Clinical and microbiological characteristics of Cryptococcus gattii isolated from 7 hospitals in China

Background Infection, even outbreak, caused by Cryptococcus gattii (C. gattii) has been reported in Canada and the United States, but there were sparsely-reported cases of C. gattii in China. Our interest in occurrence, clinical manifestation, laboratory identification and molecular characterization of Chinese C. gattii strains leads us to this research. Results Out of 254 clinical isolates, initially identified as Cryptococcus neoformans (C. neoformans), eight strains were re-identified as C. gattii. Multi-locus sequence typing (MLST) showed genotype VGI accounted for the most (6 / 8), the other two strains were genotype VGII (VGIIa and VGIIb respectively) with 3 specific spectra of molecular weight about 4342, 8686, 9611 Da by MALDI-TOF MS. The minimal inhibitory concentrations (MICs) of Fluconazole with Yeast one was 2~4 times higher than that with ATB fungus 3 and MICs of antifungal agents against VGII strains were higher than against VGI strains. Comparative proteome analysis showed that 329 and 180 proteins were highly expressed by C. gattii VGI and VGII respectively. The enrichment of differentially expressed proteins was directed to Golgi complex. Conclusions Infection by C. gattii in China occurred sparsely. Genotype VGI was predominant but VGII was more resistant to antifungal agents. There was significant difference in protein expression profile between isolates of VGI and VGII C. gattii.


Results
Identification and antifungal susceptibility profile of C. gattii isolates Among 254 collected Cryptococcal strains, eight showed blue pigmentation on canavanine glycine bromothymol blue (CGB) agar and were characterized as C.gattii by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with scores from 2.043 to 2.275, otherwise they were all identified as C.neoformans by VITEK 2 compact as seen in Table 1. The mass spectra obtained from eight C. gattii isolates were characterized by diverse spectra in the range between 2000 and 10,000 Da. Three specific spectra with molecular weight of about 4342, 8686, 9611 Da could be seen in two isolates of C. gattii VGII, they were absent in six isolates of C. gattii VGI, as shown in Fig.1. The dendrogram created by MALDI Biotyper divided eight isolates into two groups of C. gattii VGI and VGII as shown in Fig.2. The MICs of six antifungal drugs to eight strains of C. gattii were listed in Table 1.

MLST analysis and genotyping
MLST confirmed six strains of C. gattii VGI and two strains of C. gattii VGII as indicated in supplementary  Table 1. The 2 strains of isolate number 1 and 6 with C.gattii genotype VGII could be subtyped into VGIIa and VGIIb respectively by further comparison with Vancouver Island reference stains of R265 and R272, as in supplementary Table 2. Some gene mutation existed in unlinked gene loci such as FTR1 and RAS1 as indicated in Fig.3.

Clinical information of patients infected with C. gattii
As shown in Table 1, the eight strains of C. gattii were isolated from eight patients. Two patients with isolate numbers of 2 and 6 were from Hainan and Yunnan Province, which were recognized as tropical areas. Three patients with isolate number 3 (from Chongqing city), 4 and 7 (from Fujian Province) were recognized as being from subtropical area. The other three patients with isolate numbers of 1, 5 and 8 were from Shandong Province, Inner Mongolia Autonomous Region and Henan Province, which were recognized as temperate regions. Information of patient infected with isolate number 3 was unavailable, the other 7 patients were immunocompetent young male adults, with age range of 21~60 years old. Their clinical and laboratory information were summarized in Table 2.

Differential protein analysis between C. gattii VGI and VGII
In this study, the proteome expression profiles of two strains of VGII and four strains of VGI C. gattii were created. A total of 3628 proteins were identified, of which, 2436 proteins were found in both VGII and VGI C. gattii, however, C. gattii genotype VGI and VGII possessed 774 and 418 specific proteins respectively. Comparative proteome analysis showed that 329 and 180 proteins were highly expressed in C. gattii VGI and VGII  Fig.4. A cluster analysis of the differentially expressed proteins was shown in Fig. 5. The information of GO term and KEGG metabolic pathway analysis indicated that the enrichment of differential expressed proteins between VGI and VGII was mainly directed to Golgi apparatus, Golgi membrane and Golgi vesicle as shown in Table 3. The most significant enriched metabolic pathways were oxidative phosphorylation, ribosome and metabolic pathway as shown in Fig.6.

Discussion
Selection of identification method and drug resistance of Chinese C. gattii strains C. neoformans and C. gattii are two important fungal pathogens for humans and animals. Both of them are round with capsule and positive urease test. These routine laboratory identification methods are unable to distinguish the two species [7]. Although there were several reports about infection of C. gattii from China [8][9][10], the number of C. gattii infections may have been underestimated because most laboratories utilize ink staining and biochemical methods, like VITEK 2 compact, for routine identificatio. CGB agar was recommended to identify C. gattii from C. neoformans due to its convenience and low cost [7], but it took longer time to get the result. Molecular methods would be faster and more accurate for differentiation of the two pathogenic Cryptococci species. However, they are not available in all clinical laboratories, where an accurate identificaction of species should be made in order to install the proper treatment. In this study, eight strains of C. gattii were not only correctly identified but also grouped by MALDI-TOF MS with Bruker software 3.0. Of course, eight strains was not enough and more strains should be studied to obtain the spectrum difference between groups of C. gattii.
The results of antifungal susceptibility test by two kits were in good agreement, except that MICs of Fluconazole were 2~4 times higher by Yeast one than by ATB fungus 3. All the C.gattii isolates were susceptible to 5-Flucytosine, Amphotericin B, Itraconazole, Voriconazole and Posaconazole, only one strain was resistant to Fluconazole with MIC of 128 by Yeast one. However, interpretation of MICs of antifungal agents including Fluconazole for category of "susceptibility" or "resistance" was hampered by the lack of clinical breakpoints for C. gattii. Some previous studies have shown that C. gattii may be less susceptible to antifungal agents compared with C. neoformans. In a study from Taiwan, C. gattii was less susceptible to 5-Flucytosine and amphotericin B, and in Spain, MICs of Fluconazole, Voriconazole and Posaconazole to C. gattii were significantly higher [11][12][13]. The correlation between susceptibility profile and genotype of C. gattii has rarely been studied [14], our data indicated that antifungal agents exhibited higher MICs against isolates of genotype VGII than genotype VGI, which agreed with the data of Hagen et al. and Lockhart et al. [14][15][16][17]. However, Clinical relevance between MIC breakpoint and epidemiological cut-off value (ECV) based on MIC distributions of wild-type strains has currently been studied on both C. neoformans and C. gattii isolates from Europe, USA, Australia, Brazil, Canada, India and South Africa. ECVs of Amphotericin B (0.5~1 μg/ml), 5-Flucytosine (4~16 μg/ml), Fluconazole (8~32 μg/ml) and other azoles varied similarly by molecular type for both C. neoformans and C. gattii [15,16].

Molecular and epidemical characteristics of Chinese C. gattii strains
Up to now, four genotypes of C. gattii have been detected, they were VGI, VGII, VGIII and VGIV. Lockhart and colleagues had reported that VGII and VGIII were the most-frequently identified isolates in America, VGIV was almost exclusively in Africa, and VGI predominated in Europe, Australia and Asia [18]. C. gattii, as an important pathogen, caused outbreak in British Columbia, Canada and the Pacific Northwest, the United States. But 8 strains in our study were pathogens causing sporadic infections according to the strain origination and their isolation time.

Patient information and clinical characteristics
All the patients in our study had no recent travel history, the C. gattii infection occurred regionally and domestically. They were immunocompetent young male adults with age range of 21~60 years old, which was considered as the reason for increased exposure to environmental Fig. 2 The dendrogram of 8 strains of C. gattii and 2 strains of C.neoformans sources and increased chance for infection [19] . . Most patients had headache and fever, three patients showed neck stiffness, positive Kernig's sign and meningeal lesions, meningeal enhancement or lacunar infarction by brain MRI. Most of them also demonstrated irregular nodule, consolidation and mass by lung CT, which was consistent with the research made by Ngamskulrungroj who indicated that C. gattii could cause fatal lung infection [20].
It was reported that host response varied based on the genotype of the organism and concomitant illnesses [21]. There was also study which revealed that C. gattii VGII strains were more virulent than VGI strains and VGIIa were even more virulent than VGIIb independent of their clinical or environmental origin [22]. In our study, all patients were treated by Fluconazole plus amphotericin B, most of them improved effectively without severe neurological sequelae. This might be explained by the fact that these patients infected by C. gattii VGI more than C. gattii VGII on the one hand, and on the other hand, two of them were infected with C. gattii VGII which showed sequence mutations in the gene location of RAS1 and FTR1 compared with reference strains of R265 and R272 respectively. Whether the mutations were relevant to virulence decrease, further work needs to be done.

Analysis of differentially expressed protein in two genotypes of C. gattii
Our study showed significant difference in protein expression spectra between VGI and VGII. GO is a very important tool of biological information. By establishing a set of controlled words with dynamic form, the attributes of genes and gene products in organisms can be described comprehensively. KEGG is the main public database with which the most important biochemical metabolic pathways and signal transduction pathways can be determined. Our results suggested that the differential protein of C. gattii VGI and VG II was mainly located on the organelles associated with the Golgi body, which meant energy metabolism of C. gattii might be involved in the difference of pathogenesis mechanism for different genotype of C. gattii. Study on the secretory

Conclusions
In spite that only 8 strains were available for analysis, our results demonstrated that C. gattii genotype VGI was predominant in China and C. gattii genotype VGII was more resistant to antifungal agents. C. gattii VGII isolates possessed obvious protein peaks with molecular weight of approximate 4342, 8686, 9611 Da. The full protein spectrum data of C.gattii were provided for the first time, a total of 3628 proteins were identified, 329 and 180 proteins were highly expressed by C. gattii VGI and VGII respectively. The enrichment of differentially expressed proteins between VGI and VGII was mainly directed to Golgi complex. It will lay a foundation for better understanding and further research on the pathogenic mechanism for different genotype of C. gattii.

Collection and initial identification of clinical isolates
A total of 254 clinical isolates initially identified as C. neoformans by biochemical methods were collected from seven hospitals in China, they were all stored at − 80°C.
All the isolates were sub-cultured onto Sabouraud dextrose agar medium at 25°C for 48~72 h and identified by VITEK 2 Compact (bioMérieux SA, France). Each isolate was also inoculated on canavanine-glycinebromthymol blue (CGB) agar [23] at 25°C for 24~120 h, The isolates which showed blue pigmentation on CGB agar were then subjected to further identification.

Identification by MALDI-TOF MS
Isolates were re-identified and analyzed by MALDI-TOF MS (Bruker, Daltonik, Bremen, Germany). Briefly, one single colony was smeared directly on the MALDI-TOF MS analysis plate and formic acid was added. After 3~5 min, matrix was added and the plate was put into the MALDI-TOF MS instrument with MALDI Biotyper software 3.0 (Bruker Daltonik GmbH) for species

Clinical information of patients
Under authorization of Medical Record Department, Clinical information of patients infected by C. gattii was reviewed retrospectively, including age, sex, underlying diseases, symptoms, imaging findings, laboratory examinations and antifungal therapy.

Analysis of differential proteins
According to the regional distribution and the integrity of clinical data of the strains, six strains (4 of VGI and 2 of VGII) of C. gattii were selected for proteome analysis. All the isolates were sub-cultured onto Sabouraud dextrose agar medium, incubated at 25°C for 48~72 h , the proteins were extracted from the strains, The protein concentration was 0.5 μg/μl, after being separated by SDS-PAGE electrophoresis (One-dimensional), the proteins were hydrolyzed and analyzed. Original mass spectrum files of 6 strains were imported into Maxquatt software (version 1.3.0.5) for analysis. The database source was for C.gattii. in Uniprot (http://www. geneontology.org/). Plus or minus 15 ppm of polypeptide molecular weight, or missing 2 cutting sites were set as Maxquatt parameters, false discovery rate (FDR) for polypeptide identification was set as 0.01. A standard of p < 0.05 and 2-fold expression were considered as different proteins. The enrichment and pathway analysis was carried out by using the GO plot package of R language and KEGG online tool (https://david.ncifcrf.gov/, http:// www.kegg.jp/kegg/pathway.html). The bubble diagram was drawn according to the R language ggplot2 package.