Identification of Novel Hybrids Between Cryptococcus neoformans var. grubii VNI and Cryptococcus gattii VGII

Cryptococcus neoformans and Cryptococcus gattii are pathogenic yeasts causing meningoencephalitis in immunocompromised and immunocompetent hosts. The fungus is typically haploid, and sexual reproduction occurs normally between individuals with opposite mating types, α and a. C. neoformans var. grubii (serotype A) is comprised of molecular types VNI, VNII, and VNB, and C. neoformans var. neoformans (serotype D) contains the molecular type VNIV. Additionally, diploid or aneuploid AD hybrids (VNIII) have been reported. C. gattii contains the molecular types VGI, VGII, VGIII, and VGIV, which encompass both serotypes B and C. To identify possible hybrid strains, URA5-RFLP analysis was performed on 350 globally obtained clinical, environmental, and veterinary isolates. Four clinical isolates from cerebrospinal fluid showed combination patterns of C. neoformans var. grubii and C. gattii: Brazil (n = 2), Colombia (n = 1), and India (n = 1). These strains were monokaryotic and diploid or aneuploid. M13 PCR fingerprinting showed that they contained fragments of both proposed parental groups. Luminex IGS genotyping identified these isolates as hybrids with two different molecular type combinations: three VNI/VGII and one VNI/VGI. Blue color development on CGB agar was delayed in three isolates and absent in one. C. gattii-specific PCR confirmed the presence of C. gattii in the hybrids. CAP59 allele-specific PCR revealed that all the hybrids contained both serotype A and B alleles. Determination of mating-type allelic patterns by PCR revealed that the isolates were αA aB. This is the first study discovering novel natural hybrids between C. neoformans molecular type VNI and C. gattii molecular type VGII.     

Regional pattern of the molecular types of Cryptococcus neoformans and Cryptococcus gattii in Brazil

The molecular types of 443 Brazilian isolates of Cryptococcus neoformans and Cryptococcus gattii were analyzed to determine their geographic distribution within Brazil and their underlying host conditions. The following data, imported from previous epidemiological studies as well as two culture collections, were analyzed for: place of isolation, source (clinical or environmental), host risk factors, species, serotype, mating type, and molecular type. Molecular typing by PCR-fingerprinting using primers for the minisatellite-specific core sequence of the wild-type phage M13 or microsatellites [(GACA)4, (GTG)5], restriction fragment length polymorphism of URA5 gene analysis, and/or amplified fragment length polymorphism (AFLP) identified eight major genotypes: VNI/AFLP1, VNII/AFLP1A, VNIII/AFLP2, and VNIV/AFLP3 for C. neoformans, and VGI/AFLP4, VGII/AFLP6, VGIII/AFLP5, and VGIV/AFLP7 for C. gattii. The most common molecular type found in Brazil was VNI (64%), followed by VGII (21%), VNII (5%), VGIII (4%), VGI and VNIV (3% each), and VNIII (< 1%). Primary cryptococcosis caused by the molecular type VGII (serotype B, MAT) prevails in immunocompetent hosts in the North and Northeast regions, disclosing an endemic regional pattern for this specific molecular type in the Northern Brazil.     

Genotype and mating type analysis of Cryptococcus neoformans and Cryptococcus gattii isolates from China that mainly originated from non-HIV-infected patients

Cryptococcosis has been reported to be mostly associated with non-HIV-related patients in China. However, little is known about the molecular characteristics of clinical isolates from the Cryptococcus neoformans species complex in this country. In this study, 115 clinical isolates were included. Molecular type VNI was the most representative ( n =103), followed by VGI ( n =8), VNIII ( n =2), VNIV ( n =1), and VGII ( n =1). With the exception of a serotype D mating type a isolate, all possessed the MAT α locus. Multilocus sequence typing (MLST) revealed that most Cryptococcus gattii isolates from China shared identical MLST profiles with the most common MLST genotype reported in the VGI group, and the only one VGII isolate resembled the Vancouver Island outbreak minor genotype. The C. gattii strains involved in this study were successfully grouped according to their molecular type and mating types by PCR-restriction fragment length polymorphism (RFLP) analysis of the GEF1 gene. Our results suggest that (1) in China, cryptococcosis is mostly caused by C. neoformans var. grubii (molecular type VNI), and mating type α; (2) The most common causative agents of C. gattii infection in China are closely related to a widely distributed MLST genotype; and (3) The PCR-RFLP analysis of the GEF1 gene has the potential to identify the molecular and mating types of C. gattii simultaneously.     

Many globally isolated AD hybrid strains of Cryptococcus neoformans originated in Africa

Interspecific and intervarietal hybridization may contribute to the biological diversity of fungal populations. Cryptococcus neoformans is a pathogenic yeast and the most common fungal cause of meningitis in patients with AIDS. Most patients are infected with either of the two varieties of C. neoformans, designated as serotype A (C. neoformans var. grubii) or serotype D (C. neoformans var. neoformans). In addition, serotype AD strains, which are hybrids of these two varieties, are commonly isolated from clinical and environmental samples. While most isolates of serotype A and serotype D are haploid, AD strains are diploid or aneuploid, and contain two sets of chromosomes and two mating type alleles, MATa and MATalpha, one from each of the serotypes. The global population of serotype A is dominated by isolates with the MATalpha mating type (Aalpha); however, about half of the globally analyzed AD strains possess the extremely rare serotype A MATa allele (Aa). We previously described an unusual population of serotype A in Botswana, in which 25% of the strains contain the rare MATa allele. Here we utilized two methods, phylogenetic analysis of three genes and genotyping by scoring amplified fragment length polymorphisms, and discovered that AD hybrid strains possessing the rare serotype A MATa allele (genotype AaDalpha) cluster with isolates of serotype A from Botswana, whereas AD hybrids that possess the MATalpha serotype A allele (AalphaDa and AalphaDalpha) cluster with cosmopolitan isolates of serotype A. We also determined that AD hybrid strains are more resistant to UV irradiation than haploid serotype A strains from Botswana. These findings support two hypotheses: (i) AaDalpha strains originated in sub-Saharan Africa from a cross between strains of serotypes A and D; and (ii) this fusion produced hybrid strains with increased fitness, enabling the Botswanan serotype A MATa genome, which is otherwise geographically restricted, to survive, emigrate, and propagate throughout the world.     

我国新生隐球菌临床株基因型分析

为了解中国大陆地区新生隐球菌临床株基因型分布情况, 我们对来自我国中东部地区的18个省、直辖市的120株血清A型和9株血清B型新生隐球菌临床株采用PCR指纹分析、IGS序列测定和MLST(Multilocus sequencing test)方法进行分析。结果显示血清A型菌株的M13指纹图谱中主要条带与VNI型标准株相同, 但次要条带与VNI现有各亚型均不相符, 我们将这种新VNI亚型命名为VNIc型, 但VNIc型并不只分布在中国大陆, MLST分析显示中国大陆地区的VNIc型菌株同来自美国、日本、马拉维和巴西的7株菌株亲缘关系高达75, 提示VNIc型可能是一种世界范围内分布的基因型; 对血清A型菌株采用(GACA)4和URA-5 RFLP指纹分析则呈现与VNI标准株完全一致的主次条带; 9株血清B型菌株基因型为VGI型。本研究初步揭示了我国大陆地区新生隐球菌临床株的基因型分布。     

Genetic Diversity of the Cryptococcus Species Complex Suggests that Cryptococcus gattii Deserves to Have Varieties

The Cryptococcus species complex contains two sibling taxa, Cryptococcus neoformans and Cryptococcus gattii. Both species are basidiomycetous yeasts and major pathogens of humans and other mammals. Genotyping methods have identified major haploid molecular types of C. neoformans (VNI, VNII, VNB and VNIV) and of C. gattii (VGI, VGII, VGIII and VGIV). To investigate the phylogenetic relationships among these haploid genotypes, we selected 73 strains from 2000 globally collected isolates investigated in our previous typing studies, representing each of these genotypes and carried out multigene sequence analyses using four genetically unlinked nuclear loci, ACT1, IDE, PLB1 and URA5. The separate or combined sequence analyses of all four loci revealed seven clades with significant support for each molecular type. However, three strains of each species revealed some incongruence between the original molecular type and the sequence-based type obtained here. The topology of the individual gene trees was identical for each clade of C. neoformans but incongruent for the clades of C. gattii indicating recent recombination events within C. gattii. There was strong evidence of recombination in the global VGII population. Both parsimony and likelihood analyses supported three major clades of C. neoformans (VNI/VNB, VNII and VNIV) and four major clades of C. gattii (VGI, VGII, VGIII and VGIV). The sequence variation between VGI, VGIII and VGIV was similar to that between VNI/VNB and VNII. MATa was for the first time identified for VGIV. The VNIV and VGII clades are basal to the C. neoformans or the C. gattii clade, respectively. Divergence times among the seven haploid monophyletic lineages in the Cryptococcus species complex were estimated by applying the hypothesis of the molecular clock. The genetic variation found among all of these haploid monophyletic lineages indicates that they warrant varietal status.     

Comparative hybridization reveals extensive genome variation in the AIDS-associated pathogen Cryptococcus neoformans

Background

Genome variability can have a profound influence on the virulence of pathogenic microbes. The availability of genome sequences for two strains of the AIDS-associated fungal pathogen Cryptococcus neoformans presented an opportunity to use comparative genome hybridization (CGH) to examine genome variability between strains of different mating type, molecular subtype, and ploidy.

Results

Initially, CGH was used to compare the approximately 100 kilobase MAT a and MATα mating-type regions in serotype A and D strains to establish the relationship between the Log2 ratios of hybridization signals and sequence identity. Subsequently, we compared the genomes of the environmental isolate NIH433 (MAT a) and the clinical isolate NIH12 (MATα) with a tiling array of the genome of the laboratory strain JEC21 derived from these strains. In this case, CGH identified putative recombination sites and the origins of specific segments of the JEC21 genome. Similarly, CGH analysis revealed marked variability in the genomes of strains representing the VNI, VNII, and VNB molecular subtypes of the A serotype, including disomy for chromosome 13 in two strains. Additionally, CGH identified differences in chromosome content between three strains with the hybrid AD serotype and revealed that chromosome 1 from the serotype A genome is preferentially retained in all three strains.

Conclusion

The genomes of serotypes A, D, and AD strains exhibit extensive variation that spans the range from small differences (such as regions of divergence, deletion, or amplification) to the unexpected disomy for chromosome 13 in haploid strains and preferential retention of specific chromosomes in naturally occurring diploids.     

Isolates of Cryptococcus neoformans from infected animals reveal genetic exchange in unisexual, alpha mating type populations

Sexual reproduction and genetic exchange are important for the evolution of fungal pathogens and for producing potentially infective spores. Studies to determine whether sex occurs in the pathogenic yeast Cryptococcus neoformans var. grubii have produced enigmatic results, however: basidiospores are the most likely infective propagules, and clinical isolates are fertile and genetically diverse, consistent with a sexual species, but almost all populations examined consist of a single mating type and have little evidence for genetic recombination. The choice of population is critical when looking for recombination, particularly when significant asexual propagation is likely and when latency may complicate assessing the origin of an isolate. We therefore selected isolates from infected animals living in the region of Sydney, Australia, with the assumption that the relatively short life spans and limited travels of the animal hosts would provide a very defined population. All isolates were mating type α and were of molecular genotype VNI or VNII. A lack of linkage disequilibrium among loci suggested that genetic exchange occurred within both genotype groups. Four diploid VNII isolates that produced filaments and basidium-like structures when cultured in proximity to an a mating type strain were found. Recent studies suggest that compatible α-α unions can occur in C. neoformans var. neoformans populations and in populations of the sibling species Cryptococcus gattii. As a mating type strains of C. neoformans var. grubii have never been found in Australia, or in the VNII molecular type globally, the potential for α-α unions is evidence that α-α unisexual mating maintains sexual recombination and diversity in this pathogen and may produce infectious propagules.     

Genotypic Diversity Is Associated with Clinical Outcome and Phenotype in Cryptococcal Meningitis across Southern Africa

Cryptococcal meningitis is a major cause of mortality throughout the developing world, yet little is known about the genetic markers underlying Cryptococcal virulence and patient outcome. We studied a cohort of 230 Cryptococcus neoformans (Cn) isolates from HIV-positive South African clinical trial patients with detailed clinical follow-up using multi-locus sequence typing and in vitro phenotypic virulence assays, correlating these data with clinical and fungal markers of disease in the patient. South African Cn displayed high levels of genetic diversity and locus variability compared to globally distributed types, and we identified 50 sequence types grouped within the main molecular types VNI, VNII and VNB, with 72% of isolates typed into one of seven ''high frequency'' sequence types. Spatial analysis of patients’ cryptococcal genotype was not shown to be clustered geographically, which might argue against recent local acquisition and in favour of reactivation of latent infection. Through comparison of MLST genotyping data with clinical parameters, we found a relationship between genetic lineage and clinical outcome, with patients infected with the VNB lineage having significantly worse survival (n=8, HR 3.35, CI 1.51-7.20, p=0.003), and this was maintained even after adjustment for known prognostic indicators and treatment regimen. Comparison of fungal genotype with in vitro phenotype (phagocytosis, laccase activity and CSF survival) performed on a subset of 89 isolates revealed evidence of lineage-associated virulence phenotype, with the VNII lineage displaying increased laccase activity (p=0.001) and ex vivo CSF survival (p=0.0001). These findings show that Cryptococcus neoformans is a phenotypically heterogeneous pathogen, and that lineage plays an important role in cryptococcal virulence during human infection. Furthermore, a detailed understanding of the genetic diversity in Southern Africa will support further investigation into how genetic diversity is structured across African environments, allowing assessment of the risks different ecotypes pose to infection.     

Serotyping of Dutch Staphylococcus aureus strains from carriage and infection

International epidemiological studies have shown that clinical isolates of Staphylococcus aureus are usually capsulated with either type 5 or 8 capsular polysaccharides (CPs). Because all noncapsulated strains were found to be cross-reactive with polysaccharide 336 (336PS) antibodies, the noncapsulated strains were denoted as type 336PS. The capsular types of 162 Dutch methicillin-susceptible S. aureus strains derived from individuals living in the Rotterdam area were determined. The serotype distribution was 28.4% serotype 5, 53.7% type 8, and 17.9% type 336PS. Serotyping was in agreement with genotyping by amplified fragment length polymorphism (AFLP) and multi locus sequence typing (MLST). Among 49 nasal carriage isolates from healthy children 24.5% belonged to serotype 5, 67.3% were type 8 and 8.2% were type 336PS. For 28 adult patients on chronic ambulatory peritoneal dialysis (CAPD) the serotype incidences among carriage isolates obtained from the nose, catheter exit-site, and abdominal skin were 45.1%, 41.2% and 13.7%, respectively. Among S. aureus strains deriving from blood cultures, the serotype incidences were 17.7% serotype 5, 53.2% type 8, and 29.0% type 336PS. Apparently, type 336PS strains are more prevalent (P=0.017) among bacteraemia isolates as compared with the nasal carriage isolates obtained from healthy children and CAPD patients. In conclusion, all Dutch S. aureus isolates belonged to types 5, 8, or 336PS, which is in agreement with data from other countries. Thus, addition of the 336PS conjugate to a type 5- and type 8-CP protein conjugate vaccine would significantly extend the vaccine coverage.     

Molecular epidemiology of clinical and environmental isolates of the Cryptococcus neoformans species complex reveals a high genetic diversity and the presence of the molecular type VGII mating type a in Colombia

The aim of this study was to investigate the epidemiological relationships of clinical and environmental isolates of the Cryptococcus neoformans species complex in Colombia. The current study reflects data from 1987 to 2004. In Colombia serotypes A and B are most frequently recovered from patients and the environment. Of the 178 clinical isolates studied, 91.1% were of serotype A, 8.4% serotype B and 0.5% serotype C. Of the 247 environmental isolates, 44.2% were of serotype A, 42.6% serotype B and 13.2% serotype C. No serotype D isolates were isolated. Serotype AD has not been recovered in Colombia. PCR fingerprinting with the primers M13, (GACA)4 and (GTG)5 and URA5 gene restriction fragment length polymorphism analysis grouped the majority of clinical serotype A and environmental serotype B isolates into the molecular types VNI (98.1%) and VGII (100%), respectively. Mating type alpha was determined in 99.3% of serotype A isolates, but 96.6% of serotype B isolates were of mating type a. Similar profiles between clinical and environmental isolates suggest that the patients may have acquired the infection from the environment. The data presented form part of the Colombian contribution to the ongoing global survey of the C. neoformans species complex.     

Evidence of sexual recombination among Cryptococcus neoformans serotype A isolates in sub-Saharan Africa

The most common cause of fungal meningitis in humans, Cryptococcus neoformans serotype A, is a basidiomycetous yeast with a bipolar mating system. However, the vast majority (>99.9%) of C. neoformans serotype A isolates possess only one of the two mating type alleles (MATα). Isolates with the other allele (MATa) were recently discovered and proven to mate in the laboratory. It has been a mystery whether and where C. neoformans strains undergo sexual reproduction. Here, we applied population genetic approaches to demonstrate that a population of C. neoformans serotype A clinical isolates from Botswana contains an unprecedented proportion of fertile MATa isolates and exhibits evidence of both clonal expansion and recombination within two partially genetically isolated subgroups. Our findings provide evidence for sexual recombination among some populations of C. neoformans serotype A from sub-Saharan Africa, which may have a direct impact on their evolution.     

Analysis of Clostridium botulinum serotype E strains by using multilocus sequence typing, amplified fragment length polymorphism, variable-number tandem-repeat analysis, and botulinum neurotoxin gene sequencing

A total of 41 Clostridium botulinum serotype E strains from different geographic regions, including Canada, Denmark, Finland, France, Greenland, Japan, and the United States, were compared by multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) analysis, variable-number tandem-repeat (VNTR) analysis, and botulinum neurotoxin (bont) E gene sequencing. The strains, representing environmental, food-borne, and infant botulism samples collected from 1932 to 2007, were analyzed to compare serotype E strains from different geographic regions and types of botulism and to determine whether each of the strains contained the transposon-associated recombinase rarA, involved with bont/E insertion. MLST examination using 15 genes clustered the strains into several clades, with most members within a cluster sharing the same BoNT/E subtype (BoNT/E1, E2, E3, or E6). Sequencing of the bont/E gene identified two new variants (E7, E8) that showed regions of recombination with other E subtypes. The AFLP dendrogram clustered the 41 strains similarly to the MLST dendrogram. Strains that could not be differentiated by AFLP, MLST, or bont gene sequencing were further examined using three VNTR regions. Both intact and split rarA genes were amplified by PCR in each of the strains, and their identities were confirmed in 11 strains by amplicon sequencing. The findings suggest that (i) the C. botulinum serotype E strains result from the targeted insertion of the bont/E gene into genetically conserved bacteria and (ii) recombination events (not random mutations) within bont/E result in toxin variants or subtypes within strains.     

Low diversity Cryptococcus neoformans variety grubii multilocus sequence types from Thailand are consistent with an ancestral African origin

The global burden of HIV-associated cryptococcal meningitis is estimated at nearly one million cases per year, causing up to a third of all AIDS-related deaths. Molecular epidemiology constitutes the main methodology for understanding the factors underpinning the emergence of this understudied, yet increasingly important, group of pathogenic fungi. Cryptococcus species are notable in the degree that virulence differs amongst lineages, and highly-virulent emerging lineages are changing patterns of human disease both temporally and spatially. Cryptococcus neoformans variety grubii (Cng, serotype A) constitutes the most ubiquitous cause of cryptococcal meningitis worldwide, however patterns of molecular diversity are understudied across some regions experiencing significant burdens of disease. We compared 183 clinical and environmental isolates of Cng from one such region, Thailand, Southeast Asia, against a global MLST database of 77 Cng isolates. Population genetic analyses showed that Thailand isolates from 11 provinces were highly homogenous, consisting of the same genetic background (globally known as VNI) and exhibiting only ten nearly identical sequence types (STs), with three (STs 44, 45 and 46) dominating our sample. This population contains significantly less diversity when compared against the global population of Cng, specifically Africa. Genetic diversity in Cng was significantly subdivided at the continental level with nearly half (47%) of the global STs unique to a genetically diverse and recombining population in Botswana. These patterns of diversity, when combined with evidence from haplotypic networks and coalescent analyses of global populations, are highly suggestive of an expansion of the Cng VNI clade out of Africa, leading to a limited number of genotypes founding the Asian populations. Divergence time testing estimates the time to the most common ancestor between the African and Asian populations to be 6,920 years ago (95% HPD 122.96 - 27,177.76). Further high-density sampling of global Cng STs is now necessary to resolve the temporal sequence underlying the global emergence of this human pathogen.     

Presence of pathogenic cryptococci on trees situated in two recreational areas in South Africa

Knowledge of the environmental prevalence of members of the Cryptococcus neoformans/Cryptococcus gattii species complex is important, since cryptococcal infection is acquired from the environment. We determined whether trees located in two South African recreational areas harboured pathogenic cryptococci and compared the isolates to clinical isolates obtained from Western Cape hospitals with molecular typing techniques. The majority of isolates originating from trees in a public park in Cape Town (PPCT) were C. gattii sensu stricto, followed by C. neoformans sensu stricto genotype AFLP1/VNI. The PPCT trees might be a source of infection, since all genotype AFLP1/VNI isolates from these trees and one clinical isolate belonged to the same sequence type (ST), i.e. ST23. Recombination and basidiospore production might be occurring in PPCT trees that contained C. gattii s.s. isolates belonging to both mating types. The presence of C. gattii s.s. in PPCT trees might therefore pose a risk to human health.     

Comparative Genotyping of Campylobacter jejuni Strains from Patients with Guillain-Barré Syndrome in Bangladesh

Background

Campylobacter jejuni is a common cause of acute gastroenteritis and is associated with post-infectious neuropathies such as the Guillain-Barré syndrome (GBS) and the Miller Fisher syndrome (MFS). We here present comparative genotyping of 49 C. jejuni strains from Bangladesh that were recovered from patients with enteritis or GBS. All strains were serotyped and analyzed by lipo-oligosaccharide (LOS) genotyping, amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE).

Methodology/Principal Findings

C. jejuni HS:23 was a predominant serotype among GBS patients (50%), and no specific serotype was significantly associated with GBS compared to enteritis. PCR screening showed that 38/49 (78%) of strains could be assigned to LOS classes A, B, C, or E. The class A locus (4/7 vs 3/39; p<0.01) was significantly associated in the GBS-related strains as compared to enteritis strains. All GBS/oculomotor related strains contained the class B locus; which was also detected in 46% of control strains. Overlapping clonal groups were defined by MLST, AFLP and PFGE for strains from patients with gastroenteritis and GBS. MLST defined 22 sequence types (STs) and 7 clonal complexes including 7 STs not previously identified (ST-3742, ST-3741, ST-3743, ST-3748, ST-3968, ST-3969 and ST-3970). C. jejuni HS:23 strains from patients with GBS or enteritis were clonal and all strains belonged to ST-403 complex. Concordance between LOS class B and ST-403 complex was revealed. AFLP defined 25 different types at 90% similarity. The predominant AFLP type AF-20 coincided with the C. jejuni HS:23 and ST-403 complex.

Conclusion/Significance

LOS genotyping, MLST, AFLP and PFGE helped to identify the HS:23 strains from GBS or enteritis patients as clonal. Overall, genotypes exclusive for enteritis or for GBS-related strains were not obtained although LOS class A was significantly associated with GBS strains. Particularly, the presence of a clonal and putative neuropathogenic C. jejuni HS:23 serotype may contribute to the high prevalence of C. jejuni related GBS in Bangladesh.     

Molecular Epidemiology Reveals Genetic Diversity amongst Isolates of the Cryptococcus neoformans/C. gattii Species Complex in Thailand

To gain a more detailed picture of cryptococcosis in Thailand, a retrospective study of 498 C. neoformans and C. gattii isolates has been conducted. Among these, 386, 83 and 29 strains were from clinical, environmental and veterinary sources, respectively. A total of 485 C. neoformans and 13 C. gattii strains were studied. The majority of the strains (68.9%) were isolated from males (mean age of 37.97 years), 88.5% of C. neoformans and only 37.5% of C. gattii strains were from HIV patients. URA5-RFLP and/or M13 PCR-fingerprinting analysis revealed that the majority of the isolates were C. neoformans molecular type VNI regardless of their sources (94.8%; 94.6% of the clinical, 98.8% of the environmental and 86.2% of the veterinary isolates). In addition, the molecular types VNII (2.4%; 66.7% of the clinical and 33.3% of the veterinary isolates), VNIV (0.2%; 100% environmental isolate), VGI (0.2%; 100% clinical isolate) and VGII (2.4%; 100% clinical isolates) were found less frequently. Multilocus Sequence Type (MLST) analysis using the ISHAM consensus MLST scheme for the C. neoformans/C. gattii species complex identified a total of 20 sequence types (ST) in Thailand combining current and previous data. The Thai isolates are an integrated part of the global cryptococcal population genetic structure, with ST30 for C. gattii and ST82, ST83, ST137, ST141, ST172 and ST173 for C. neoformans being unique to Thailand. Most of the C. gattii isolates were ST7 = VGIIb, which is identical to the less virulent minor Vancouver island outbreak genotype, indicating Thailand as a stepping stone in the global spread of this outbreak strain. The current study revealed a greater genetic diversity and a wider range of major molecular types being present amongst Thai cryptococcal isolates than previously reported.     

Cryptococcus neoformans var. grubii - Pathogenicity of environmental isolates correlated to virulence factors, susceptibility to fluconazole and molecular profile

The pathogenicity of Cryptococcus neoformans is heterogeneous and is associated with the expression of virulence factors. This study aimed to correlate the pathogenicity of C. neoformans var. grubii in BALB/c mice with in vitro virulence factors, fluconazole minimal inhibitory concentrations (MICs) and molecular profiles, before and after animal passage. Ten environmental isolates and one ATCC strain of C. neoformans var. grubii mating type α were evaluated. Most isolates (91%) killed 50% or more of the infected animals by day 24 postinfection and were recovered from the lungs and brains of surviving animals on days 7 and 14 postinfection. The burden of yeast in the lungs was more variable than that in the brain. The differences in the expression of virulence factors (growth at 37oC, presence and size of the capsule and production of melanin, urease, proteinase and phospholipase) by most isolates pre and postpassage in animals were not statistically significant. The fluconazole MICs in postpassaged lines differed by a one-dilution from the MIC of the corresponding prepassaged line for six isolates. Using molecular typing [polymerase chain reaction-fingerprinting with (GACA)4 and M13], eight isolates were identified as VNI and three as VNII. We concluded that different isolates with the same molecular and phenotypic profiles, including isolates that are markedly hypervirulent, span a wide range of virulence and there were no changes in virulence factors in the postpassaged lines when compared with the corresponding nonpassaged lines.     

Genotypic characterization of Salmonella by multilocus sequence typing, pulsed-field gel electrophoresis and amplified fragment length polymorphism

Molecular typing is an important tool in surveillance and outbreak investigations of human Salmonella infections. In this study, three molecular typing methods were used to investigate the discriminatory ability, reproducibility and the genetic relationship between 110 Salmonella enterica subspecies enterica isolates. A total of 25 serotypes were investigated that had been isolated from humans or veterinary sources in Denmark between 1995 and 2001. All isolates were genotyped by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP). When making genetic trees, all three methods resulted in similar clustering that often corresponded with serotype, although some serotypes displayed more diversity than others. Of the three techniques, MLST was the easiest to interpret and compare between laboratories. Unfortunately the seven housekeeping genes used in this MLST scheme lacked diversity and the ability to discriminate between isolates were higher with both PFGE and AFLP. The discriminatory power of AFLP and PFGE were similar but PFGE fingerprints were both easier to reproduce, interpret and less time-consuming to analyze when compared to AFLP. PFGE is the therefore the preferred molecular typing method for surveillance and outbreak investigations, whereas AFLP is most useful for local outbreak investigations.     

不同毒力差异基因型的新生隐球菌MLST分型及交配型鉴定研究

目的：了解及比较两组毒力差异明显的新生隐球菌格鲁比变种的多位点序列分型（MLST）的特点并进行交配型鉴定。方法采用多位点序列分型（MLST）的方法,设计7个看家基因（CAP59,GPD1,LAC1,PLB1,SOD1,URA5和IGS1）的引物,扩增并分析来源分别为环境和临床的各10株新生隐球菌格鲁比变种的基因型,并鉴定实验菌株交配型,与多位点微卫星分型（MLMT）结果对比,比较不同基因分型方法在分类中的稳定性和可靠性。结果在微卫星分型中为MLMT-36的10株环境分离株,MLST分型为ST-15,而在微卫星分型中为MLMT-13型的10株临床分离株,MLST分型为ST-32,所有菌株交配型均为MAT-α。结论MLST分型结果与MLMT分型结果高度一致,提示以上两种分子分型技术在真菌分类鉴定研究中可显示对于其分离背景及进化来源的高分辨率及稳定性。