Generation and Characterization of Single Chain Variable Fragment against Alpha-Enolase of Candida albicans.

Candida albicans (C. albicans) is an opportunistic human pathogen responsible for approximately a half of clinical candidemia. The emerging Candida spp. with resistance to azoles is a major challenge in clinic, suggesting an urgent demand for new drugs and therapeutic strategies. Alpha-enolase (Eno1) is a multifunctional protein and represents an important marker for invasive candidiasis. Thus, C. albicans Eno1 (CaEno1) is believed to be an important target for the development of therapeutic agents and antibody drugs. Recombinant CaEno1 (rCaEno1) was first used to immunize chickens. Subsequently, we used phage display technology to construct two single chain variable fragment (scFv) antibody libraries. A novel biopanning procedure was carried out to screen anti-rCaEno1 scFv antibodies, whose specificities were further characterized. The polyclonal IgY antibodies showed binding to rCaEno1 and native CaEno1. A dominant scFv (CaS1) and its properties were further characterized. CaS1 attenuated the growth of C. albicans and inhibited the binding of CaEno1 to plasminogen. Animal studies showed that CaS1 prolonged the survival rate of mice and zebrafish with candidiasis. The fungal burden in kidney and spleen, as well as level of inflammatory cytokines were significantly reduced in CaS1-treated mice. These results suggest CaS1 has potential of being immunotherapeutic drug against C. albicans infections.

Clonality of Fluconazole-Nonsusceptible Candida tropicalis in Bloodstream Infections, Taiwan, 2011-2017.

Candida tropicalis is the leading cause of non-C. albicans candidemia in tropical Asia and Latin America. We evaluated isolates from 344 patients with an initial episode of C. tropicalis candidemia. We found that 58 (16.9%) patients were infected by fluconazole-nonsusceptible (FNS) C. tropicalis with cross resistance to itraconazole, voriconazole, and posaconazole; 55.2% (32/58) of patients were azole-naive. Multilocus sequence typing analysis revealed FNS isolates were genetically closely related, but we did not see time- or place-clustering. Among the diploid sequence types (DSTs), we noted DST225, which has been reported from fruit in Taiwan and hospitals in Beijing, China, as well as DST376 and DST505-7, which also were reported from hospitals in Shanghai, China. Our findings suggest cross-boundary expansion of FNS C. tropicalis and highlight the importance of active surveillance of clinical isolates to detect dissemination of this pathogen and explore potential sources in the community.

Prevalence, mechanisms and genetic relatedness of the human pathogenic fungus Aspergillus fumigatus exhibiting resistance to medical azoles in the environment of Taiwan.

Emerging azole resistance in Aspergillus fumigatus poses a serious threat to human health. This nationwide surveillance study investigated the prevalence and molecular characteristics of azole-resistant A. fumigatus environmental isolates in Taiwan, an island country with increasing use of azole fungicides. Of the 2760 air and soil samples screened from 2014 to 2016, 451 A. fumigatus isolates were recovered from 266 samples and 34 isolates from 29 samples displayed resistance to medical azoles (itraconazole, voriconazole or posaconazole). The resistance prevalence was 10.9% and 7.5% in A. fumigatus-positive samples and isolates respectively. Most (29, 85.3%) azole-resistant isolates harboured TR34 /L98H mutations, which were widely distributed, clustered genetically with clinical isolates, and had growth rates that were similar to those of the wild-type isolates. Microsatellite genotyping revealed both the global spread of the TR34 /L98H isolates and the occurrence of TR34 /L98H/S297T/F495I isolates belonging to local microsatellite genotypes. AfuMDR3 and atrF, two efflux transporter genes, were constitutively upregulated in two individual resistant isolates without cyp51A mutations, highlighting their potential roles in azole resistance. These results emphasize the need for periodic environmental surveillance at the molecular level in regions in which azole fungicides are applied, and agricultural fungicide management strategies that generate less selective pressure should be investigated.

DST659 genotype of Candida albicans showing positive association between biofilm formation and dominance in Taiwan.

Based on multiple locus sequence typing, we previously found that DST659 and DST693 were dominant genotypes of Candida albicans among the bloodstream isolates at Chang-Gung Memorial Hospital at Linkou. Biofilm-forming activity, which is critical for C. albicans virulence, probably contributed to the dominance of antifungal sensitive isolates in hospital. Both in vitro membrane weighting and in vivo zebrafish egg infection assays were used to evaluate the biofilm-forming activity of DST659 and DST693 genotypes. Medical records of the patients infected by these two genotypes were retrospectively reviewed. High biofilm-forming activity of DST659 isolates was demonstrated in vitro and further proved with the zebrafish egg infection model, which showed a positive correlation between the biofilm-forming extent on chorion and the in vitro biofilm activity. Moreover, significantly less embryos survived when infected with DST659 isolates than those with DST693 (1.25% vs. 11.43%), and the high-biofilm subset of DST659 showed a greater reduction in survival of embryos at 48 h post-infection than the low-biofilm subset (0 vs. 1.92%). Patients infected with DST659 seemed to survive slightly worse than those infected with DST693, although the difference was insignificant. It is noteworthy that DST659-infected patients were associated with a higher incidence in renal insufficiency as compared to those with DST693, the low biofilm genotype. We suggest that a strong biofilm activity of DST659 contributed to a high mortality rate in zebrafish hosts and poor renal function in patients, as well as gaining the dominance in the northern Taiwan.

Mixed yeast infections in Taiwan.

Clinically significant yeast isolates were collected via Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) in 2014, and mixed infections were investigated. Among 44 out of 1092 specimens containing multiple species, 17, 11, 5, 3, and 8 were from urine, sputum, blood, ascites, and 6 others, respectively. There predominant combinations of mixed infection were 14 Candida albicans/Candida glabrata, 13 C. albicans/Candida tropicalis, and 9 C. glabrata/C. tropicalis. Furthermore, we also detected fluconazole resistant isolates Candida norvegensis and Candida krusei. Hence, it is important to accurately identify the species with different drug susceptibilities when they are in the same specimen.

The survey of tinea capitis and scalp dermatophyte carriage in nursing home residents.

Tinea capitis is a contagious dermatophyte infection of scalp and associated hairs. On the other hand, asymptomatic carriage is a status of positive dermatophyte scalp culture, but without signs or symptoms of tinea capitis, and no evidence of hair shaft invasion confirmed by direct microscopy. Tinea capitis and asymptomatic carriage mostly occur in children, but adult females are becoming another population in recent decades. In this study, we focused on the prevalence and related fungi of tinea capitis and asymptomatic carriage in elderly by the shampoo brush method, as well as the source of transmission, in 10 nursing home residents. Two hundred and thirteen residents were screened, and 186 isolates were identified, of which only three were dermatophytes (1.4%). The scalp dermatophyte isolates were identified as Trichophyton rubrum by morphological characters and sequences comparisons in all three cases. After revisiting, these cases were proved to be asymptomatic carriers by negative microscopic and culture examination; however, two cases were found to have concurrent tinea pedis and onychomycosis, which were identified as T. rubrum and Trichophyton interdigitale. The source of the T. rubrum scalp carriage may come from tinea elsewhere on the body of the same subject or from other people in the same institute. Finding and treating the source of carriage, as well as treating scalp carriage patients according to the colony counts, may help prevent disease spreading.

Are we ready for the global emergence of multidrug-resistant Candida auris in Taiwan?

Candida auris is a recently identified multi-resistant Candida species, first reported in Japan in 2009, and poses a serious global health threat. Lack of awareness of this new Candida species and difficulties with laboratory identification have impacted significantly on outbreak detection and management, and compromised patient outcome. Accordingly, there is an urgent need to raise awareness of healthcare personnel to this emerging pathogen and determine its prevalence, impact, and challenges to the Taiwan healthcare system. Enhanced laboratory testing strategies are needed to differentiate C. auris from other Candida species to provide accurate diagnosis and implement control measures early enough to prevent hospital outbreaks. In this report, we review the key epidemiological, microbiological and clinical characteristics of C. auris and provide the results of a multicenter surveillance study of C. auris in Taiwan. We also conducted a web-based survey to determine awareness of the medical community to C. auris and the capability of Taiwanese hospital laboratories to identify this microorganism. C. auris has not yet been isolated from humans in Taiwan, but the unique features of this microorganism and its ability to reach across international boundaries justify the importance of these initiatives in Taiwan.

Ndt80p is involved in L-sorbose utilization through regulating SOU1 in Candida albicans.

Ndt80p, a known transcriptional factor, regulates various targets involved in stress responses, filamentous growth, and virulence in Candida albicans. Potential targets of Ndt80p have been identified at the transcriptional level. The present study was conducted to identify genes regulated by Ndt80p from the protein level. We found that the levels of Ahp1p, Fma1p, Hsp21p, Rfa2p, Snz1p, Sod1p, Sou1p, Trp99p, orf19.251, orf19.1862, and orf19.5620, were affected by the null mutation of NDT80 by two-dimensional polyacrylamide gel-electrophoresis analysis. Among the 11 proteins, all but Sou1p and Rfa2p are suggested to be involved in known functions of Ndt80p. Here, we demonstrate that Ndt80p plays a role in l-sorbose utilization through regulating SOU1 in C. albicans.

Molecular epidemiology of invasive Candida albicans at a tertiary hospital in northern Taiwan from 2003 to 2011.

Candida albicans is a common cause of bloodstream fungal infections in hospitalized patients. To investigate its epidemiology, multilocus sequence typing (MLST) was performed on 285 C. albicans bloodstream isolates from patients in Chang Gung Memorial Hospital at Linkou (CGMHL), Taiwan from 2003 to 2011. Among these isolates, the three major diploid sequence types (DSTs) were 693, 659, and 443 with 19, 16, and 13 isolates, respectively. The 179 DSTs were classified into 16 clades by unweighted pair-group method using arithmetic averages (UPGMA). The major ones were clades 1, 4, 3, and 17 (54, 49, 31, and 31 isolates, respectively). Further analyses with eBURST clustered the 285 isolates into 28 clonal complexes (CC). The most common complexes were CC8, CC20, and CC9. DST 693 that had the highest number of isolates was determined to be the cluster founder of CC20, which belonged to clade 3. So far, 33 isolates worldwide including 29 from Taiwan and 4 from Korea, are CC20, suggesting that CC20 is an Asian cluster. Two fluconazole-resistant isolates belonging to CC12 and CC19 were detected. All other CGMHL isolates were susceptible to 5-flucytosine, amphotericin B, anidulfungin, caspofungin, fluconazole, itraconazole, micafungin, posaconazole, and voriconazole. However, CC20 isolates exhibited significantly lower susceptibility to fluconazole. In conclusion, the 285 CGMHL C. albicans isolates displayed geographically clustering with Asian isolates, and most of them are susceptible to common antifungal drugs. Isolates of DST 693, a Taiwanese major genotype belonging to MLST clade 3, were more resistant to fluconazole than other isolates.

Azole-resistant Aspergillus fumigatus isolates carrying TR34/L98H mutations in Taiwan.

Cumulative evidence described the emergence and geographical expansion of azole-resistant A. fumigatus associated with azole treatment failure. To investigate the status of azole resistance in A. fumigatus in Taiwan, we studied 38 A. fumigatus clinical isolates cultivated from 31 patients at two teaching hospitals from 2011 to 2014. Three isolates obtained from respiratory samples of two azole-naïve patients with pulmonary aspergillosis were found to display multi-azole resistance and cross resistance to agricultural azole fungicides, and all carried TR34/L98H mutations in cyp51A gene. The prevalence rates of azole resistance were 7.9% and 6.5% based on isolates and patients respectively. A phylogenetic analysis suggested genetic diversity of the TR34/L98H isolates in Taiwan, including a unique genotype distinct from strains outside Taiwan. The result underlines the emergence of such isolates in Taiwan as well, emphasising the importance of further surveillance for azole-resistant A. fumigatus and implementation of strategies that prevent fungicide-driven resistance selection.

Yeast oropharyngeal colonization in human immunodeficiency virus-infected patients in central taiwan.

A prospective, cross-sectional study was conducted at a medical center in central Taiwan to understand the prevalence, associated factors, and microbiologic features for oropharyngeal yeast colonization in human immunodeficiency virus-infected outpatients. Oral yeast colonization was detected in 127 (45 %) patients, including 21 (16.5 %) colonized by more than one species. Of the 154 isolates, Candida albicans was the most common species (114, 74 %), followed by Candida dubliniensis (10, 6.5 %), Candida glabrata (10, 6.5 %), Candida tropicalis (7, 4.5 %), and 13 others. We found that receiving antituberculous drug (p = 0.046) or atazanavir (p = 0.045) was two predictors for patients colonized by non-C. albicans species (p = 0.005) and risking mixed yeast colonization (p = 0.009). Even though our data showed that clinical antifungal drugs remained effective in vitro against the colonizing yeasts, the increased mixed yeast colonization indicates a potential issue for controlling mixed infections in hospital settings.

Genotype analysis based on intergenic spacer 1 sequences of Trichosporon asahii collected in Taiwan.

Among 32 Trichosporon asahii isolates collected in four rounds of the Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) studies, conducted in 1999, 2002, 2006, and 2010, five different intergenic spacer 1 (IGS1) genotypes were detected. Genotype 1 was the most common (43.8%), followed by genotypes 3 (28.1%), 7 (12.5%), 5 (9.4%), and 4 (6.3%). Interestingly, genotype 7 was more prevalent in Taiwan than in other areas (P = 0.01); while we did not find a significant association between IGS1 genotype and susceptibility to antifungal drugs, we did note that the majority of isolates of T. asahii were susceptible to both fluconazole and voriconazole, consistent with previous reports. A higher proportion of isolates (P = 0.05) collected in 2010 (4/12, 33.3%) had high amphotericin B MICs (≥ 2 mg/l) than those collected in the previous three TSARYs (1/21, 5%). Hence, the new data of genotypes and drug susceptibilities in the present study may contribute to the epidemiology of T. asahii.

R432 is a key residue for the multiple functions of Ndt80p in Candida albicans.

Ndt80p is an important transcription modulator to various stress-response genes in Candida albicans, the most common human fungal pathogen in systemic infections. We found that Ndt80p directly regulated its target genes, such as YHB1, via the mid-sporulation element (MSE). Furthermore, the ndt80(R432A) allele, with a reduced capability to bind MSE, failed to complement the defects caused by null mutations of NDT80. Thus, the R432 residue in the Ndt80p DNA-binding domain is involved in all tested functions, including cell separation, drug resistance, nitric oxide inactivation, germ tube formation, hyphal growth, and virulence. Hence, the importance of the R432 residue suggests a novel approach for designing new antifungal drugs by blocking the interaction between Ndt80p and its targets.

Distribution and drug susceptibilities of Candida species causing candidemia from a medical center in central Taiwan.

Invasive fungal infections have increased significantly in the past few decades because of the increase in high-risk populations. To investigate the distribution and drug susceptibilities of such infections, we analyzed all 152 Candida isolates causing candidemia from 2004 to 2006 at the China Medical University Hospital, a medical center in central Taiwan. Candida albicans was the most common species, accounting for 52.6% of the isolates, followed by C. tropicalis (19.7%), C. parapsilosis (14.5%), C. glabrata (8.6%), C. guilliermondii (3.9%), and C. pelliculosa (0.7%). All isolates were susceptible to amphotericin B, anidulafungin, micafungin, and voriconazole according to minimum inhibitory concentrations (MICs) after a 24-h incubation; 0.7%, 6.6%, and 7.9% of isolates were resistant to amphotericin B, fluconazole, and voriconazole, respectively, after 48-h incubation. Both C. albicans and C. parapsilosis had high degrees of agreement for azoles between 24- and 48-h incubation periods, whereas C. glabrata (38.5-46.2%) and C. tropicalis (56.7-63.3%) did not. The majority of the isolates with high azole MICs displayed a trailing growth phenotype. Hence, the MICs of different drugs after 24-h incubation may be considered for prognosis of candidemia.

Microbiota signatures associated with invasive Candida albicans infection in the gastrointestinal tract of immunodeficient mice.

Candida albicans is a commensal microorganism in the human gut but occasionally causes invasive C. albicans infection (ICA), especially in immunocompromised individuals. Early initiation of antifungal therapy is associated with reduced mortality of ICA, but rapid diagnosis remains a challenge. The ICA-associated changes in the gut microbiota can be used as diagnostic and therapeutic targets but have been poorly investigated. In this study, we utilized an immunodeficient Rag2γc (Rag2-/-il2γc-/-) mouse model to investigate the gut microbiota alterations caused by C. albicans throughout its cycle, from its introduction into the gastrointestinal tract to invasion, in the absence of antibiotics. We observed a significant increase in the abundance of Firmicutes, particularly Lachnospiraceae and Ruminococcaceae, as well as a significant decrease in the abundance of Candidatus Arthromitus in mice exposed to either the wild-type SC5314 strain or the filamentation-defective mutant (cph1/cph1 efg1/efg1) HLC54 strain of C. albicans. However, only the SC5314-infected mice developed ICA. A linear discriminate analysis of the temporal changes in the gut bacterial composition revealed Bacteroides vulgatus as a discriminative biomarker associated with SC5314-infected mice with ICA. Additionally, a positive correlation between the B. vulgatus abundance and fungal load was found, and the negative correlation between the Candidatus Arthromitus abundance and fungal load after exposure to C. albicans suggested that C. albicans might affect the differentiation of intestinal Th17 cells. Our findings reveal the influence of pathogenic C. albicans on the gut microbiota and identify the abundance of B. vulgatus as a microbiota signature associated with ICA in an immunodeficient mouse model.

Fruits are vehicles of drug-resistant pathogenic Candida tropicalis.

IMPORTANCE: Of 123 identified isolates from the fruit surface, C. tropicalis was the most frequently found species, followed by Meyerozyma caribbica and Candida krusei. All three fluconazole-resistant C. tropicalis were non-susceptible to voriconazole and belonged to the same predominant genotype of azole-resistant C. tropicalis causing candidemia in patients in Taiwan. Our findings provide evidence that fruit should be washed before eaten not only to remove chemicals but also potential drug-resistant pathogenic microbes, especially for immunocompromised individuals. To keep precious treatment options in patients, we not only continuously implement antimicrobial stewardship in hospitals but also reducing/stopping the use of agricultural fungicide classes used in human medicine.

Susceptibilities to amphotericin B, fluconazole and voriconazole of Trichosporon clinical isolates.

A total of 35 Trichosporon isolates were collected from the Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) project from 1999 to 2006, and their identifications as well as drug susceptibilities were determined. The most frequently isolated species was T. asahii (62.9%), and the most common clinical sample that yielded Trichosporon isolates was urine (37.1%). The etiology of all seven invasive trichosporonosis was T. asahii. For the 22 T. asahii isolates, the MIC(50) and MIC(90) for amphotericin B were 0.25 and 1 µg/mL, respectively. Those for fluconazole were 2 and 4 µg/mL, respectively, and for voriconazole 0.031 and 0.063 µg/mL, respectively. When the intraclass correlation coefficients (ICCs) and agreements were calculated, we found that the MICs of fluconazole obtained from different methods were similar and the inter-method discrepancies were low. Nevertheless, no unanimous MIC of amphotericin B and voriconazole was obtained among different methods.

Distribution of Yeast Species and Risk Factors of Oral Colonization after Oral-Care Education among the Residents of Nursing Homes.

Most yeasts causing infections in humans are part of commensal microflora and etiological agents of different infections when hosts become susceptible, usually due to becoming immunocompromised. The colonization of potentially pathogenic microbes in the oral cavity is increased by poor oral hygiene. This follow-up survey was conducted approximately two months after providing information on proper oral care at 10 nursing homes in Taiwan. Among the 117 of 165 residents colonized by yeasts, 67 were colonized by more than one yeast species. A total of 231 isolates comprising eight fungal genera and 25 species were identified. Candida albicans (44.6%) was the dominant species, followed by Candida glabrata (17.7%), Candida parapsilosis (8.7%), Candida tropicalis (7.8%), and Candida pararugosa (7.3%). Residents having a yeast colony-forming unit >10 (OR, 8.897; 95% CI 2.972−26.634; p < 0.001) or using a wheelchair (OR, 4.682; 95% CI 1.599−13.705; p = 0.005) were more likely to be colonized by multiple species. By comparing before and after oral-care education, dry mouth (OR, 3.199; 95% CI 1.448−7.068; p = 0.011) and having heart disease (OR, 2.681; 95% CI 1.068−6.732; p = 0.036) emerged as two independent risk factors for increased density of colonizing yeast.

Genomic Surveillance of Listeria monocytogenes in Taiwan, 2014 to 2019.

Listeria monocytogenes is a life-threatening foodborne pathogen. Here, we report the genomic characterization of a nationwide dataset of 411 clinical and 82 food isolates collected in Taiwan between 2014 and 2019. The observed incidence of listeriosis increased from 0.83 to 7 cases per million population upon implementation of mandatory notification in 2018. Pregnancy-associated cases accounted for 2.8% of human listeriosis and all-cause 7-day mortality was of 11.9% in nonmaternal-neonatal listeriosis. L. monocytogenes was isolated from 90% of raw pork and 34% of chicken products collected in supermarkets. Sublineages SL87, SL5, and SL378 accounted for the majority (65%) of clinical cases. SL87 and SL378 were also predominant (57%) in food products. Five cgMLST clusters accounted for 57% clinical cases, suggesting unnoticed outbreaks spanning up to 6 years. Mandatory notification allowed identifying the magnitude of listeriosis in Taiwan. Continuous real-time genomic surveillance will allow reducing contaminating sources and disease burden. IMPORTANCE Understanding the phylogenetic relationship between clinical and food isolates is important to identify the transmission routes of foodborne diseases. Here, we performed a nationwide study between 2014 and 2019 of both clinical and food Listeria monocytogenes isolates and sequenced their genomes. We show a 9-fold increase in listeriosis reporting upon implementation of mandatory notification. We found that sublineages SL87 and SL378 predominated among both clinical (50%) and food (57%) isolates, and identified five cgMLST clusters accounting for 57% of clinical cases, suggestive of potential protracted sources of contamination over up to 6 years in Taiwan. These findings highlight that mandatory declaration is critical in identifying the burden of listeriosis, and the importance of genome sequencing for a detailed characterization of the pathogenic L. monocytogenes genotypes circulating in Asia.