Degradation of human immunoglobulins by proteases from Streptococcus pneumoniae obtained from various human sources

The ability of Streptococcus pneumoniae to degrade human secretory immunoglobulin A (S-IgA), IgG, and IgM was tested in 102 strains by use of the thin-layer enzyme assay cultivation technique. The strains were isolated from patients with acute phases of otitis media, meningitis, and pneumonia as well as from symptomless carriers. An ability to degrade S-IgA, IgG, and IgM was revealed in 50, 84, and 96 strains, respectively. An IgG- and IgM-degrading ability of S. pneumoniae has not previously been reported. A concurrent degradation of the three immunoglobulins was revealed in 38 strains; degradation of two of them was revealed in 54 strains, and degradation of only one of them was revealed in 9 strains. One strain failed to degrade any of the immunoglobulins. Correlations were not found between the ability of the S. pneumoniae strains to degrade S-IgA, IgG, or IgM and the serotype affiliation or between the ability to degrade IgG or IgM and the origin of strains. However, the ability to degrade S-IgA was evident more often in strains isolated from symptomless carriers and from bronchial secretions of patients with acute pneumonia than it was in strains from patients with acute meningitis or acute otitis media or from the blood of patients with acute pneumonia. These latter findings may indicate a biological significance of S-IgA-degrading ability in bacterial colonization of mucosal surfaces.     

In vivo efficacy of cefotaxime and amoxicillin against penicillin-susceptible, penicillin-resistant and penicillin-cephalosporin-resistant strains of Streptococcus pneumoniae in a mouse pneumonia model

Objective: To compare cefotaxime (CTX) to amoxicillin (AMO) (usually considered the definitive therapy for penicillinsusceptible Streptococcus pneumoniae infections) in an immunocompromised mouse pneumonia model.
Methods: Three S. pneumoniae clinical isolates were used: two serotype 19 strains, a penicillin-susceptible (P^s) strain (penicillin MIC = 0.03 μ/mL) and a highly penicillin-resistant (P^r) strain (penicillin MIC = 4 μ/mL), and one serotype 23F strain, a penicillin-cephalosporin-resistant (CFTR) strain (CTX MIC = 4 μ/mL).
Results: CTX activity in this mouse model of pneumonia induced by the highly penicillin-resistant strain of S. pneumoniae was lower than expected from its low MIC against this organism. Furthermore, AMO had greater efficacy than CTX against a CFTR S. pneumoniae strain.
Conclusion: Our data suggest that there is no major difference in the in vivo efficacy of the two agents, cefotaxime and amoxicillin, against penicillin-resistant and penicillin-cephalosporin-resistant S. pneumoniae.     

A locus contained within a variable region of pneumococcal pathogenicity island 1 contributes to virulence in mice

We have previously described a 27-kb pathogenicity island of Streptococcus pneumoniae, termed pneumococcal pathogenicity island 1 (PPI1), which contains iron uptake locus piaABCD, required for full virulence in mice, and a further 28 previously uncharacterized genes. We have investigated one of these, Sp1051, which encodes a protein of unknown function. Disruption of Sp1051 does not affect growth in laboratory broth, serum, or blood but impairs virulence in mouse models of infection. When S. pneumoniae capsular serotypes were analyzed by PCR and Southern hybridization, it was found that 33% did not contain Sp1051. Analysis of other genes within PPI1 demonstrated that, compared to the serotype 4 genome published by The Institute for Genome Research (TIGR), the genomes of many strains contain deletions of a variable number of genes between Sp1046 and Sp1064, conforming to one of six different patterns. Amplification by PCR of this PPI1 variable region from a capsular serotype 17 strain and comparison of the sequence to TIGR serotype 4 strain sequence showed that Sp1051 is contained within an 11.3-kb segment of DNA flanked by 7-bp direct repeats within the serotype 4 strain which is not present in the serotype 17 strain. Further comparison of the sequences of this region between the three published S. pneumoniae genomes demonstrated that serotype 19F and strain R6 contain novel complements of genes not present in the serotype 4 strain. These data indicate that there is striking variation in gene content and structure of the 3' region of PPI1 among strains and that this region includes at least one virulence determinant. Gene variation within horizontally acquired DNA such as that of PPI1 may be one factor modulating differences in virulence among strains.     

Contribution of serotype and genetic background to virulence of serotype 3 and serogroup 11 pneumococcal isolates

The capsular serotype has long been associated with the virulence of Streptococcus pneumoniae. Here we present an in-depth study of phenotypic and genetic differences between serotype 3 and serogroup 11 S. pneumoniae clinical isolates from both the general and indigenous populations of Australia. Both serotypes/groups included clonally unrelated strains with differences in well-known polymorphic virulence genes, such as nanA and pspA, as demonstrated by multilocus sequence typing and Western blot analysis. Nonetheless, the serotype 3 strains were consistently and significantly more virulent in mice than the serogroup 11 strains. Despite extensive genomic analysis, noncapsular genes common to one serotype/group but not the other were not identified. Nevertheless, following the conversion of a serotype 11A isolate to serotype 3 and subsequent analysis in an intranasal infection model, it was evident that both capsular and noncapsular factors determine the virulence phenotype in mice. However, it appears that these noncapsular factors vary from strain to strain.     

A common clone of erythromycin-resistant Streptococcus pneumoniae in Greece and the UK

Objective  To investigate the possible genetic relationship among erythromycin-resistant Streptococcus pneumoniae strains isolated in Greece and the UK.
Methods  During 1995–97, 140 S. pneumoniae strains were isolated from clinical specimens submitted to the microbiology departments of the two main children's hospital in Athens. All erythromycin-resistant strains were further studied with respect to the presence of genes encoding for the two major mechanisms of macrolide resistance, their serotypes, and pulsed-field gel electrophoresis (PFGE) types, in comparison to a previously characterized UK erythromycin-resistant clone.
Results  Eleven of the 140 isolates (7.9%) were resistant to erythromycin; nine of these were susceptible to penicillin. Serotyping allocated seven, three and one isolates to serotypes 14, 19F and serogroup 6, respectively. The mef A gene was detected in seven isolates (five serotype 14 and two serotype 19F), erm B in two (one serotype 19F and the serogroup 6 isolate), whilst in the remaining two isolates no resistance gene could be detected by polymerase chain reaction (PCR). Pulsed-field gel electrophoresis of genomic DNA showed that five Greek serotype 14 isolates belonged to the same chromosomal type as the serotype 14 erythromycin-resistant UK clone.
Conclusions  The present study showed that erythromycin resistance among the S. pneumoniae isolates was mostly owing to the efflux mechanism and suggested a possible clonal spread of serotype 14 erythromycin-resistant S. pneumoniae strains between Greece and the UK.     

Enzyme electrophoresis, sero- and subtyping, and outer membrane protein characterization of two Neisseria meningitidis strains involved in laboratory-acquired infections.

Two cases of laboratory-acquired infections due to Neisseria meningitidis were suspected to have occurred in two French hospitals. The first case occurred shortly, i.e., 3 days, after one strain had been handled by a laboratory technician, and the link between this strain and the strain causing meningitis was easily established. In the second case, infection occurred 3 weeks after 10 strains had been handled by a technician. In this case, it was necessary to use high-resolution markers in order to establish the link between the infecting strain and 1 of the 10 strains handled. The antigenic formulae of the two infecting strains (serogroup:serotype:subtype) were, respectively, C:NT:P1.12 and B:2a:P1.2. Outer membrane protein profile analysis and multilocus enzyme electrophoresis unequivocally confirmed the identity of the respective strains.     

Serotypes and clonal types of penicillin-susceptible streptococcus pneumoniae causing invasive disease in children in five Latin American countries

We used multilocus sequencing typing (MLST) to determine the genetic backgrounds of 185 recent penicillin susceptible Streptococcus pneumoniae isolates with serotypes that most frequently cause invasive disease in preschool age children in five Latin American countries-Argentina, Brazil, Colombia, Mexico, and Uruguay. Most of the isolates were associated with pneumonia (90/185), meningitis (74/185), and bacteremia (17/185). The collection of strains included seven serotypes-14, 6B, 5, 1, 23 F-which represent the serotypes of S. pneumoniae most frequently associated with sterile site infections in children. Also included were strains expressing serotypes 7F and 3. Comparison of serotype and multilocus sequence type allowed division of the isolates into two groups: strains expressing serotypes 1, 5, 3, and 7 were represented by a relatively few sequence types while strains expressing serotypes 6B, 14, and 23 F showed great genetic diversity. The genetic diversity of serotypes 14, 6B, and 23 F may be related to the capacity of these serotypes to colonize the nasopharynx of healthy carriers during which opportunities for diversification through genetic exchanges can occur. The findings present an interesting contrast with the results of an earlier study in which over 80% of invasive penicillin- resistant serotype 14 and 23 isolates from the same countries were found to belong to as few as two pandemic clones of S. pneumoniae.     

Identification of epidemic strains of Streptococcus suis by genomic fingerprinting.

A natural outbreak of Streptococcus suis meningitis in two closed swine herds was studied. DNA fingerprinting, serotyping, and biochemical profiles were assessed. Multiple serotypes were recovered from these herds. In farm A, 50 S. suis strains were isolated from 330 swabs collected. Eighteen strains belonged to serotype 2, and 32 strains belonged to serotypes 3, 5, 6, 8, 9, and 11. In farm B, 16 S. suis strains were recovered from a total of 70 samples. Eight strains belonged to serotype 7 and eight belonged to serotypes 2, 3, 5, and 8. In each epidemiological situation, a single strain characterized by a distinctive restriction fragment pattern predominated among affected penmates. The epidemic serotype 2 strain was detected in farm A in weaned pigs between the ages of 5 and 7 weeks. In contrast, the pathogenic strain in farm B belonged to serotype 7 and was isolated from pigs up to 3 weeks of age. The results from both farms strongly suggest a lateral spread of these organisms. No vertical transmission could be shown in either herd. It was concluded that genomic fingerprinting is an appropriate method to distinguish outbreak isolates of S. suis from nonoutbreak strains, within the same serotype or from epidemiologically unrelated clusters of strains.     

Characterization of Streptococcus agalactiae strains by multilocus enzyme genotype and serotype: identification of multiple virulent clone families that cause invasive neonatal disease.

The chromosomal genotypes of 277 isolates of 16 serotypes of Streptococcus agalactiae were characterized by analysis of electrophoretically demonstrable allele profiles at 12 metabolic enzyme loci. The collection comprised the type strain and 276 strains recovered from French symptomatic and asymptomatic subjects. Sixty-one distinctive electrophoretic types (ETs), representing multilocus clonal genotypes, were identified. Cluster analysis of the ETs revealed two primary phylogenetic divisions separated by a genetic distance of 0.62, Division I contained 67 isolates which could be assigned to 13 ETs. Twenty-seven of these isolates were from samples of cerebrospinal fluid (CSF) from neonatal meningitis patients. Two ETs, separated by a genetic distance of 0.217, contained 26 of these 27 isolates. Division II contained 210 isolates, of which 27 were isolated from CSF. This division was more polymorphic and included 48 ETs. Spanning a genetic distance of 0.3, three clusters and one ET were identified within this group. Twenty-four of 27 strains isolated from CSF belonged to one cluster, and 19 of them belonged to two adjacent ETs with a genetic distance of 0.083. Fifty-five of the 68 serotype Ia strains and 24 of the 26 serotype Ib strains were each confined to one of the evolutionary lineages, and 85 of the 86 strains which carried protein antigen c belonged to phylogenetic division II. Most of the type III organisms were assigned to two clone families. The characteristics of this French population argue for the existence of particular groups of strains responsible for neonatal meningitis and demonstrate that serotyping can supply information about the genetic distribution of strains.     

Molecular epidemiology of Klebsiella pneumoniae strains that produce SHV-4 beta-lactamase and which were isolated in 14 French hospitals.

Preliminary results suggested that the diffusion in France of the SHV-4 extended-spectrum beta-lactamase was probably due to the spread of one single epidemic strain of Klebsiella pneumoniae. In this study, we tested various phenotypic and genotypic markers to compare K. pneumoniae strains producing this enzyme isolated in 14 French hospitals between 1987 and 1989. All of the strains were of the same capsule serotype, K25. Twelve of them were of the same biotype: weak urease activity and no sucrose fermentation. Among the six plasmid profiles observed, one accounted for eight strains. Large plasmids of 170 kb encoding SHV-4 beta-lactamase were present in all strains of K. pneumoniae and could be transferred by conjugation with high frequency to Escherichia coli J53-2 or HB101 from all except one strain. Plasmid EcoRI restriction patterns suggested that these plasmids were closely related and similar to pUD18 encoding SHV-3 beta-lactamase, originally described in France and differing from SHV-4 by one amino acid substitution. Ribotyping with EcoRI and HindIII and genomic fingerprinting with XbaI by pulsed-field gel electrophoresis were concordant and suggested that 12 of the isolates recovered from the 14 hospitals were probably the same strain. Dissemination in France of the SHV-4 extended-spectrum beta-lactamase was thus essentially due to the diffusion of a single K. pneumoniae clone.     

Outer Membrane Protein Composition in Disease Isolates of Haemophilus influenzae: Pathogenic and Epidemiological Implications

The outer membrane protein composition of 50 disease isolates of Haemophilus influenzae has been determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All strains, including 28 strains of serotype b, one strain each of serotypes a, c, d, e, and f, and 17 untypable strains, had an outer membrane protein composition typical of gram-negative bacteria, i.e., these membranes contained two to three dozen proteins with four to six proteins accounting for most of their protein content. Variation in the mobility of these major outer membrane proteins from strain to strain was common but not universal; the observed patterns provided useful data and new insight into the epidemiology of type b disease. The basic findings can be summarized as follows: (i) All 50 strains possessed three proteins (one minor and two major) each having identical mobilities. The other proteins, both major and minor, varied in mobility. (ii) All type b strains possessed a fourth (major) protein of identical mobility. (iii) The 28 type b strains, on the basis of the mobility of the six major outer membrane proteins, could be divided into eight subtypes. Of all the other strains examined, both typable and untypable, only the serotype a strain belonged to one of these subtypes. (iv) The untypable strains showed considerable variation in the mobilities of their major outer membrane proteins. Of these 17 strains, 13 had an additional major outer membrane protein not present in encapsulated strains. (v) The outer membrane protein composition of a single strain remained unchanged after many passages on solid media, but varied with the growth phase. (vi) The outer membrane protein composition of isolates obtained from nine patients during an epidemic of type b meningitis varied, indicating that a single strain was not responsible for the epidemic. At least five different strains were responsible for these nine cases. (vii) Identical outer membrane protein compositions were observed in the following: in a type b strain and a mutant of this strain deficient in capsule production, indicating that the level of capsule synthesis is not obviously related to outer membrane protein composition; in type b strains isolated from different anatomic sites of patients acutely ill with meningitis, indicating that the strain associated with bacteremia is the same as that isolated from the cerebrospinal fluid; in type b strains isolated from siblings who contracted meningitis at about the same time, indicating infection with the same strain; and in type b strains isolated from the initial and repeat infection of a single patient, suggesting that reinfection was due to the same strain.     

Molecular typing and virulence analysis of serotype K1 Klebsiella pneumoniae strains isolated from liver abscess patients and stool samples from noninfectious subjects in Hong Kong, Singapore, and Taiwan

Serotype K1 Klebsiella pneumoniae with multilocus sequence type 23 (ST23) has been strongly associated with liver abscess in Taiwan. Few data regarding the strain types and virulence of this serotype from other Asian countries are available. Serotype K1 K. pneumoniae strains isolated from liver abscess and stool samples from subjects hospitalized in Hong Kong, Singapore, and Taiwan hospitals were examined. Forty-seven serotype K1 isolates were identified: 26 from liver abscess samples and 21 from stool samples. MLST revealed 7 sequence types: 85.1% (40 of 47 isolates) belonged to ST23, 1 isolate belonged to ST163 (a single-locus variant of ST23), and 2 isolates were ST249 (a 3-locus variant of ST23). New STs, namely, ST367, ST425, and ST426, were allocated to 3 of 4 isolates from stool samples. The virulence of these strains was determined by neutrophil phagocytosis and mouse infection models. Except for two ST23 isolates, all Klebsiella pneumoniae isolates were resistant to phagocytosis. Resistance to serum killing varied in isolates of ST23, while all non-ST23 strains were susceptible to serum killing except one with ST249 from a liver abscess. All hypervirulent isolates with a 50% lethal dose of <10(2) CFU were from ST23, were resistant to phagocytosis and serum killing, and also carried both virulence-associated genes, rmpA and aerobactin. Multilocus sequence typing genotype 23 was the most prevalent sequence type among serotype K1 K. pneumoniae isolates from both liver abscess and stool samples in the Asia Pacific region. Serotype K1 K. pneumoniae isolates with capsule expression leading to phagocytic resistance and with the aerobactin gene were associated with hypervirulence.     

Sexual cycle of Cryptococcus neoformans var. grubii and virulence of congenic a and alpha isolates

Cryptococcus neoformans is a human-pathogenic fungus that has evolved into three distinct varieties that infect most prominently the central nervous system. A sexual cycle involving haploid cells of a and alpha mating types has been reported for two varieties (C. neoformans var. neoformans, serotype D, and C. neoformans var. gattii, serotypes B and C), yet the vast majority of infections involve a distinct variety (C. neoformans var. grubii, serotype A) that has been thought to be clonal and restricted to the alpha mating type. We recently identified the first serotype A isolate of the a mating type which had been thought to be extinct (strain 125.91). Here we report that this unusual strain can mate with a subset of pathogenic serotype A strains to produce a filamentous dikaryon with fused clamp connections, basidia, and viable recombinant basidiospores. One meiotic segregant mated poorly with the serotype A reference strain H99 but robustly with a crg1 mutant that lacks a regulator of G protein signaling and is hyperresponsive to mating pheromone. This meiotic segregant was used to create congenic a and alpha mating type serotype A strains. Virulence tests with rabbit and murine models of cryptococcal meningitis showed that the serotype A congenic a and alpha mating type strains had equivalent virulence in animal models, in contrast to previous studies linking the alpha mating type to increased virulence in congenic serotype D strains. Our studies highlight a role for sexual recombination in the evolution of a human fungal pathogen and provide a robust genetic platform to establish the molecular determinants of virulence.     

Population structure of Australian isolates of Streptococcus suis.

The genetic diversity of 109 isolates of Streptococcus suis, which were recovered mainly from Australian pigs, was examined by multilocus enzyme electrophoresis. The collection was genetically diverse. Sixty-five electrophoretic types (ETs) were recognized, with a mean genetic diversity per enzyme locus of 0.512, or 0.431 when the number of isolates in each ET was considered. Serotype diversity varied, being greatest for isolates of capsular serotype 15 (0.364), and then diminishing in the order of serotypes 9, 1, 4, 1/2, 2, 7, and 3 (0.120). On average, isolates from these eight serotypes represented 4.13 separate clonal groups per serotype. This diversity indicated that serotyping of S. suis for subspecific differentiation is not a reliable technique for identifying specific strains and is not a good predictor of the genetic background of a given isolate. No tendency for isolates recovered from healthy pigs to be genetically distinct from those from diseases animals was found, nor were there consistent differences between isolates recovered from animals with different disease syndromes (meningitis, pneumonia, and septicemia). Danish reference strains of serotypes 1, 2, and 7 each belonged to one of the same clonal groupings of these types found in Australia, but Danish strains of serotypes 3, 4, 6, and 8 and a strain of serotype 1 from the United Kingdom were each genetically distinct from the Australian isolates. Generally, isolates in the same ET belonged to the same serotype, but one ET contained isolates of types 6 and 6/16, and three were made up of isolates of types 2 and 1/2. One isolate of serotype 2, which was recovered from a human with meningitis, belonged to the same ET as two isolates of serotype 2 that were recovered from pigs. The human infection was therefore likely to have been zoonotic.     

Novel penicillin-, cephalosporin-, and macrolide-resistant clones of Streptococcus pneumoniae serotypes 23F and 19F in Taiwan which differ from international epidemic clones

A cluster (14 of 18) of Streptococcus pneumoniae serotype 23F isolates that were resistant to penicillin (PEN), cephalosporin, and macrolide was found in one day care center in Kaohsiung, Taiwan. We analyzed the 18 isolates by pulsed field gel electrophoresis (PFGE). All but one serotype 23F isolate demonstrated identical PFGE patterns, which were different from the established pattern of the internationally spread Spanish 23F clone. The three strains of serotype 19F also showed a uniform pattern. These data strongly suggest that two novel clones of PEN-, cephalosporin-, and macrolide-resistant S. pneumoniae serotypes 23F and 19F are present in Taiwan.     

DNA fingerprinting of Streptococcus pneumoniae strains by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis of genomic DNA was carried out on Streptococcus pneumoniae strains to determine its value in the epidemiological survey of pneumococcal infections. Twenty-one clinical strains were chosen to cover a broad range of diversity according to geographic location, penicillin susceptibility, serotype, and multilocus enzyme electrophoresis (MLEE) pattern. The restriction endonucleases ApaI and SmaI were used to digest intact chromosomes, and the fragments were resolved by field inversion gel electrophoresis (FIGE). Each digest produced 10 to 19 fragments for comparison between strains. All the strains, including strains of the same serotype and strains with the same MLEE profile, had different FIGE patterns. In some cases, the restriction patterns differed by only a few fragment bands, and two isolates differed only in the location of a single DNA fragment. The polymorphism obtained with FIGE was greater than those obtained with serotyping and MLEE analysis. The stability of the FIGE profiles was established by testing of two independent clones derived from pneumococcus strain R36A. These results indicated that pulsed-field gel electrophoresis should be an effective tool for the typing of S. pneumoniae strains, capable of subdividing serotypes or MLEE types and of tracing the origin of pneumococcal strains.     

Contribution of the ATP-dependent protease ClpCP to the autolysis and virulence of Streptococcus pneumoniae

The ATP-dependent caseinolytic proteases (Clp) are fundamental for stress tolerance and virulence in many pathogenic bacteria. The role of ClpC in the autolysis and virulence of Streptococcus pneumoniae is controversial. In this study, we tested the role of ClpC in a number of S. pneumoniae strains and found that the contribution of ClpC to autolysis is strain dependent. ClpC is required for the release of autolysin A and pneumolysin in serotype 2 S. pneumoniae strain D39. In vivo, ClpC is required for the growth of the pneumococcus in the lungs and blood in a murine model of disease, but it does not affect the overall outcome of pneumococcal disease. We also report the requirement of ClpP for the growth at elevated temperature and virulence of serotype 4 strain TIGR4 and confirm its contribution to the thermotolerance, oxidative stress resistance, and virulence of D39.     

Genetic relatedness within serotypes of penicillin-susceptible Streptococcus pneumoniae isolates

The molecular epidemiological characteristics of all Streptococcus pneumoniae strains isolated in a nationwide manner from patients with meningitis in The Netherlands in 1994 were investigated. Restriction fragment end labeling analysis demonstrated 52% genetic clustering among these penicillin-susceptible strains, a value substantially lower than the percentage of clustering among Dutch penicillin-nonsusceptible strains. Different serotypes were found within 8 of the 28 genetic clusters, suggesting that horizontal transfer of capsular genes is common among penicillin-susceptible strains. The degree of genetic clustering was much higher among serotype 3, 7F, 9V, and 14 isolates than among isolates of other serotypes, i.e., 6A, 6B, 18C, 19F, and 23F. We further studied the molecular epidemiological characteristics of pneumococci of serotype 3, which is considered the most virulent serotype and which is commonly associated with invasive disease in adults. Fifty epidemiologically unrelated penicillin-susceptible serotype 3 invasive isolates originating from the United States (n = 27), Thailand (n = 9), The Netherlands (n = 8), and Denmark (n = 6) were analyzed. The vast majority of the serotype 3 isolates (74%) belonged to two genetically distinct clades that were observed in the United States, Denmark, and The Netherlands. These data indicate that two serotype 3 clones have been independently disseminated in an international manner. Seven serotype 3 isolates were less than 85% genetically related to the other serotype 3 isolates. Our observations suggest that the latter isolates originated from horizontal transfer of the capsular type 3 gene locus to other pneumococcal genotypes. In conclusion, epidemiologically unrelated serotype 3 isolates were genetically more related than those of other serotypes. This observation suggests that serotype 3 has evolved only recently or has remained unchanged over long periods.     

Mosaic genes and mosaic chromosomes-genomic variation in Streptococcus pneumoniae

The genome sequences of two strains of Streptococcus pneumoniae, one of the major human pathogens, are currently available: that of the nonencapsulated laboratory strain R6, the origin of which dates back to the early 20th century, and of the serotype 4 TIGR strain isolated recently. The two genomes are not only different in size (2 versus 2.16 Mb) but differ also by approximately 10% of their genes, many of which being organized in large clusters. Their strain-specific genes and gene clusters are described here. The R6 genome contains 69 kb organized in six large regions that are absent from the TIGR strain, which in turn contains an extra 157kb in twelve clusters compared to R6. In addition, the TIGR strain contains 13 clusters of 4 kb and larger that are not shared by a variety of genetically different S. pneumoniae strains. Many regions bear signs of gene transfer events such as the presence of insertion sequences, transposable elements, and putative site-specific integrases/recombinases. Three strain-specific regions are devoted to genes encoding proteins with the cell wall anchor motif LPXTG which are important for the interaction with host cells and appear to be highly variable, similar to cell wall-associated choline-binding proteins.     

Investigation of the basis of virulence in serotype A strains of <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> from apparently immunocompetent individuals

Cryptococcus neoformans serotype A strains commonly infect immunocompromised patients to cause fungal meningitis. To understand the basis of serotype A cryptococcal infections in apparently immunocompetent patients, we tested two hypotheses: the strains were naturally occurring hypervirulent pkr1 (PKA regulatory subunit) mutants, or the strains were hybrids with C. neoformans var. gattii strains that normally infect immunocompetent individuals. Analysis of clinical isolates obtained from apparently immunocompetent individuals from three continents revealed that none were pkr1 mutants, but several exhibited phenotypes consistent with perturbations in cAMP signaling. Additionally, none of the strains were unusual hybrids with gattii strains. Except for one strain that was an AD hybrid, all others were serotype A (var. grubii) isolates. Taken together, our findings indicate that the ability of these clinical isolates to infect apparently normal individuals may be attributable to mutations other than pkr1 and/or underlying immune system impairment in patients.