Determining the Frequency of Resistance of Streptococcus pneumoniae to Ciprofloxacin, Levofloxacin, Trovafloxacin, Grepafloxacin, and Gemifloxacin

Newer fluoroquinolones have good activity against Streptococcus pneumoniae and may be useful clinically for the treatment of pneumonia. Although resistance among Streptococcus pneumoniae has been reported, it is rare. The frequency of single-step resistance and the emergence of resistance were compared in serial transfer of 49 clinical isolates of penicillin-sensitive and -resistant Streptococcus pneumoniae to ciprofloxacin, levofloxacin, trovafloxacin, grepafloxacin, and gemifloxacin. Single-step resistance frequencies to four times the minimum inhibitory concentration were 2.73×10^-6 (±8.46×10^-6) for ciprofloxacin, 1.78×10^-7 (±4.62×10^-7) for trovafloxacin, 5.45×10^-7 (±1.24×10^-6) for grepafloxacin, 6.78×10^-7 (±1.38×10^-6) for gemifloxacin, and 9.23×10^-8 (±4.47×10^-7) for levofloxacin. In serial transfer experiments, all isolates became resistant to clinically relevant levels of all fluoroquinolones after eight passages. The resistance occurred most rapidly with ciprofloxacin followed by grepafloxacin, gemifloxacin, trovafloxacin, and levofloxacin. These results show that strains with decreased susceptibility to fluoroquinolones occur frequently in cultures of Streptococcus pneumoniae, and this organism can readily become resistant to clinically relevant concentrations of fluoroquinolones in vitro. Electronic Publication     

In Vitro Activity of Gemifloxacin (SB-265805) Compared to Eleven Other Antimicrobial Agents Against Streptococcal Isolates, Excluding Streptococcus pneumoniae

 The purpose of the study presented here was to determine the in vitro activity of gemifloxacin compared with that of 11 other antimicrobial agents (5 of them quinolones) against 400 isolates of β-haemolytic and viridans group streptococci. The minimum inhibitory concentration values for gemifloxacin against 90% of the streptococci tested were as follows: Lancefield groups A, C and G, 0.06 μg/ml; Lancefield group B, Streptococcus mitis, Streptococcus mutans and Streptococcus bovis, 0.125 μg/ml; and Streptococcus milleri, 0.03 μg/ml. Resistance to penicillin, ampicillin and erythromycin was found mainly in the Streptococcus mitis isolates; tetracycline showed variable results, and no vancomycin resistance was encountered. Higher rates of ciprofloxacin resistance were identified in the Streptococcus bovis, mitis and mutans isolates. In conclusion, gemifloxacin was the most active quinolone tested followed by trovafloxacin, sparfloxacin, grepafloxacin, ciprofloxacin and levofloxacin, especially against isolates resistant to β-lactam agents, macrolides and tetracycline.     

Intermediate Susceptibility to Ciprofloxacin in Salmonella typhi Strains in India

European Journal of Clinical Microbiology &; Infectious Diseases -     

Characterization of erythromycin-resistant Streptococcus pneumoniae in Korea, and In Vitro activity of telithromycin against erythromycin-resistant Streptococcus pneumoniae

To characterize the phenotypes and genotypes of erythromycin-resistant clinical isolates of Streptococcus pneumoniae in Korea and to evaluate the in vitro activity of telithromycin against these erythromycin-resistant isolates, we tested a total of 676 isolates of S. pneumoniae collected from 1997 to 2002 in a tertiary hospital in Seoul, Republic of Korea. MICs for erythromycin and telithromycin were determined by the agar dilution method. The macrolide resistance phenotypes of erythromycin-resistant isolates were determined by the erythromycin- clindamycin-rokitamycin triple disk (ECRTD) and MIC induction tests, whereas their macrolide resistance genotypes were determined by PCR for the erm(B), erm(A), subclass erm(TR), and mef genes. To discriminate between mef(A) and mef(E), PCR-restriction fragment length polymorphism (RFLP) analyses were performed. Of the 676 S. pneumoniae isolates, 459 (67.9%) were resistant to erythromycin. Of the 459 erythromycin-resistant isolates, 343 (74.7%) were assigned to the cMLS phenotype, 48 (10.4%) to the iMcLS phenotype, 4 (0.9%) to the iMLS phenotype, and 64 (14.0%) to the M phenotype. The erm(B) gene was detected in 251 (54.6%) isolates, the mef gene was detected in 64 (14.0%), and both the erm(B) and mef genes were detected in 144 (31.4%) isolates. All of the mef genes detected were identified as mef(E). Of the 459 erythromycin- resistant isolates, all but one were susceptible to telithromycin. The MIC(50)/MIC(90) to telithromycin of isolates carrying erm(B), mef(E), and both genes was 0.06/0.5 microg/ml, 0.03/0.125 microg/ml, and 0.5/1.0 microg/ml, respectively. Although the MICs of telithromycin for the erythromycin-resistant isolates varied according to genotype, telithromycin was very active against these erythromycin-resistant S. pneumoniae.     

In Vitro Plaque-Forming Cell Responses Induced by Streptococcus pneumoniae in Humans

When peripheral blood lymphocytes (PBL) were stimulated in vitro with the rough form of type 2 Streptococcus pneumoniae R36a, the resulting plaque-forming cells (PFC) did not produce antibodies directed against phosphorylcholine, a major antigenic determinant of the cell wall C-polysaccharide. Instead, R36a stimulated polyclonal PFC in PBL and splenic lymphocytes. We compared the polyclonal responses stimulated by R36a with those induced by two well-characterized polyclonal activators (PA), Staphylococcus aureus Cowan I and pokeweed mitogen (PWM). We found that R36a was a poor mitogen for PBL, whereas the other two PA were potent mitogens; that the predominant isotype produced in response to all three PA was IgM; that adherent cells strongly inhibited the polyclonal PFC response to both R36a and Staph. aureus but not PWM; and that T cells were necessary for induction of polyclonal antibody-secreting cells by all three stimuli.     

Quality Control Limits for Dilution and Disk Diffusion Susceptibility Tests of Trovafloxacin against Eight Quality Control Strains

A 10-laboratory collaborative effort was designed to generate data to propose quality control limits for susceptibility tests of trovafloxacin. Broth microdilution, agar dilution, and disk diffusion tests were evaluated with eight different control strains. All tests were reproducible, and control limits are proposed.     

In Vitro Response of Human Lymphocytes to Mycoplasma pneumoniae

In vitro culture and stimulation of human peripheral lymphocytes were employed to investigate the role of cellular immunity in Mycoplasma pneumoniae disease. Subjects with documented natural infections served as donors. The lymphocyte response to whole M. pneumoniae organisms was determined as incorporation of tritiated thymidine in a semimicro culture system. The range of cellular reactivity stimulated by specific antigen was within the range stimulated by phytohemagglutinin. The difference between responses of subjects with documented infection and serologically negative controls was highly significant. Specific reactivity of peripheral lymphocytes correlated closely with the presence of serum growth-inhibiting antibodies, and both persisted for several years following infection. Serum complement-fixing titers correlated well with lymphocyte stimulability during the first year but antibody, as measured by this technique, tended to disappear in later convalescence. In light of previous studies, which revealed a lack of correlation between humoral antibodies and resistance to reinfection, these results suggest that immunity to M. pneumoniae infection is mediated by circulating small lymphocytes.     

Evaluation of the Dade MicroScan MICroSTREP Antimicrobial Susceptibility Testing Panel with Selected Streptococcus pneumoniae Challenge Strains and Recent Clinical Isolates

The MicroScan MICroSTREP panel is a recently marketed frozen broth microdilution panel for susceptibility testing of various streptococci, including Streptococcus pneumoniae. The panel contains 10 antimicrobial agents in cation-adjusted Mueller-Hinton broth supplemented with 3% lysed horse blood, similar in concept to the National Committee for Clinical Laboratory Standards (NCCLS) reference broth microdilution method for testing streptococci. A group of 210 isolates of S. pneumoniae were selected to include isolates with previously documented resistance to agents incorporated in the MICroSTREP panel, as well as recent invasive clinical isolates. All isolates were tested simultaneously with the MICroSTREP panel and an NCCLS reference panel whose drug concentrations were prepared to coincide with those of the MICroSTREP panel. Of the 210 isolates, 5 failed to grow in the MICroSTREP panel; 3 of those also did not grow in the reference panel. Essential agreement of MICs determined by the two methods (test MIC ± one dilution of the reference MIC) was 99.6% overall and ranged from 98.0% with chloramphenicol to 100% with penicillin, ceftriaxone, erythromycin, tetracycline, and vancomycin. There were no very major or major interpretive category errors resulting from the MICroSTREP panel tests. Minor interpretive category errors ranged from 12.2% with cefotaxime and 9.8% with ceftriaxone (due mainly to clustering of MICs for the selected strains near the breakpoints) to 0% with chloramphenicol and vancomycin. These results indicate that the MicroScan MICroSTREP frozen panels provide susceptibility results with pneumococci that are essentially equivalent to results derived by the NCCLS reference broth microdilution procedure.     

In Vitro Antimicrobial Susceptibility of Aerococcus urinae

Aerococcus urinae may cause urinary tract infections, bacteremia, and endocarditis. No standardized susceptibility test methods or interpretive criteria have been proposed for this organism. This study reports the MIC results for 128 A. urinae isolates tested by broth microdilution. The isolates had low MICs to amoxicillin, cefotaxime, ceftriaxone, doxycycline, linezolid, meropenem, penicillin, rifampin, tetracycline, trimethoprim-sulfamethoxazole, and vancomycin. However, 55% of the isolates had MICs to clindamycin of >0.25 μg/ml, 44% had MICs to erythromycin of >0.25 μg/ml, and 16% had MICs to levofloxacin of >2 μg/ml.     

Serotype-Related Variation in Susceptibility to Complement Deposition and Opsonophagocytosis among Clinical Isolates of Streptococcus pneumoniae

The polysaccharide capsule is a major virulence factor of Streptococcus pneumoniae; it affects complement resistance and shields the bacterium from phagocytes. Certain capsular serotypes appear to be better able to cause invasive disease than others. Serotypes 1 and 5 are common causes of invasive disease but are rarely isolated from healthy carriers, whereas serotypes 6B and 23F are more frequently isolated from carriage than invasive disease. We have recently shown that serotypes 6B and 19F differ in resistance to complement C3 deposition and opsonophagocytic killing. In this study we assessed the complement resistance and susceptibility to opsonophagocytosis of several other serotypes targeted by the pneumococcal conjugate vaccines. Clinical isolates of serotypes 1, 4, 5, 14, 18C, and 23F were tested along reference strains of corresponding capsular types. The concentration of anticapsular antibodies required for opsonophagocytic killing correlated inversely with C3 deposition on the serotype. Serotype 1 was the most resistant of the clinical isolates to C3 deposition and, along with serotypes 5 and 19F, required the highest concentration of capsule antibodies for opsonophagocytic killing, whereas serotype 23F was the most sensitive to opsonophagocytosis. Sensitivity to C3 deposition and opsonophagocytosis was associated with serotype-specific mortality of invasive pneumococcal disease, suggesting that the primary pathogens, such as serotypes 1 and 5, are more resistant to complement and require a higher concentration of capsule antibodies for opsonophagocytic killing than the opportunistic serotypes such as 6B and 23F, which are associated with a more severe disease outcome.Streptococcus pneumoniae colonizes the nasopharynx (NP) of healthy individuals but occasionally breaks from its carriage habitat and causes diseases ranging from acute otitis media to more severe diseases such as pneumonia, sepsis, and meningitis. Isolates that lack the capsule rarely cause invasive disease in humans, and loss of the capsule greatly attenuates virulence in animal models of disease (31, 54). Each of the more than 90 known pneumococcal serotypes produces a biochemically distinct polysaccharide structure, which is a major determinant of pneumococcal virulence since only a small number of serotypes account for the majority of infections (18). The polysaccharide capsule shields the bacterium from phagocytosis by inhibiting recognition of opsonins adhered to the bacterial cell wall.Certain serotypes are relatively common causes of invasive pneumococcal disease (IPD) with respect to their prevalence in carriage, whereas others are common colonizers of the NP but rarely cause disease (6, 16). The disease potential, or relative invasiveness, is a measure of the ability of pneumococci to progress from nasopharyngeal carriage to invasive disease in humans, similar to the attack rate, which is the risk of disease as a result of pathogen acquisition (6, 50). Most of the invasive property of pneumococci seems to be determined by their capsular serotype rather than genetic background. However, different clones of the same serotype can vary in an ability to cause IPD (6, 16, 45).Variation in disease potential among serotypes has been reported in a number of epidemiological studies. In young children in England, the relative invasiveness of more prevalent carriage serotypes—6B, 19F, and 23F—was low compared to the high disease potential of serotypes 1, 4, 14, 18C, and 7F (6). In another United Kingdom study in infants <2 years old, serotypes 23F, 6A, 19F, 16F, 6B, and 15B/C were associated with low attack rates; serotypes 4, 14, 7F, 9V, and 18C were associated with relatively high attack rates; and serotypes 1, 5, and 9A were only isolated from IPD (50). In a study of Finnish children <2 years of age serotypes 19F and 23F showed a tendency to be more common in carriage and serotypes 14, 18C, 19A, and 6B were significantly more common in IPD (16). Even in populations with a high proportion of disease caused by serotypes 1 and 5, such as in the Gambia (2), serotypes 1 and 5 are rarely detected in nasopharyngeal carriage (19, 29, 47). In a meta-analysis of seven data sets, serogroups 1, 5, and 7 had the highest invasive disease potential (7).The duration of carriage varies by capsular type and is inversely correlated with the attack rate (50). Capsular serotypes carried for a short duration and with a high attack rate, such as serotypes 1, 5, and 7F, behave like “primary pathogens,” which affect previously healthy individuals and are associated with lower mortality. Meanwhile, serotypes that are carried for a long duration, such as 6B, 19F, and 23F, behave like opportunistic pathogens causing disease in patients with an underlying disease and are associated with a more severe disease and higher mortality (49). This observation correlates with the outcome of analysis of the largest ever population-based study on the severity of IPD published recently by Harboe et al. Odds ratios (ORs) for the death as an outcome of IPD were higher for opportunistic serotypes compared to invasive capsular types (17).Comparison of pneumococcal strains isolated from carriage and IPD suggests that carriage isolates are more heterogeneous and invasiveness is associated with clonality (6, 42). Isolates of serogroups 7 and 14 are clonal and rare among carriers, whereas isolates of serogroups 6, 19, and 23 are heterogeneous, suggesting that the clonality may represent an advantage for invasiveness (51). Molecular epidemiology studies reveal that IPD caused by serotype 1 in the Gambia (3) and serotypes 1 and 5 in Israel (37) resulted from expansion of single, virulent clones, in contrast to serotypes 6B and 23F, which showed a large diversity in their genotypic characteristics (37). Studies of serotype 1 disease cases in different geographic regions did not identify clones with distinct virulence properties (8), which could reflect either a high virulence potential of the serotype 1 capsule or selection of genotypes advantageous for invasiveness.Variations in susceptibility to host immune defense mechanisms can contribute to the differences between capsular serotypes in invasiveness. Immune response to pneumococcus strongly depends on opsonization of the bacteria with complement C3 molecules (C3b and iC3b), the deposition of which may be influenced by the capsular polysaccharides (20). We have previously shown that significantly more C3 is deposited on serotype 6B than 19F isolates and that a significantly higher concentration of polysaccharide-specific antibodies is required for opsonophagocytic killing of serotype 19F isolates (33). The fact that less C3 was deposited on serotype 19F pneumococci in the presence or absence of antibodies suggests innate differences between capsular serotypes in resistance to the complement. Comparison of C3 deposition on capsule-switched mutants of TIGR4 demonstrated large differences between serotypes (22), which indicates that the capsular serotype alone can have a significant impact on the complement resistance of strains.Weinberger et al. described recently an association between serotype prevalence in carriage and the amount of capsular polysaccharide produced in vitro by isolates of a particular serotype (55). The association linked capsule chemistry to the success of the serotype as a colonizer. This observation suggested that a similar correlation can be present between capsule and the invasiveness of the serotype. In the present study we compared the resistance to complement and opsonophagocytosis of clinical isolates of several different pneumococcal serotypes. Each serotype was represented by multiple different clones. Isolates from blood or cerebrospinal fluid, from serotypes 1, 4, 5, 14, 18C, and 23F, as well as mucosal isolates from serotypes 1, 14, and 23F, were analyzed, along with reference strains used in the standard opsonophagocytic assay. We analyzed the results in a context of the serotype-specific severity of invasive disease and the chemical structure of the polysaccharides.     

Use of Amplified Fragment Length Polymorphism To Identify 42 Cladophialophora Strains Related to Cerebral Phaeohyphomycosis with In Vitro Antifungal Susceptibility

The amplified fragment length polymorphism technique has been applied to identify neurotropic chaetothyrialean black yeasts and relatives from clinical sources. Cladophialophora bantiana, C. emmonsii, C. arxii, C. devriesii, and C. modesta, previously identified on the basis of sequencing and phenotypic and physiological criteria, were confirmed by cluster analysis, demonstrating the clear separation of C. bantiana as a rather homogeneous group from the other species. C. bantiana is a neurotropic fungus causing cerebral abscesses with a mortality of up to 70%. Successful therapy consists of neurosurgical intervention and optimal antifungal therapy. Since the latter is not clearly defined in a large series, we tested the in vitro activities of eight antifungal drugs against clinical isolates of C. bantiana (n = 37), C. modesta (n = 2), C. arxii (n = 1), C. emmonsii (n = 1), and C. devriesii (n = 1), all of which had caused invasive infections. The resulting MIC₉₀s for all neurotropic C. bantiana strains were as follows, in increasing order: posaconazole, 0.125 μg/ml; itraconazole, 0.125 μg/ml; isavuconazole, 0.5 μg/ml; amphotericin B, 1 μg/ml; voriconazole, 2 μg/ml; anidulafungin, 2 μg/ml; caspofungin, 4 μg/ml; and fluconazole, 64 μg/ml. On the basis of these in vitro results and the findings of previous clinical and animal studies, posaconazole seems to be a good alternative to the standard treatment, amphotericin B, for C. bantiana cerebral infections. The new agent isavuconazole, which is also available as an intravenous preparation, has adequate activity against C. bantiana.The genus Cladophialophora represents anamorph members of the ascomycetes in the order Chaetothyriales in the family Herpotrichiellaceae comprising the black yeasts and relatives (10). These dematiaceous fungi are normally associated with soil or vegetative matter; however, they are increasingly being seen as causative agents of mycoses in humans (27, 37, 48) and domestic (14, 23) and wild (29) animals. Cladophialophora carrionii is the type species and an agent of chromoblastomycosis, a cutaneous and subcutaneous disease. The genus Cladophialophora encompasses several other clinically significant species which are potentially able to cause severe fungal infections in otherwise immunocompetent patients. In human infections, the brain is frequently involved (27, 37, 48). Within the genus, the majority of brain abscesses with fatal outcomes are associated with Cladophialophora bantiana (formerly Cladosporium bantianum, Cladosporium trichoides, Cladosporium trichoides var. chlamydosporum, Torula bantiana, and Xylohypha bantiana), a neurotropic fungus, although severe phaeohyphomycotic infections are also caused by novel Cladophialophora species like C. modesta (40, 44), C. arxii (53, 56), C. emmonsii (Xylohypha emmonsii) (45), C. devriesii (22, 28, 42) C. saturnica (4), and C. boppii (34). Moreover, Exophiala dermatitidis and Rhinocladiella mackenziei, other members of the black yeast group, are also frequently isolated from cerebral infections (8, 27, 37). Central nervous system infection due to C. bantiana is reported worldwide, though a general preference for warmer climates with high humidity is apparent (27). Indeed, many cases are reported from India (19, 30, 33, 55), as opposed to arid climatic zones (8). The first case of C. bantiana (Cladosporium trichoides) infection was reported in 1952, when the fungus was isolated from a human brain abscess and was demonstrated to be neurotropic in laboratory animals (6). A review of 17 cases of brain abscess, published in the English language literature by the mid-1970s, reported that the majority of patients had no underlying disease (41). The most recent series of 48 patients with brain abscess due to C. bantiana showed that 35 patients (72%) had no risk factors and that only 13 patients (28%) survived the infection, despite combined surgical and antifungal treatment (48). The minority of immunocompromised patients are transplant recipients, intravenous drug abusers, or individuals on steroids (2, 13, 15, 24, 31, 35, 36, 51, 54, 57, 59).The mode of infection is either by hematogenous spread from an unrecognized pulmonary focus, through direct extension from adjacent paranasal sinuses, or by penetrating trauma to the head. However, the majority of patients had had no recent evidence of pulmonary or sinus infections (27). Cladophialophora species are prone to identification problems (3, 20). Due to the high degree of phenotypic similarity between recently described new Cladophialophora species and C. bantiana, identification problems are imminent. For most cases published in the older literature, identification down to the species level cannot be repeated or confirmed by molecular methods due to the absence of the original isolates; hence, the etiological agent described in older publications may often have been misidentified. It is now well established that molecular identification methods, which have driven new developments in fungal taxonomy, are more reliable than classical morphological methods (3, 20). Amplified fragment length polymorphism (AFLP) is a technique based on the detection of genomic restriction fragments by PCR amplification, which can be used with the DNA of any organism (58). The purpose of this study was to study the inter- and intraspecific genomic variations of 42 Cladophialophora isolates stored in the CBS collection and recovered from cases with cerebral phaeohyphomycosis and other infections. Antifungal therapy is mainly based on the experience gathered from and published in isolated case reports and mostly involved amphotericin B, itraconazole, and flucytosine singly or in different combinations (48). Animal studies (1) and human experience suggested that amphotericin B has no value in treating cerebral infections, probably due to poor penetration of the central nervous system (CNS), but that triazoles might be of value (26, 38). A recent study of antifungal therapy in a murine model of disseminated infection by C. bantiana confirmed the poor activity of amphotericin B but found that posaconazole and flucytosine extended survival (39). This suggests that new antifungal drugs with broad-spectrum activity and suitable pharmacokinetic profiles compared with those of conventional antifungal agents might be more effective against C. bantiana (1, 39). Only limited data on in vitro antifungal activities against the neurotropic fungus C. bantiana are available. Therefore, with no standard therapy available, unfavorable results in animal experiments, and only a small published series of susceptibility testing with itraconazole and voriconazole (47), the second objective of this study was in vitro testing of this large collection of Cladophialophora strains for their susceptibilities to eight antifungal drugs, including the new triazole isavuconazole.(Part of this work was presented as a poster at Trends in Medical Mycology, Athens, Greece, October 2009 [4a].)     

Predicting Susceptibility of Streptococcus pneumoniae to Ceftriaxone and Cefotaxime by Cefuroxime and Ceftizoxime Disk Diffusion Testing

In this study, disk diffusion testing with ceftizoxime and cefuroxime was evaluated for use in predicting the susceptibility of Streptococcus pneumoniae to ceftriaxone and cefotaxime. Of the 194 isolates included in this study, 138 were susceptible, 34 were intermediate, and 22 were resistant to cefotaxime by MIC testing; 138 isolates were susceptible, 35 were intermediate, and 21 were resistant to ceftriaxone by MIC testing. A zone of inhibition around the cefuroxime disk of >/=32 mm correctly categorized 101 of 138 isolates as susceptible to cefotaxime and ceftriaxone. A zone of inhibition around the ceftizoxime disk of >/=26 mm correctly categorized 111 of 138 isolates as susceptible to cefotaxime and 114 of 138 as susceptible to ceftriaxone. We conclude that disk diffusion can separate S. pneumoniae isolates susceptible to ceftriaxone and cefotaxime from those that are not susceptible. Isolates not falling into the susceptible category by disk diffusion require additional testing to determine the MIC.     

In vitro activity of pristinamycin against Streptococcus pneumoniae

The activity of the pristinamycin was investigated using disk diffusion agar or ATB PNEUMO system and MIC determination using reference liquid medium method (NCCLS) on 749 S. pneumoniae strains isolated in Aquitaine in 1999. We have realized the killing curves against 10 isolates selected from erythromycin-susceptible and resistant S. pneumoniae strains. All the strains tested by ATB PNEUMO system were susceptible to pristinamycin, using disk diffusion agar, 6.8% of strains were intermediate or resistant. However the MIC's of pristinamycin determined by liquid dilution method against these strains were < 1 mg/L. These data suggest that the zone of inhibition around the disk was not correlated with MIC for erythromycin pneumococci and MIC testing must be performed. The results of killing curves showed a very good and rapid bactericidal activity of pristinamycin within two hours for concentration equal to 4 x MIC and four hours for 2 x MIC.     

Decreased virulence of a pneumolysin-deficient strain of Streptococcus pneumoniae in murine meningitis

Pneumolysin, neuraminidases A and B, and hyaluronidase are virulence factors of Streptococcus pneumoniae that appear to be involved in the pathogenesis of meningitis. In a murine model of meningitis after intracerebral infection using mutants of S. pneumoniae D39, only mice infected with a pneumolysin-deficient strain were healthier at 32 and 36 h, had lower bacterial titers in blood at 36 h, and survived longer than the D39 parent strain. Cerebellar and spleen bacterial titers, meningeal inflammation, and neuronal damage scores remained uninfluenced by the lack of any of the virulence factors.