Persistence of an epidemic cluster of Rhodotorula mucilaginosa in multiple geographic regions in China and the emergence of a 5-flucytosine resistant clone

Figures & data

Table 1. Clinical characteristics of 43 invasive infection cases caused by R. mucilaginosa.

Characteristics	Value
Mean age (range)	55.0 (23–88)
Male sex, n (%)	27 (62.8)
Underlying disease, n (%)
Hypoproteinemia	12 (27.9)
Tumour	12 (27.9)
Diabetes	9 (20.9)
Neutropenia (<10^9/L)	2 (4.7)
Severe anaemia (<60 g/L)	2 (4.7)
COPD	2 (4.7)
Autoimmune disease	1 (2.3)
ICU history, n (%)	24 (55.8)
Prophylaxis with antimicrobial agents, n (%)
Broad-spectrum antibiotics	29 (67.4)
Broad-spectrum antibiotics + Azoles	5 (11.6)
Broad-spectrum antibiotics + Azoles + Echinocandins	3 (7.0)
Broad-spectrum antibiotics + Echinocandins	1 (2.3)
None	3 (7.0)
Not recorded	2 (4.7)
Treatment, n (%)
Fluconazole	9 (20.9)
Caspofungin	4 (9.3)
Fluconazole + Caspofungin	3 (7.0)
Voriconazole + Caspofungin	2 (4.7)
Itraconazole + Caspofungin	1 (2.3)
Vorconazle	1 (2.3)
None	22 (51.2)
Not recorded	1 (2.3)
CVC removal	6 (14.0)
Mortality, n (%)	5 (11.6)

Abbreviations: COPD: chronic obstructive pulmonary disease; CVC: central venous catheter; n: number.

Figure 1. Hospital course, history and distribution of microsatellite types included in the study. Different colours represent different clinical departments. Number in colourful square means the serial number of cases in each hospital. The red dot on the right side of the number indicates that the strain of the case belongs to the epidemic cluster. The red rectangle shows that patients underwent a transfer between two wards. *, the area where the colour block is located shows the part time of the patient in hospital because of unavailable clinical records. **, the case was diagnosed in an outpatient clinic.

Table 2. Polymorphism of rDNA types and sources of 54 R. mucilaginosa strains included in the study.

rDNA type (No. of strains)	SNPs in the region of rDNA		No. (%) of strains in hospital:				No. (%) of strains from:
	ITS region	D1/D2 domain	H01	H02	H03	H04	Environment	Pets
Type 1 (8)	None	None	2 (7.7)	1 (11.1)		2 (33.3)	3 (75.0)
Type 2 (38)	T503C	None	24 (92.3)	2 (22.2)	6 (75.0)	4 (66.7)	1 (25.0)	1 (100.0)
Type 3 (1)	C398T	None			1 (12.5)
Type 4 (3)	None	C399T		3 (33.3)
Type 5 (2)	T503C	C399T		2 (22.2)
Type 6 (2)	C91T A120G C408G T503C	530insT		1 (11.1)	11 (12.5)

Figure 2. Genetic relationship/Minimum spanning tree of all strains based on microsatellite genotypes under: (A) different sources, (B) hospitals, (C) rDNA types, and (D) profile of 5-flucytosine. Each circle represents a genotype, and the size of the circle is correlated with the number of strains. The strains belonging to the epidemic cluster were highlighted in yellow.

Table 3. Primers and information related to the microsatellite loci for R. mucilaginosa.

Locus	Primer sequences (5′-3′)	Repeat type	Size range (bp)	No. of alleles	DP
RM14	GGTGTAGCCCCGGTACTTCC AGCGGCATCTGCTTCTCCTC	TCC	164–194	5	0.546
RM54	CATCGCTGTCGGTCGTCTCT	AG	164–170	4	0.507
	CTTACAGTAGGCGGACGCGA
RM57	CGCGCAAGACTCTCTGCAAG CAGCGCCAACTACATGCCAG	CT	184–188	3	0.314
RM60	ACAGCGTCAAGAGCCCTTCT GCCTCCACGGTGCCTGTAAT	TC	183–193	4	0.662
RM71	CCCTTCCCTCCCATTCGGTT GCAAAAGCTAGCGTGGGGAC	TC	197–201	3	0.073
RM72	TGTGCGTTGACGTGTTTGGG CCGACAGCGCTACAGTCAGT	AG	123–125	2	0.107
RM83	CAGCGCGGTCTCAACAACTG GTCCTCATCCTCGGGGTTCG	TC	182–190	4	0.511
RM113	GTCTCCAACCACGAGATGCA ACGCAAACCTGCCTCTCCTT	AG	179–205	6	0.589
RM116	GTACGCTCGGCTGTTCGTTG ACAAGATCGGAACCTCGGGC	AG	185–193	3	0.174
RM119	CTCCTCCGCGATCCCAACTC TCTTCTTGGGTCGTCCTCGC	AGC	141–162	7	0.416
RM125	CTTGGTGGTGGTTGGCGC ACCCGGTCGAAGCGTAATCC	TGC	126–183	7	0.601
RM127	GAGAGAGGCCTCGGAAGCAG GCTGTTGGTCGTGCACAGG	AG	168–180	4	0.269
RM131	GACGGGGTTCGAGAGTTGGT ACGGCGATAGGAGGGGTACT	AAG	194–257	7	0.616
RM134	CTGCACTCTGTGTGGCATGC CGAGGTGCGTGATCAGGGAA	TC	215–227	6	0.386
RM139	CGACACCGCTCGAGACTCTT TGCATCTCTCCCGCTTTGGT	TC	187–199	5	0.210

Abbreviations: DP: discriminatory power.

Figure 3. Phylogenetic relationship of selected clinical, environmental, and animal strains of R. mucilaginosa inferred based on genomic SNPs. Isolates are coloured according to their source (human [black], environment [blue], and Pet [red]).

Figure 4. Clustering heatmap of comparison between microsatellite genotypes and genomic SNPs of all representative strains (A), strains belonging to epidemic cluster (B). Gradient colour represents the numbers of pairwise genomic SNPs (bp). MT, microsatellite.

Table 4. Antifungal susceptibility profiles of R. mucilaginosa clinical strains to 9 antifungal agents.

Antifungal agent	MIC range	MIC50	MIC90	GM MIC	Mode ^a
Azoles
Fluconazole	64 to >256	>256	>256	>256	>256
Voriconazole	0.25–8	4	4	3.33	4
Itraconazole	0.12–16	1	2	1.10	1
Posaconazole	0.25–2	2	2	1.62	2
Flucytosine
5-Flucytosine	<0.06 to >64	<0.06	<0.06	0.12	<0.06
Echinocandins
Anidulafungin	>8	>8	>8	>8	>8
Micafungin	>8	>8	>8	>8	>8
Caspofungin	8 to >8	>8	>8	>8	>8
Polyenes
Amphotericin B	0.25–1	0.5	1	0.52	0.5

Abbreviations: GM: Geometric Mean; a: Most frequent MIC.

Data availability

Genome raw reads are available at National Centre for Biotechnology Information (NCBI) under BioProject accession number PRJNA794005.

Microsatellite genotypes with fragment sizes of 15 tandem repeat loci were listed in Table S4. Minimum inhibitory concentration of 9 antifungal agents against R. mucilaginosa clinical strains was listed in Table S5. Phenotypic characteristics of colonies of R. mucilaginosa after 7 days incubation were showed in Figure S1.