Capsular polysaccharide gene diversity of pneumococcal serotypes 6A, 6B, 6C,and 6D

This study was performed to better understand the genetic diversity and evolutionary relatedness of pneumococcal serotypes 6A, 6B, 6C, and 6D. Multi-locus sequence typing (MLST) was performed for 160 serogroup 6 isolates from clinical specimens collected from children between 1991 and 2010. We identified 38 sequence types (STs) comprising five clonal complexes with 12 singletons. Although most STs were confined to a single serotype, some STs were shared by two serotypes, and one ST was shared by three serotypes. Many STs of serotype 6A showed genetic relatedness with those of serotype 6C or 6D in eBURST analysis. Five capsular polysaccharide (cps) genes – wchA, wciO, wciP, wzy, and wzx – were analysed in 74 isolates from our clinical samples and in 36 isolates from GenBank. There were several profiles and clades in each serotype on the analysis of the concatenated sequences of the five cps genes. Small genetic distances between serotypes 6A and 6B and between serotypes 6C and 6D were observed while serotype 6B with an indel sequence formed a distinct clade. When comparing the individual cps genes between the serotypes, there was also a high level of similarity in the wchA and wciO gene sequences between serotype 6C and serotype 6D. On the other hand, serotypes 6A and 6D had the most highly similar wzy and wzx gene sequences. The wzy sequences of serotype 6C were nearly identical (99.6%) to those of serotype 6A clade II strains. In conclusion, we revealed the diversity of the genetic background and cps sequences in each pneumococcal serotype of serogroup 6. Pneumococcal serotype diversity might be attributable to complex serial mutation and recombination events.     

Use of pyrosequencing to differentiate Streptococcus pneumoniae serotypes 6A and 6B

Accurate serotyping of Streptococcus pneumoniae remains important to monitor the changes in seroepidemiology of the organism over time. Though several PCR-based systems have been developed for this purpose, the cross-reactivity within serogroups often limits discrimination between types. All serogroup 6 isolates can be identified using a multiplex PCR system; however, due to the high sequence homology between the cps-6B and cps-6A loci, serotypes 6A and 6B cannot be differentiated by this method. We describe the use of pyrosequencing to reliably differentiate between serotypes 6A and 6B using a previously described single nucleotide polymorphism at codon 195 of the cps locus wciP gene. We observed complete concordance between capsular serotyping results and wciP pyrosequencing among 210 isolates examined, indicating that pyrosequencing is a rapid and accurate technique for deducing serotypes 6A and 6B.     

Genetic basis for the new pneumococcal serotype, 6C

We have recently reported a new pneumococcal serotype (6C), which is closely related to serotype 6A (I. H. Park et al., J. Clin. Microbiol. 45:1225-1233, 2007). To investigate the genetic basis for serotype 6C, we studied the capsule gene loci of 14 6C isolates from three different continents, including one isolated in Alabama 27 years ago. The wciN region of all 6C isolates has a 1,029-bp-long sequence that replaces the 1,222-bp-long sequence of the 6A wciN region. This recombination event has created a new 1,125-bp-long open reading frame which encodes a product that is also homologous to glycosyl transferases. Flanking this introduced gene is 300 bp upstream and 100 bp downstream with only about 90% homology with 6A and which is identical in all 6C isolates. Transfer of the wciN region converts 6A to 6C. Determination of the DNA sequence of the entire capsule gene locus of one 6C isolate showed that the 6C capsule gene locus is almost identical (>98% homologous) to that of 6A except for the wciN region. These findings indicate that the 6C capsule type originated more than 27 years ago by a single recombination event in a 6A locus in which 6A wciN was replaced by a gene of unknown origin.     

Multidrug-resistant Streptococcus pneumoniae serotype 6D clones in South Korea

To investigate the characteristics of main Streptococcus pneumoniae clones of serotype 6D (ST282 and ST3171) in South Korea, antimicrobial susceptibility testing was performed, and 11 genes around the cps locus were sequenced on ST282(6D), ST3171(6D), and ST81(6A) isolates. The antimicrobial susceptibility patterns were very similar between clones belonging to the same clonal complex, ST81(6A) and ST282(6D); nonsusceptibilities to penicillin and cefuroxime, high MICs of ceftriaxone, and high resistance rates to trimethoprim-sulfamethoxazole. However, ST3171(6D) isolates showed resistance to only macrolides and clindamycin. The sequences of 11 genes around the cps locus indicated the same genetic backgrounds between the ST81(6A) and ST282(6D) isolates. On the other hand, ST3171(6D) isolates showed nucleotide and amino acid differences from ST81(6A) and ST282(6D) isolates in most genes, indicating a different genetic background. The mosaic structure of dexB gene in ST282(6D) isolates indicated that recombination might occur in the dexB gene. Our results suggest that the multidrug-resistant ST282(6D) pneumococcal clone has emerged by serial genetic recombination, including capsular switch.     

Genetic basis for the structural difference between Streptococcus pneumoniae serotype 15B and 15C capsular polysaccharides

In a search for the genetic basis for the structural difference between the related Streptococcus pneumoniae capsular serotypes 15B and 15C and for the reported reversible switching between these serotypes, the corresponding capsular polysaccharide synthesis (cps) loci were investigated by keeping in mind that at the structural level, the capsules differ only in O acetylation. The cps locus of a serotype 15B strain was identified, partially PCR amplified with primers based on the related serotype 14 sequence, and sequenced. Sequence analysis revealed, among other open reading frames, an intact open reading frame (designated cps15bM) whose product, at the protein level, exhibited characteristics of previously identified acetyltransferases. Genetic analysis of the corresponding region in a serotype15C strain indicated that the same gene was present but had a premature stop in translation. Closer analysis indicated that the serotype 15B gene contained a short tandem TA repeat consisting of eight TA units. In serotype 15C, this gene contained nine TA units that resulted in a frameshift and a truncated product. Genetic analysis of 17 serotype 15B and 15C clinical isolates revealed a perfect correlation between the serotype and the length of the short tandem repeat in the putative O-acetyltransferase gene. The number of TA repeating units varied between seven and nine in the various isolates. Together, the data strongly suggest that the structural difference between serotypes 15B and 15C is based on variation in the short tandem TA repeat in the O-acetyltransferase gene and that the transition between serotypes is due to slipped-strand mispairing with deletion or insertion of TA units in the cps15bM gene.     

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.     

Molecular serotyping of Klebsiella species isolates by restriction of the amplified capsular antigen gene cluster

The objective of the present work was to develop a molecular method that would enable determination of the capsular serotypes of Klebsiella isolates without the use of antiserum. PCR amplification of the capsular antigen gene cluster (cps) was followed by digestion with the restriction enzyme HincII (cps PCR-restriction fragment length polymorphism [RFLP] analysis). The profiles (C patterns) obtained for 224 strains representing the 77 known K serotypes showed 3 to 13 fragments ranging in size from 0.2 to 4.4 kb. A total of 97 distinct C patterns were obtained; 100% of 61 pairs of samples tested twice showed reproducible C patterns. The C patterns were K-type specific; i.e., the C pattern(s) of any K serotype was distinct from the C patterns of all other K serotypes, with the only exceptions being serotypes K22 and K37, which are known to cross-react. For 12 of 17 K types for which at least two strains were included, C-pattern variations were found among strains with the same K serotype. Therefore, cps PCR-RFLP analysis has a higher discriminatory power than classical K serotyping. C-pattern identity was observed among strains with a given K type that were collected many years apart and from distinct sources, indicating C-pattern stability. Only 4.5% of the strains were nontypeable, because of unsuccessful PCR amplification (whereas 8 to 23% are nontypeable by classical K serotyping). Three of four noncapsulated strains analyzed showed recognizable C patterns. The K serotypes of 18 (82%) of 22 recent Klebsiella pneumoniae clinical isolates could be deduced from their C patterns. In conclusion, cps PCR-RFLP analysis allows determination of the K serotype, while it is easier to perform and more discriminatory than classical serotyping.     

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.     

Streptococcus pneumoniae type determination by multiplex polymerase chain reaction

The purpose of this study was to develop pneumococcal typing by multiplex PCR and compare it with conventional serotyping by quellung reaction. Pneumococcal strains used in this study included 77 isolates from clinical specimens collected from children at Seoul National University Children's Hospital from 2006 to 2010. These strains were selected as they represented 26 different serotypes previously determined by quellung reaction. Molecular type was determined by 8 sequential multiplex PCR assays. Bacterial DNA extracted from cultured colonies was used as a template for PCR, and primers used in this study were based on cps operon sequences. Types 6A, 6B, 6C, and 6D were assigned based on the presence of wciN(β) and/or wciP genes in 2 simplex PCRs and sequencing. All 77 isolates were successfully typed by multiplex PCR assays. Determined types were as follows: 1, 3, 4, 5, 6A, 6B, 6C, 6D, 7C, 7F, 9V, 10A, 11A, 12F, 13, 14, 15A, 15B/15C, 19A, 19F, 20, 22F, 23A, 23F, 34, 35B, and 37. The results according to the PCR assays were in complete concordance with those determined by conventional quellung reaction. The multiplex PCR assay is highly reliable and potentially reduces reliance upon conventional serotyping.     

Acquisition of new capsular genes among clinical isolates of antibiotic-resistant Streptococcus pneumoniae

Antibiotic-resistant clones of Streptococcus pneumoniae are recognizable through a combination of unique molecular, microbiological, and serological properties. In the course of surveillance of epidemic clones of S. pneumoniae, several isolates were identified that shared the clone-specific pulsed-field gel electrophoretic (PFGE) pattern and antibiotype but expressed serotypes atypical for the particular clone. A selected group of isolates belonging to the Spanish/USA clone but expressing serotypes 19, 14, or 3, instead of the expected serotype 23F, were tested using DNA probes for each of the 18 open reading frames (ORFs) of the 23F capsular locus. In no case were there any 23F-specific genes retained, with the possible exception of genes already known to be common to the capsular loci involved. Analysis of the sequence of the capsular locus of a penicillin-resistant serotype 23F isolate from Mexico showed that part of the cpsA gene of this strain, as well as genes cpsQ and cpsR, had high degrees of identity to the sequence of the homologous genes in isolates expressing serotype 19F. The capsular locus of this Mexican strain may have originated from an in vivo capsular switch event in which the original 19F locus was replaced by 23F-specific capsular genes.     

Stability of serotypes during nasopharyngeal carriage of Streptococcus pneumoniae

Serotype changes among natural isolates of Streptococcus pneumoniae are well documented and occur by recombinational exchanges at the capsular biosynthetic locus. However, the frequency with which this phenomenon occurs within the nasopharynx of children is not clear and is likely to be highest in the nasopharynx of children, who have high rates of pneumococcal carriage. A birth cohort of 100 infants was studied, and pneumococci were recovered from nasopharyngeal samples taken at monthly intervals during the first 6 months of life and then at 2-monthly intervals until the age of 2 years. Among the 1,353 nasopharyngeal samples were 523 that contained presumptive pneumococci, and three colonies from each were serotyped. A total of 333 isolates, including all isolates of differing serotypes from the same child, were characterized by multilocus sequence typing. Sixty-eight children carried multiple serotypes during the first 2 years of life. Two children carried a typeable and a nonserotypeable pneumococcus of identical genotype, and five children carried genetically indistinguishable isolates of serotypes 15B and 15C. These isolates were considered, respectively, to be due to loss of capsule expression and the known ability of serotype 15B and 15C pneumococci to interconvert by loss or gain of an acetyl group on the capsular polysaccharide. In all other cases, isolates from the same children that differed in serotype also differed in genotype, indicating the acquisition of a different pneumococcal strain rather than a change in capsular type. There was therefore no evidence in this study for any change of serotype due to recombinational replacements at the capsular locus among the pneumococci carried within the nasopharynges of the children.     

Multilocus sequence typing system for group B streptococcus

A multilocus sequence typing (MLST) system was developed for group B streptococcus (GBS). The system was used to characterize a collection (n = 152) of globally and ecologically diverse human strains of GBS that included representatives of capsular serotypes Ia, Ib, II, III, V, VI, and VIII. Fragments (459 to 519 bp) of seven housekeeping genes were amplified by PCR for each strain and sequenced. The combination of alleles at the seven loci provided an allelic profile or sequence type (ST) for each strain. A subset of the strains were characterized by restriction digest patterning, and these results were highly congruent with those obtained with MLST. There were 29 STs, but 66% of isolates were assigned to four major STs. ST-1 and ST-19 were significantly associated with asymptomatic carriage, whereas ST-23 included both carried and invasive strains. All 44 isolates of ST-17 were serotype III clones, and this ST appeared to define a homogeneous clone that was strongly associated with neonatal invasive infections. The finding that isolates with different capsular serotypes had the same ST suggests that recombination occurs at the capsular locus. A web site for GBS MLST was set up and can be accessed at http://sagalactiae.mlst.net. The GBS MLST system offers investigators a valuable typing tool that will promote further investigation of the population biology of this organism.     

Development of Rapid Serotype-Specific PCR Assays for Eight Serotypes of Streptococcus suis

Streptococcus suis is an emerging zoonotic pathogen causing severe infections in pigs and humans. Thirty-three serotypes of S. suis have been identified using serum agglutination. The capsular polysaccharides synthesis (cps) locus is usually conserved among different strains of the same serotype. The cps loci of 15 serotypes have been sequenced, while the loci of the other serotypes remain unknown. In the present study, two to six serotype-specific genes of each of eight serotypes, i.e., serotypes 3, 4, 5, 8, 10, 19, 23, and 25, were identified using cross-hybridization with 93 nucleic acid probes specific to genes in the cps locus, and serotype-specific PCR assays for rapid and sensitive detection of the eight serotypes were then developed. The PCR typing results of the 148 serologically typeable isolates were completely consistent with agglutination results. Furthermore, some autoagglutinating, acapsular, and multiagglutinating strains which could not be differentiated by traditional serum agglutination assays were positive in the PCR assays. Use of the PCR assays with clinical tonsillar specimens showed that the assays are sensitive and able to identify samples with autoagglutinating isolates. To our knowledge, this is the first study to identify the serotype-specific genes of the eight Streptococcus suis serotypes and develop rapid and sensitive PCR assays for the eight serotypes which can be identified only by serum agglutination.     

The cps Genes of Streptococcus suis Serotypes 1, 2, and 9: Development of Rapid Serotype-Specific PCR Assays

We developed three type-specific PCR assays for the rapid and sensitive detection of Streptococcus suis serotype 1 (plus 14), serotype 2 (plus 1/2), and serotype 9 strains in tonsillar specimens from pigs. The PCR primers were based on the sequences of type-specific capsular genes of S. suis serotype 1, 2, and 9 strains. We recently characterized a major part of the capsular biosynthesis (cps) locus of S. suis serotype 2. Here we extended these studies and characterized major parts of the cps loci of S. suis serotypes 1 and 9. Type-specific genes were identified by cross-hybridization experiments between the individual cps genes and chromosomal DNAs from the 35 different serotypes. Four genes of S. suis serotype 1 specifically hybridized with serotype 1 and 14 strains only. Five genes of S. suis serotype 2 specifically hybridized with serotype 2 and 1/2 strains only, and two genes of S. suis serotype 9 specifically hybridized with serotype 9 strains. Until now rapid and sensitive diagnostic tests were available only for pathogenic strains of serotype 2 and highly pathogenic strains of serotype 1. The serotype-specific PCR assays can therefore be useful tools for the identification of serotype 1, 14, 2, 1/2, and 9 strains both for diagnostic purposes and in epidemiological and transmission studies. Therefore, these tests may facilitate control and eradication programs.     

Pneumococcus with the “6E” cps Locus Produces Serotype 6B Capsular Polysaccharide

Genetic studies of serogroup 6 isolates of Streptococcus pneumoniae identified putative serotype 6E. Although its capsular polysaccharide structure has not been elucidated, putative serotype 6E is described in an increasing number of studies as a potentially new serotype. We show here that SPEC6B, which is widely used as a target strain for serotype 6B opsonophagocytosis assays, has the genetic features of the putative serotype 6E but produces capsular polysaccharide identical to 6B capsular polysaccharide as determined by one-dimensional (1D) and 2D nuclear magnetic resonance (NMR). Thus, putative serotype 6E is a mere genetic variant of serotype 6B. Also, SPEC6B is appropriate as a target strain for serotype 6B opsonophagocytosis assays. This example illustrates the difficulties of assigning new bacterial serotypes based on genetic findings alone.     

Molecular epidemiology of penicillin-resistant Streptococcus pneumoniae in a university hospital, Ankara, Turkey

Penicillin-resistant Streptococcus pneumoniae isolates (n = 76) from clinical samples of patients admitted to Hacettepe University Hospital between January 1997 and December 2001 were included in the study. MICs of penicillin G, erythromycin A, clindamycin, cefaclor, cefotaxime, vancomycin, chloramphenicol, tetracycline, ciprofloxacin and rifampicin were determined by agar dilution. The isolates were serogrouped on the basis of the Neufeld Quellung reaction and were typed by BOX-PCR. Genetic polymorphism of the penicillin resistance genes pbp2b and pbp2x was investigated by restriction fragment length polymorphism (RFLP) analysis. Of the 76 isolates tested, 64 (84.2%) showed intermediate resistance to penicillin, while 12 (15.8%) were resistant to higher levels of penicillin (MIC > or = 2 mg/L). The resistance patterns of the isolates revealed six different resistance profiles. There were 22 different serotypes, with c. 55% of the isolates belonging to serotypes 23B, 19A, 19F, 14, 6 A and 9V. Five distinct patterns for pbp2b and 12 distinct patterns for pbp2x were obtained by RFLP analysis of penicillin-binding protein genes. The combination of these patterns allowed isolates to be classified into 22 fingerprint subgroups. BOX-PCR analysis showed that the isolates fell into 14 distinct BOX genotypes, with 33 subtypes. Serotype 9V isolates with pbp genotype 2-6 and BOX-PCR type 4, 4.1 or 4.2 were related to the pandemic clone Spain(9V)-3. No relatedness to other international clones was detected among the other study strains, but genetic relatedness was observed among some of the serotype 19A and 23B isolates. Overall, the results demonstrated that most of the penicillin-resistant pneumococcal isolates in Turkey, other than those belonging to serotypes 9V, 19A and 23B, were derived from several independent clones, possibly resulting from multiple importation of strains originating from outside the country. Differences in pbp patterns, serotypes and resistance profiles among isolates that showed similar BOX-PCR patterns supported the hypothesis that horizontal transfer of capsular genes, pbp genes and other genetic determinants between S. pneumoniae and viridans group streptococci may have occurred.     

Characterization of penicillin intermediate serotypes of Streptococcus pneumoniae carried by human immunodeficiency virus-infected adults and healthy children in Uganda

There are little data on the genetic relatedness between antibiotic-resistant pneumococcal isolates colonizing the Ugandan population. Penicillin-intermediate pneumococci of serogroups or serotypes rarely or not previously reported as being penicillin nonsusceptible were selected out of 166 isolates representing 26 capsular serogroups or serotypes isolated from Ugandan children in 1995 and human immunodeficiency virus (HIV) infected Ugandan adults in 2004-2005. Pairs of penicillin-intermediate pneumococci of the same serogroup or serotype present in both patient populations were characterized further by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Seven such pairs of isolates were found and included serogroups 7, 11, 15B/C, and 16 as well as serotypes 13, 21, and 35B. PFGE of these seven pairs showed no clonality between serogroups or serotypes, and clonality only within serogroup 11 and serotype 13. MLST of the 14 individual isolates revealed 13 different sequence types (STs), 11 of which had not previously been recorded. Comparisons with all known STs revealed that most of these strains were related only to strains of the same serotype in other countries, with these related strains frequently also being penicillin intermediate. These findings suggest that penicillin nonsusceptibility in Uganda is likely due to the introduction of antibiotic-resistant pneumococcal clones into Uganda rather than development of resistance within the country.     

Antimicrobial susceptibility and serotype distribution of Streptococcus pneumoniae and molecular characterization of multidrug-resistant serotype 19F, 6B,and 23F Pneumococci in northern Thailand

Penicillin-resistant Streptococcus pneumoniae is widely spread worldwide. Our study was undertaken to examine the susceptibility and serotypes of S. pneumoniae in northern Thailand. Ninety-three S. pneumoniae strains were isolated from 93 patients at Chiang Mai University Hospital, Chiang Mai, Thailand, from September 1999 to June 2000. The strains were isolated from sputum (n = 51), blood (n = 15), nasopharynges (n = 14), and other sources (e.g., pus, ears, ascites, and cerebrospinal fluid) (n = 13). Of the 93 isolates, 29 (31.2%) were susceptible, 24 (25.8%) showed intermediate resistance (MIC, 0.12 to 1.0 micro g/ml), and 40 (43.0%) were fully resistant (MIC, >/=2.0 micro g/ml) to penicillin G. Seven (46.7%) from blood, 5 (35.7%) from nasopharynges, 15 (29.4%) from sputum, and 2 (15.4%) from other sources were susceptible isolates. Serotyping with the use of antiserum revealed differences in the predominant types that were susceptible (6A, 11A, and 19A), intermediately resistant (6B and 23F), and fully resistant (6B, 19F, and 23F). Molecular typing by pulsed-field gel electrophoresis of multidrug-resistant pneumococci showed four patterns (A, B, C, and D) for 16 isolates of serotype 19F, with pattern B being predominant (12 isolates). This finding was different from that with the Taiwan multidrug-resistant serotype 19F clone. Eleven isolates of serotype 6B all showed pattern E, and nine isolates of serotype 23F showed two patterns (F and G), with pattern F being predominant (seven isolates). This finding was similar to that with the Spanish multidrug-resistant serotype 23F clone. Our results indicated that the resistance of pneumococci to antibiotics in northern Thailand is progressing rapidly and that effort should be intensified to prevent any spread of pandemic multidrug-resistant serotypes 19F, 6B, and 23F.     

Serotype AD strains of Cryptococcus neoformans are diploid or aneuploid and are heterozygous at the mating-type locus

Cryptococcus neoformans is a pathogenic basidiomycete with a defined sexual cycle involving mating between haploid yeast cells with a transient diploid state. C. neoformans occurs in four predominant serotypes (A, B, C, and D), which represent different varieties or species. Rare clinical and environmental isolates with an unusual AD serotype have been reported and suggested to be diploid. We found by fluorescence-activated cell sorter analysis that serotype AD strains are aneuploid or diploid. PCR analysis with primers specific for serotype A or D alleles of the CNA1, CLA4, and GPA1 genes revealed that both alleles are often present in serotype AD strains. PCR analysis with primers specific for genes in the MATa or MATalpha mating-type loci revealed that serotype AD strains are heterozygous for the mating-type locus. Interestingly, in several serotype AD strains, the MATalpha locus was derived from the serotype D parent and the MATa locus was inherited from a serotype A parent that has been thought to be extinct. Basidiospores from a self-fertile serotype AD strain bearing the putative serotype A MATa locus showed a very low viability ( approximately 5%), and no fertile serotype A MATa strain could be recovered. Serotype AD strains were virulent in a murine model. Hybrid AD strains could readily be isolated following a laboratory cross between a serotype A strain and a serotype D strain. In summary, serotype AD strains of C. neoformans are unusual aneuploid or diploid strains that result from matings between serotype A and D strains. Self-fertile isolates fail to undergo normal meiosis because of genetic divergence. Our findings further suggest that serotype A MATa strains may exist in nature.     

Specificity of Cryptococcus neoformans factor sera determined by enzyme-linked immunosorbent assay and dot enzyme assay.

An indirect enzyme-linked immunosorbent assay (ELISA) and a dot enzyme assay (DEA) were used to determine the specificities of Cryptococcus neoformans factor sera to serotype type-specific capsular polysaccharides, glucuronoxylomannans (GXMs). Pure and chemically characterized GXMs were obtained from representative isolates of C. neoformans serotypes A, B, C, and D. Distinctive specificity patterns and quantitative differences were observed for each factor serum when the selected GXMs were studied by ELISA. The specificity patterns for each factor serum determined by DEA almost completely paralleled the ELISA results. The serotype specificities demonstrated by ELISA and DEA were similar to previously reported results that were obtained by slide agglutination studies of whole cells. On the basis of the ELISA and DEA activity patterns, factor sera 5, 6, and 8 were specific for serotypes B, C, and D, respectively; factor serum 1 was strongly reactive to all serotypes; factor serum 2 was specific for serotypes A, B, and D; factor serum 3 was specific for serotypes A and D; and factor serum 4 was specific for serotypes B and C. The specificity of factor serum 7 for serotype A was demonstrated by DEA only. Structural variation was indicated among the serotype C isolates studied because a unique activity pattern versus factor serum 6 was observed for each isolate. The quantitative differences in the activity of the GXMs from five serotype C isolates suggest that mannopyranoside residues substituted O-2 and O-4 with xylose are essential elements of the determinant responsible for the observed activity of factor 6. No significant differences in activity patterns and specificities of factor serum 6 were observed when O-deacetylated GXMs were substituted for the native GXMs. Our results show that ELISA and DEA are valuable techniques for the serological analysis of cryptococcal factor sera and GXMs.