Emergence and Pathogenicity of Highly Virulent Cryptococcus gattii Genotypes in the Northwest United States

Cryptococcus gattii causes life-threatening disease in otherwise healthy hosts and to a lesser extent in immunocompromised hosts. The highest incidence for this disease is on Vancouver Island, Canada, where an outbreak is expanding into neighboring regions including mainland British Columbia and the United States. This outbreak is caused predominantly by C. gattii molecular type VGII, specifically VGIIa/major. In addition, a novel genotype, VGIIc, has emerged in Oregon and is now a major source of illness in the region. Through molecular epidemiology and population analysis of MLST and VNTR markers, we show that the VGIIc group is clonal and hypothesize it arose recently. The VGIIa/IIc outbreak lineages are sexually fertile and studies support ongoing recombination in the global VGII population. This illustrates two hallmarks of emerging outbreaks: high clonality and the emergence of novel genotypes via recombination. In macrophage and murine infections, the novel VGIIc genotype and VGIIa/major isolates from the United States are highly virulent compared to similar non-outbreak VGIIa/major-related isolates. Combined MLST-VNTR analysis distinguishes clonal expansion of the VGIIa/major outbreak genotype from related but distinguishable less-virulent genotypes isolated from other geographic regions. Our evidence documents emerging hypervirulent genotypes in the United States that may expand further and provides insight into the possible molecular and geographic origins of the outbreak.     

Characterization of environmental sources of the human and animal pathogen Cryptococcus gattii in British Columbia, Canada, and the Pacific Northwest of the United States

Cryptococcus gattii has recently emerged as a primary pathogen of humans and wild and domesticated animals in British Columbia, particularly on Vancouver Island. C. gattii infections are typically infections of the pulmonary and/or the central nervous system, and the incidence of infection in British Columbia is currently the highest reported globally. Prior to this emergence, the environmental distribution of and the extent of colonization by C. gattii in British Columbia were unknown. We characterized the environmental sources and potential determinants of colonization in British Columbia. C. gattii was isolated from tree surfaces, soil, air, freshwater, and seawater, and no seasonal prevalence was observed. The C. gattii concentrations in air samples were significantly higher during the warm, dry summer months, although potentially infectious propagules (<3.3 microm in diameter) were present throughout the year. Positive samples were obtained from many different areas of British Columbia, and some locations were colonization "hot spots." C. gattii was generally isolated from acidic soil, and geographic differences in soil pH may influence the extent of colonization. C. gattii soil colonization also was associated with low moisture and low organic carbon contents. Most of the C. gattii isolates recovered belonged to the VGIIa genetic subtype; however, sympatric colonization by the VGIIb strain was observed at most locations. At one sampling site, VGIIa, VGIIb, VGI, and the Cryptococcus neoformans serotype AD hybrid all were coisolated. Our findings indicate extensive colonization by C. gattii within British Columbia and highlight an expansion of the ecological niche of this pathogen.     

A Decade of Experience: Cryptococcus gattii in British Columbia

It has been over a decade since Cryptococcus gattii was first recognized as the causative organism of an outbreak of cryptococcosis on Vancouver Island, British Columbia. A number of novel observations have been associated with the study of this emergent pathogen. A novel genotype of C. gattii, VGIIa was described as the major genotype associated with clinical disease. Minor genotypes, VGIIb and VGI, are also responsible for disease in British Columbians, in both human and animal populations. The clinical major genotype VGIIa and minor genotype VGIIb are identical to C. gattii isolated from the environment of Vancouver Island. There is more heterogeneity in VGI, and a clear association with the environment is not apparent. Between 1999 and 2010, there have been 281 cases of C. gattii cryptococcosis. Risk factors for infection are reported to be age greater than 50 years, history of smoking, corticosteroid use, HIV infection, and history of cancer or chronic lung disease. The major C. gattii genotype VGIIa is as virulent in mice as the model Cryptococcus, H99 C. neoformans, although the outbreak strain produces a less protective inflammatory response in C57BL/6 mice. The minor genotype VGIIb is significantly less virulent in mouse models. Cryptococcus gattii is found associated with native trees and soil on Vancouver Island. Transiently positive isolations have been made from air and water. An ecological niche for this organism is associated within a limited biogeoclimatic zone characterized by daily average winter temperatures above freezing.     

Single-nucleotide polymorphism phylotyping of Escherichia coli

We describe a rapid and easily automated phylogenetic grouping technique based on analysis of bacterial genome single-nucleotide polymorphisms (SNPs). We selected 13 SNPs derived from a complete sequence analysis of 11 essential genes previously used for multilocus sequence typing (MLST) of 30 Escherichia coli strains representing the genetic diversity of the species. The 13 SNPs were localized in five genes, trpA, trpB, putP, icdA, and polB, and were selected to allow recovery of the main phylogenetic groups (groups A, B1, E, D, and B2) and subgroups of the species. In the first step, we validated the SNP approach in silico by extracting SNP data from the complete sequences of the five genes for a panel of 65 pathogenic strains belonging to different E. coli pathovars, which were previously analyzed by MLST. In the second step, we determined these SNPs by dideoxy single-base extension of unlabeled oligonucleotide primers for a collection of 183 commensal and extraintestinal clinical E. coli isolates and compared the SNP phylotyping method to previous well-established typing methods. This SNP phylotyping method proved to be consistent with the other methods for assigning phylogenetic groups to the different E. coli strains. In contrast to the other typing methods, such as multilocus enzyme electrophoresis, ribotyping, or PCR phylotyping using the presence/absence of three genomic DNA fragments, the SNP typing method described here is derived from a solid phylogenetic analysis, and the results obtained by this method are more meaningful. Our results indicate that similar approaches may be used for a wide variety of bacterial species.     

Single-Nucleotide Polymorphism Phylotyping of Escherichia coli

We describe a rapid and easily automated phylogenetic grouping technique based on analysis of bacterial genome single-nucleotide polymorphisms (SNPs). We selected 13 SNPs derived from a complete sequence analysis of 11 essential genes previously used for multilocus sequence typing (MLST) of 30 Escherichia coli strains representing the genetic diversity of the species. The 13 SNPs were localized in five genes, trpA, trpB, putP, icdA, and polB, and were selected to allow recovery of the main phylogenetic groups (groups A, B1, E, D, and B2) and subgroups of the species. In the first step, we validated the SNP approach in silico by extracting SNP data from the complete sequences of the five genes for a panel of 65 pathogenic strains belonging to different E. coli pathovars, which were previously analyzed by MLST. In the second step, we determined these SNPs by dideoxy single-base extension of unlabeled oligonucleotide primers for a collection of 183 commensal and extraintestinal clinical E. coli isolates and compared the SNP phylotyping method to previous well-established typing methods. This SNP phylotyping method proved to be consistent with the other methods for assigning phylogenetic groups to the different E. coli strains. In contrast to the other typing methods, such as multilocus enzyme electrophoresis, ribotyping, or PCR phylotyping using the presence/absence of three genomic DNA fragments, the SNP typing method described here is derived from a solid phylogenetic analysis, and the results obtained by this method are more meaningful. Our results indicate that similar approaches may be used for a wide variety of bacterial species.     

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.     

Population Structure of Clinical and Environmental Vibrio parahaemolyticus from the Pacific Northwest Coast of the United States

Vibrio parahaemolyticus is a common marine bacterium and a leading cause of seafood-borne bacterial gastroenteritis worldwide. Although this bacterium has been the subject of much research, the population structure of cold-water populations remains largely undescribed. We present a broad phylogenetic analysis of clinical and environmental V. parahaemolyticus originating largely from the Pacific Northwest coast of the United States. Repetitive extragenic palindromic PCR (REP-PCR) separated 167 isolates into 39 groups and subsequent multilocus sequence typing (MLST) separated a subset of 77 isolates into 24 sequence types. The Pacific Northwest population exhibited a semi-clonal structure attributed to an environmental clade (ST3, N = 17 isolates) clonally related to the pandemic O3:K6 complex and a clinical clade (ST36, N = 20 isolates) genetically related to a regionally endemic O4:K12 complex. Further, the identification of at least five additional clinical sequence types (i.e., ST43, 50, 65, 135 and 417) demonstrates that V. parahaemolyticus gastroenteritis in the Pacific Northwest is polyphyletic in nature. Recombination was evident as a significant source of genetic diversity and in particular, the recA and dtdS alleles showed strong support for frequent recombination. Although pandemic-related illnesses were not documented during the study, the environmental occurrence of the pandemic clone may present a significant threat to human health and warrants continued monitoring. It is evident that V. parahaemolyticus population structure in the Pacific Northwest is semi-clonal and it would appear that multiple sequence types are contributing to the burden of disease in this region.     

Four Genotyping Schemes for Phylogenetic Analysis of Pseudomonas aeruginosa: Comparison of Their Congruence with Multi-Locus Sequence Typing

Several molecular typing schemes have been proposed to differentiate among isolates and clonal groups, and hence establish epidemiological or phylogenetic links. It has been widely accepted that multi-locus sequence typing (MLST) is the gold standard for phylogenetic typing/long-term epidemiological surveillance, but other recently described methods may be easier to carry out, especially in settings with limited access to DNA sequencing. Comparing the performance of such techniques to MLST is therefore of relevance. A study was therefore carried out with a collection of P. aeruginosa strains (n = 133) typed by four typing schemes: MLST, multiple-locus variable number tandem repeat analysis (MLVA), pulsed-field gel electrophoresis (PFGE) and the commercial DiversiLab microbial typing system (DL). The aim of this study was to compare the results of each typing method with MLST. The Simpson''s indices of diversity were 0.989, 0.980, 0.961 and 0.906 respectively for PFGE, MLVA, DL and MLST. The congruence between techniques was measured by the adjusted Wallace index (W): this coefficient indicates the probability that a pair of isolates which is assigned to the same type by one typing method is also typed as identical by the other. In this context, the congruence between techniques was recorded as follow: MLVA-type to predict MLST-type (93%), PFGE to MLST (92%), DL to MLST (64.2%), PFGE to MLVA (63.5%) and PFGE to DL (61.7%). Conversely, for all above combinations, prediction was very poor. The congruence was increased at the clonal complex (CC) level. MLST is regarded the gold standard for phylogenetic classification of bacteria, but is rather laborious to carry out in many settings. Our data suggest that MLVA can predict the MLST-type with high accuracy, and even higher when studying the clonal complex level. Of the studied three techniques MLVA was therefore the best surrogate method to predict MLST.     

Whole Genome Sequence Typing to Investigate the Apophysomyces Outbreak following a Tornado in Joplin,Missouri, 2011

Case reports of Apophysomyces spp. in immunocompetent hosts have been a result of traumatic deep implantation of Apophysomyces spp. spore-contaminated soil or debris. On May 22, 2011 a tornado occurred in Joplin, MO, leaving 13 tornado victims with Apophysomyces trapeziformis infections as a result of lacerations from airborne material. We used whole genome sequence typing (WGST) for high-resolution phylogenetic SNP analysis of 17 outbreak Apophysomyces isolates and five additional temporally and spatially diverse Apophysomyces control isolates (three A. trapeziformis and two A. variabilis isolates). Whole genome SNP phylogenetic analysis revealed three clusters of genotypically related or identical A. trapeziformis isolates and multiple distinct isolates among the Joplin group; this indicated multiple genotypes from a single or multiple sources. Though no linkage between genotype and location of exposure was observed, WGST analysis determined that the Joplin isolates were more closely related to each other than to the control isolates, suggesting local population structure. Additionally, species delineation based on WGST demonstrated the need to reassess currently accepted taxonomic classifications of phylogenetic species within the genus Apophysomyces.     

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.     

Phylogenetic incongruence in E. coli O104: understanding the evolutionary relationships of emerging pathogens in the face of homologous recombination

Escherichia coli O104:H4 was identified as an emerging pathogen during the spring and summer of 2011 and was responsible for a widespread outbreak that resulted in the deaths of 50 people and sickened over 4075. Traditional phenotypic and genotypic assays, such as serotyping, pulsed field gel electrophoresis (PFGE), and multilocus sequence typing (MLST), permit identification and classification of bacterial pathogens, but cannot accurately resolve relationships among genotypically similar but pathotypically different isolates. To understand the evolutionary origins of E. coli O104:H4, we sequenced two strains isolated in Ontario, Canada. One was epidemiologically linked to the 2011 outbreak, and the second, unrelated isolate, was obtained in 2010. MLST analysis indicated that both isolates are of the same sequence type (ST678), but whole-genome sequencing revealed differences in chromosomal and plasmid content. Through comprehensive phylogenetic analysis of five O104:H4 ST678 genomes, we identified 167 genes in three gene clusters that have undergone homologous recombination with distantly related E. coli strains. These recombination events have resulted in unexpectedly high sequence diversity within the same sequence type. Failure to recognize or adjust for homologous recombination can result in phylogenetic incongruence. Understanding the extent of homologous recombination among different strains of the same sequence type may explain the pathotypic differences between the ON2010 and ON2011 strains and help shed new light on the emergence of this new pathogen.     

Core genome conservation of Staphylococcus haemolyticus limits sequence based population structure analysis

The notoriously multi-resistant Staphylococcus haemolyticus is an emerging pathogen causing serious infections in immunocompromised patients. Defining the population structure is important to detect outbreaks and spread of antimicrobial resistant clones. Currently, the standard typing technique is pulsed-field gel electrophoresis (PFGE). In this study we describe novel molecular typing schemes for S. haemolyticus using multi locus sequence typing (MLST) and multi locus variable number of tandem repeats (VNTR) analysis. Seven housekeeping genes (MLST) and five VNTR loci (MLVF) were selected for the novel typing schemes. A panel of 45 human and veterinary S. haemolyticus isolates was investigated. The collection had diverse PFGE patterns (38 PFGE types) and was sampled over a 20 year-period from eight countries. MLST resolved 17 sequence types (Simpsons index of diversity [SID]=0.877) and MLVF resolved 14 repeat types (SID=0.831). We found a low sequence diversity. Phylogenetic analysis clustered the isolates in three (MLST) and one (MLVF) clonal complexes, respectively. Taken together, neither the MLST nor the MLVF scheme was suitable to resolve the population structure of this S. haemolyticus collection. Future MLVF and MLST schemes will benefit from addition of more variable core genome sequences identified by comparing different fully sequenced S. haemolyticus genomes.     

Limitations of variable number of tandem repeat typing identified through whole genome sequencing of Mycobacterium avium subsp. paratuberculosis on a national and herd level

Background

Mycobacterium avium subsp. paratuberculosis (MAP), the causative bacterium of Johne’s disease in dairy cattle, is widespread in the Canadian dairy industry and has significant economic and animal welfare implications. An understanding of the population dynamics of MAP can be used to identify introduction events, improve control efforts and target transmission pathways, although this requires an adequate understanding of MAP diversity and distribution between herds and across the country. Whole genome sequencing (WGS) offers a detailed assessment of the SNP-level diversity and genetic relationship of isolates, whereas several molecular typing techniques used to investigate the molecular epidemiology of MAP, such as variable number of tandem repeat (VNTR) typing, target relatively unstable repetitive elements in the genome that may be too unpredictable to draw accurate conclusions. The objective of this study was to evaluate the diversity of bovine MAP isolates in Canadian dairy herds using WGS and then determine if VNTR typing can distinguish truly related and unrelated isolates.

Results

Phylogenetic analysis based on 3,039 SNPs identified through WGS of 124 MAP isolates identified eight genetically distinct subtypes in dairy herds from seven Canadian provinces, with the dominant type including over 80% of MAP isolates. VNTR typing of 527 MAP isolates identified 12 types, including “bison type” isolates, from seven different herds. At a national level, MAP isolates differed from each other by 1–2 to 239–240 SNPs, regardless of whether they belonged to the same or different VNTR types. A herd-level analysis of MAP isolates demonstrated that VNTR typing may both over-estimate and under-estimate the relatedness of MAP isolates found within a single herd.

Conclusions

The presence of multiple MAP subtypes in Canada suggests multiple introductions into the country including what has now become one dominant type, an important finding for Johne’s disease control. VNTR typing often failed to identify closely and distantly related isolates, limiting the applicability of using this typing scheme to study the molecular epidemiology of MAP at a national and herd-level.     

Genetic Diversity and Ecological Success of Staphylococcus aureus Strains Colonizing Humans

The genetic determinants and phenotypic traits which make a Staphylococcus aureus strain a successful colonizer are largely unknown. The genetic diversity and population structure of 133 S. aureus isolates from healthy, generally risk-free adult carriers were investigated using four different typing methods: multilocus sequence typing (MLST), amplified fragment length polymorphism analysis (AFLP), double-locus sequence typing (DLST), and spa typing were compared. Carriage isolates displayed great genetic diversity which could only be revealed fully by DLST. Results of AFLP and MLST were highly concordant in the delineation of genotypic clusters of closely related isolates, roughly equivalent to clonal complexes. spa typing and DLST provided considerably less phylogenetic information. The resolution of spa typing was similar to that of AFLP and inferior to that of DLST. AFLP proved to be the most universal method, combining a phylogeny-building capacity similar to that of MLST with a much higher resolution. However, it had a lower reproducibility than sequencing-based MLST, DLST, and spa typing. We found two cases of methicillin-resistant S. aureus colonization, both of which were most likely associated with employment at a health service. Of 21 genotypic clusters detected, 2 were most prevalent: cluster 45 and cluster 30 each colonized 24% of the carrier population. The number of bacteria found in nasal samples varied significantly among the clusters, but the most prevalent clusters were not particularly numerous in the nasal samples. We did not find much evidence that genotypic clusters were associated with different carrier characteristics, such as age, sex, medical conditions, or antibiotic use. This may provide empirical support for the idea that genetic clusters in bacteria are maintained in the absence of adaptation to different niches. Alternatively, carrier characteristics other than those evaluated here or factors other than human hosts may exert selective pressure maintaining genotypic clusters.     

High-resolution melting genotyping of Enterococcus faecium based on multilocus sequence typing derived single nucleotide polymorphisms

We have developed a single nucleotide polymorphism (SNP) nucleated high-resolution melting (HRM) technique to genotype Enterococcus faecium. Eight SNPs were derived from the E. faecium multilocus sequence typing (MLST) database and amplified fragments containing these SNPs were interrogated by HRM. We tested the HRM genotyping scheme on 85 E. faecium bloodstream isolates and compared the results with MLST, pulsed-field gel electrophoresis (PFGE) and an allele specific real-time PCR (AS kinetic PCR) SNP typing method. In silico analysis based on predicted HRM curves according to the G+C content of each fragment for all 567 sequence types (STs) in the MLST database together with empiric data from the 85 isolates demonstrated that HRM analysis resolves E. faecium into 231 "melting types" (MelTs) and provides a Simpson's Index of Diversity (D) of 0.991 with respect to MLST. This is a significant improvement on the AS kinetic PCR SNP typing scheme that resolves 61 SNP types with D of 0.95. The MelTs were concordant with the known ST of the isolates. For the 85 isolates, there were 13 PFGE patterns, 17 STs, 14 MelTs and eight SNP types. There was excellent concordance between PFGE, MLST and MelTs with Adjusted Rand Indices of PFGE to MelT 0.936 and ST to MelT 0.973. In conclusion, this HRM based method appears rapid and reproducible. The results are concordant with MLST and the MLST based population structure.     

MLST clustering of Campylobacter jejuni isolates from patients with gastroenteritis,reactive arthritis and Guillain–Barré syndrome

Aims: To determine the diversity and population structure of Campylobacter jejuni (C. jejuni) isolates from Danish patients and to examine the association between multilocus sequence typing types and different clinical symptoms including gastroenteritis (GI), Guillain–Barré syndrome (GBS) and reactive arthritis (RA). Methods and Results: Multilocus sequence typing (MLST) was used to characterize 122 isolates, including 18 from patients with RA and 8 from patients with GBS. The GI and RA isolates were collected in Denmark during 2002–2003 and the GBS isolates were obtained from other countries. In overall, 51 sequence types (STs) were identified within 18 clonal complexes (CCs). Of these three CCs, ST‐21, ST‐45 and ST‐22 clonal complexes accounted for 64 percent of all isolates. The GBS isolates in this study significantly grouped into the ST‐22 clonal complex, consistent with the PubMLST database isolates. There was no significant clustering of the RA isolates. Conclusions: Isolates from Denmark were found to be highly genetically diverse. GBS isolates grouped significantly with clonal complex ST‐22, but the absence of clustering of RA isolates indicated that the phylogenetic background for this sequela could not be reconstructed using variation in MLST loci. Possibly, putative RA‐associated genes may vary, by recombination or expression differences, independent of MLST loci. Significance and Impact of the Study: MLST typing of C. jejuni isolates from Danish patients with gastroenteritis confirmed that the diversity of clones in Denmark is comparable to that in other European countries. Furthermore, a verification of the grouping of GBS isolates compared to RA isolates provides information about evolution of the bacterial population resulting in this important sequela.     

A diverse population of Cryptococcus gattii molecular type VGIII in southern Californian HIV/AIDS patients

Cryptococcus gattii infections in southern California have been reported in patients with HIV/AIDS. In this study, we examined the molecular epidemiology, population structure, and virulence attributes of isolates collected from HIV/AIDS patients in Los Angeles County, California. We show that these isolates consist almost exclusively of VGIII molecular type, in contrast to the VGII molecular type isolates causing the North American Pacific Northwest outbreak. The global VGIII population structure can be divided into two molecular groups, VGIIIa and VGIIIb. Isolates from the Californian patients are virulent in murine and macrophage models of infection, with VGIIIa significantly more virulent than VGIIIb. Several VGIII isolates are highly fertile and produce abundant sexual spores that may serve as infectious propagules. The a and α VGIII MAT locus alleles are largely syntenic with limited rearrangements compared to the known VGI (a/α) and VGII (α) MAT loci, but each has unique characteristics including a distinct deletion flanking the 5' VGIII MATa alleles and the α allele is more heterogeneous than the a allele. Our studies indicate that C. gattii VGIII is endemic in southern California, with other isolates originating from the neighboring regions of Mexico, and in rarer cases from Oregon and Washington state. Given that >1,000,000 cases of cryptococcal infection and >620,000 attributable mortalities occur annually in the context of the global AIDS pandemic, our findings suggest a significant burden of C. gattii may be unrecognized, with potential prognostic and therapeutic implications. These results signify the need to classify pathogenic Cryptococcus cases and highlight possible host differences among the C. gattii molecular types influencing infection of immunocompetent (VGI/VGII) vs. immunocompromised (VGIII/VGIV) hosts.     

Comparison of two DNA sequence-based typing schemes for the Fusarium solani Species Complex and proposal of a new consensus method

Multilocus sequence typing (MLST) is a widely used approach for differentiating microbial isolates presenting many advantages such as easy access through online databases and straightforward interpretation. For the Fusarium solani species complex (FSSC), three gene regions have been widely used to investigate phylogenetic relationships at the interspecific level (ITS-nuLSU, EF1a, RPB2) and a nomenclature system has been proposed for the different known haplotypes. More recently, a MLST scheme was proposed for this species complex based on the polymorphisms of five housekeeping genes (ACC, ICL, GDP, MDP, SOD). Here, we compare the phylogenetic resolution and sequence discriminatory powers of these two sets of loci on 50 epidemiologically unrelated FSSC strains. Although the widely used gene set offers better phylogenetic resolution, the newly developed gene set is slightly better at discriminating isolates using a MLST method. A consensus scheme of eight loci is proposed for typing FSSC strains combining the advantages of the two previous gene sets and offering the best typing efficiency.