in this selected collection was considered a colonizing strain.
Serotype.
All strains were tested by duplicate slide
agglutination assays with capsular type-specific antisera (P. Geslin).
Susceptibility testing.
MICs were measured by the
microdilution method in Mueller-Hinton broth supplemented with 5%
sterile horse serum (24) (Diagnostics Pasteur,
Marnes-la-Coquette, France). The MIC was defined as the lowest
antibiotic concentration that inhibited all visible growth after
18 h of incubation at 37°C. Penicillin susceptibility was defined according to current breakpoints (25), i.e.,
susceptibility, MIC Experimental virulence.
Female Swiss mice weighing 20 to
22 g (Iffa-Credo Laboratories, l'Arbresle, France) were used in
virulence assays as previously described (5). In brief,
pneumococcal strains were grown at 37°C in brain-heart infusion broth
(bioMérieux, Marcy l'Etoile, France) supplemented with 5%
sterile horse serum. Mice were infected intraperitoneally with serial
10-fold dilutions of log-phase cultures in a volume of 0.5 ml per
mouse. Groups of five mice received 103 to 108
CFU/mouse. Mortality was assessed daily, and the death rates 7 days
postinfection were used to calculate the 100% lethal dose (LD100). As previously defined (5), a strain was
considered virulent when an intraperitoneal inoculum of
Statistical analysis.
Group percentages were compared by
using Fisher's exact Chi2 test. Regression analysis was
performed between log LD100 and log MIC. P
values of 0.05 or less were considered significant.
The 122 clinical strains were divided into four groups on the
basis of previous studies (6, 9) on pneumococcal strain behavior in experimental virulence models. The four groups comprised serotypes 1, 3, and 4, mainly virulent; serotype 6, both virulent and
avirulent; serotypes 9, 14, 19, and 23, all avirulent; and serotypes 7 to 38, mainly avirulent. In the four groups, the respective proportions
of clinically invasive strains were 93.8, 62.5, 66.0, and 75.0%.
Strains of invasive origin were more frequently virulent than
noninvasive strains (29.1 versus 13.9%), but this difference was not
statistically significant (P = 0.106). However, the
frequency of virulence among the 53 strains originating from blood or
cerebrospinal fluid was significantly higher than among all other
strains (35.8 versus 15.9%, P = 0.0187).
Among the 122 clinical isolates of S. pneumoniae, regardless
of the serotype, penicillin-susceptible strains (MIC
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Relationship between Capsular Type, Penicillin Susceptibility,
and Virulence of Human Streptococcus pneumoniae Isolates
in Mice
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
-lactams and multiresistance has increased alarmingly (1, 2,
11, 15, 16, 21).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
0.06 µg/ml; decreased susceptibility,
0.12 < MIC 1 µg/ml; and resistance, MIC 
2 µg/ml.
105 CFU killed 100% of mice within 1 week. A strain was
considered avirulent when the intraperitoneal LD100 was
>105 CFU. With the avirulent strains, lethal pneumonia was
only consistently induced by 107 CFU intratracheally in
leukopenic mice.
RESULTS AND DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
0.06) were
either virulent (LD100 105) or
avirulent (LD100 > 105), but all
nonsusceptible strains were avirulent (Fig.
1). By analyzing the overall serotypes, a
positive significant correlation (P < 0.0001) was
found between log MIC and log LD100. When the relationship
was analyzed individually for the most frequent serotypes, i.e., 9 (n = 13), 14 (n = 8), 19 (n = 10), and 23 (n = 19), no significant relation
was found, although there was a trend with serotype 14 (P = 0.054). Conversely, with serotype 6, a negative correlation
between penicillin resistance and virulence was shown to be significant
(P = 0.0017).
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FIG. 1.
Relationship between virulence in mice and
susceptibility to penicillin for 122 clinical S. pneumoniae
strains of various serotypes.
As shown in Fig. 2, 81.3% of strains
belonging to serotypes 1, 3, and 4 (all of which were susceptible to
penicillin) were virulent. However, these virulent isolates clustered
in the serotypes that contained no resistant strains, meaning that no
role of resistance could be inferred in this particular group.
In serotype 6 (71.9% susceptible), the proportion of
virulent strains was significantly lower (34.4 versus 81.3%,
P = 0.002). Serotype 6 was particular, as the virulent
strains (34.4%) were all penicillin susceptible, whereas the avirulent
strains (65.6%) were either susceptible (37.5%) or resistant
(28.1%). On the other hand, serotypes 9, 14, 19, and 23 were all
avirulent, whether they were penicillin susceptible (44%),
intermediate (24%), or resistant (32%), indicating that the primary
determinant of decreased virulence might be the serotype rather than
acquired penicillin resistance.
|
, avirulent; V+, virulent; S, I,
R, penicillin susceptible, intermediate, and resistant, respectively.
The potential reason for the clustering of less virulent strains among serotypes 9, 14, 19, and 23 is unclear. For instance, less-pathogenic serotypes might be more prone to long-term colonization and thus more prone to multiple antibiotic exposure. In our study, the virulent phenotype was 100% predictive of penicillin susceptibility, and the penicillin-resistant phenotype was never associated with virulence. Björkman et al. (8) studied the impact of acquired resistance to streptomycin or rifampin of Salmonella enterica serovar Typhimurium in mice and found that most resistant mutants were less virulent than their wild counterparts. Our experimental results support the clinical study by Einarsson et al. (12), which suggested that adult pneumonia caused by penicillin-nonsusceptible pneumococci was associated with milder clinical manifestations than that caused by penicillin-susceptible pneumococci, indicating either that resistance "carries a cost" or that the serotypes of penicillin-nonsusceptible pneumococci are less virulent. Moreover, Austrian (3) and Knecht et al. (18) reported a correlation between the pathogenicity of certain serotypes in humans and their virulence in rodents. Type 3 bacteremic pneumococcal pneumonia treated with penicillin has a case fatality rate of 50%, and 1 CFU may cause lethal infection in mice or rats. By contrast, type 37 pneumococci are rarely pathogenic in humans, and 107 organisms are required to cause lethal infection in rodents. Type 3 (capsule composed of glucose and glucuronic acid) is among the most invasive type and is associated with a poor prognosis, whereas type 37 (homopolymeric glucose capsule) is rarely associated with infection.
In conclusion, the relationship between susceptibility, serotype, and virulence is complex. The serotype appears to be the primary determinant of virulence and resistance, but none of the strains studied here exhibited both decreased susceptibility and virulence. In our preliminary experiments (Azoulay-Dupuis et al., 37th Intersci. Conf. Antimicrob. Agents Chemother., abstr. C35, 1997), the genetic transformation of a virulent-susceptible serotype 6 strain with genomic DNA from a penicillin-resistant isolate led to resistance and decreased virulence at the same time. Further investigations are needed to establish the precise relationship between susceptibility, serotype, and virulence in S. pneumoniae and to establish the underlying genetic mechanisms involved in each phenomenon.
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FOOTNOTES |
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* Corresponding author. Mailing address: INSERM EMI-U 9933, Bâtiment U13, Hôpital Bichat-Claude Bernard, 170 Bd Ney, 75877 Paris Cedex 18, France. Phone: 33 1 40 25 86 08. Fax: 33 1 40 25 86 02. E-mail: eazoulay{at}bichat.inserm.fr.
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Antimicrobial Agents and Chemotherapy, June 2000, p. 1575-1577, Vol. 44, No. 6
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