Mechanism of Polyhexamethylene Biguanide Resistance in Purpureocillium lilacinum Strains.

Purpureocillium lilacinum has been recently found to contaminate a 20% (200,000 µg/mL) aqueous solution of polyhexamethylene biguanide hydrochloride (PHMB) . We aimed to elucidate the mechanism underlying the resistance of P. lilacinum to PHMB. First, we induced the PHMB-resistant (IR) strains IFM 67050 (IR) and IFM 65838 (IR) from the type strain P. lilacinum CBS 284.36T via cultivation in a medium containing high concentrations of PHMB. We then analyzed the DNA sequences via Illumina sequencing to evaluate the presence of genetic mutations in IFM 65838 (IR) . Further, we established an IFM 65838 (IR) uridine/uracil auxotrophic strain, and using the orotidine-5'-decarboxylase gene, pyrG as a selection marker, we tried to knockout a mutant gene in IFM 65838 (IR) using the CRISPR-Cas9 genome-editing technique. The growth rates of IFM 67050 (IR) and IFM 65838 (IR) in medium containing PHMB increased, and the minimum inhibitory concentrations (MICs) against PHMB also increased. Based on the DNA sequence analysis, we found a nonsynonymous point mutation in the gene PLI-008146 (G779A) in IFM 67050 (IR) and IFM 65838 (IR) . This point mutation leads to site combinations of splicing changes that cause partial sequences deletion (p.Y251_G281del) in the ΔPLI-008146 locus of IFM 65838 (IR) , and deletion sequences include partial adenosine/AMP deaminase motif (PF00962) orthologous to adenosine deaminase (ADA) (GeneBank: OAQ82383.1) . Furthermore, the mutant gene ΔPLI-008146 was successfully knocked out from the resistanceinduced strain using a novel CRISPR-Cas9 gene transformation method. A considerable reduction in growth rate and MIC against PHMB was observed in the absence of the mutant gene. Therefore, ADA may represent an important resistance factor in PHMB-resistant P. lilacinum.

Molecular Phylogenetic Study of Strains Morphologically Identified as Exophiala dermatitidis from Clinical and Environmental Specimens in Japan.

In this study, we aimed to clarify the phylogenetic distribution of Exophiala dermatitidis in Japan and describe the characteristics of genotypes. We examined 67 clinical and environmental isolates that were morphologically identified and preserved as E. dermatitidis and we confirmed the identification on the basis of the ribosomal DNA internal transcribed spacer region. Genotype sequences were aligned and compared using maximum likelihood phylogenetic tree analyses of the ITS1 region. Additionally, the strains of each genotype were tested for mycological characteristics, such as growth temperature, growth rate, and drug sensitivity. The 67 strains examined were isolated from Japan, the United States, Brazil, Venezuela, and China. In accordance with the establishment of a phylogenetic tree for the ITS1 region, 45 of the 49 Japanese strains were classified as genotype A, two as genotype B, and two as genotype D (A2 according to the method of Matos et al. (2003)). Chinese strains were divided into genotypes A and D (A2), and South American strains were classified into genotypes A, B, B2, and C2, while all strains from the U.S. belonged to genotype A. New genotype groups B2 and C2 were identified in Brazilian and Venezuelan strains, respectively. There were no specific differences among the genotypes or isolated regions in the antifungal susceptibility test for all E. dermatitidis isolates. However, genotypes B2 and D (A2) exhibited growth at higher temperatures than the other genotypes.

Isolation and Characterization of the Polyhexamethylene Biguanide Hydrochloride-Resistant Fungus, Purpureocillium lilacinum.

We isolated a fungus from a 20% (= 200,000 µg/mL) aqueous solution of polyhexamethylene biguanide hydrochloride (PHMB), a widely used antimicrobial and examined its morphology and drug resistance profile. Based on the sequence of the internal transcribed spacer region of ribosomal DNA, the fungus was identified as Purpureocillium lilacinum. Although the P. lilacinum type and resistant strains showed similar morphology, the latter had extremely low PHMB susceptibility and was able to grow in 20% aqueous solution of PHMB, which eliminated the type strain. The minimum inhibitory concentration (MIC) of PHMB for the resistant strain was significantly higher than that of the type strain and other pathogenic filamentous fungi and yeasts. The susceptibility to antimicrobial agents and antifungal agents other than PHMB was similar to that of the type strain, therefore the drug resistance of the isolate was specific to PHMB. Furthermore, we sequenced the genome of the isolate to predict PHMB resistance-related genes. Despite its high resistance to PHMB, no well-known genes homologous to fungal PHMB-resistant genes were detected in the genome of the resistant strain. In summary, P. lilacinum was found to be significantly more resistant to PHMB than previously reported, via an unidentified mechanism of drug resistance.

Draft Genome Sequence of Aspergillus awamori IFM 58123NT.

Species of the Aspergillus section Nigri are taxonomically very complex. The taxonomic assignment of Aspergillus awamori is unclear. Here, we present the draft genome sequence of A. awamori strain IFM 58123NT.