Publications by authors named "Oliver Bader"

67 Publications

Colony Morphotype Forecasts Biofilm Formation of Clinical Isolates.

J Fungi (Basel) 2021 Jan 7;7(1). Epub 2021 Jan 7.

Institute for Medical Microbiology, University Medical Center Göttingen, Kreuzbergring 57, 37075 Göttingen, Germany.

is a frequent cause of fungal bloodstream infections, especially in critically ill neonates or immunocompromised patients. Due to the formation of biofilms, the use of indwelling catheters and other medical devices increases the risk of infection and complicates treatment, as cells embedded in biofilms display reduced drug susceptibility. Therefore, biofilm formation may be a significant clinical parameter, guiding downstream therapeutic choices. Here, we phenotypically characterized 120 selected isolates out of a prospective collection of 215 clinical isolates, determining biofilm formation, major emerging colony morphotype, and antifungal drug susceptibility of the isolates and their biofilms. In our isolate set, increased biofilm formation capacity was independent of body site of isolation and not predictable using standard or modified European Committee on Antimicrobial Susceptibility Testing (EUCAST) drug susceptibility testing protocols. In contrast, biofilm formation was strongly correlated with the appearance of non-smooth colony morphotypes and invasiveness into agar plates. Our data suggest that the observation of non-smooth colony morphotypes in cultures of may help as an indicator to consider the initiation of anti-biofilm-active therapy, such as the switch from azole- to echinocandin- or polyene-based strategies, especially in case of infections by potent biofilm-forming strains.
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http://dx.doi.org/10.3390/jof7010033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7827155PMC
January 2021

Rapid direct detection of pathogens for diagnosis of joint infections by MALDI-TOF MS after liquid enrichment in the BacT/Alert blood culture system.

PLoS One 2020 11;15(12):e0243790. Epub 2020 Dec 11.

Institute of Medical Microbiology, University Medical Center Göttingen, Göttingen, Germany.

Pathogen identification is a critical step during diagnosis of infectious diseases. Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry (MALDI-TOF-MS) has become the gold standard for identification of microorganisms cultured on solid media in microbiology laboratories. Direct identification of microbes from liquid specimen, circumventing the need for the additional overnight cultivation step, has been successfully established for blood culture, urine and liquor. Here, we evaluate the ability of MALDI-TOF MS for direct identification of pathogens in synovial fluid after liquid enrichment in BacT/Alert blood culture bottles. Influence of synovial specimen quality on direct species identification with the MALDI BioTyper/Sepsityper was tested with samples inoculated from pretested native synovia with concomitant inoculation of blood or pus, or highly viscous fluid. Here, we achieved >90% concordance with culture on solid medium, and only mixed-species samples posed significant problems. Performance in routine diagnostics was tested prospectively on bottles inoculated by treating physicians on ward. There, we achieved >70% concordance with culture on solid media. The major contributors to test failure were the absence of a measurable mass signal and mixed-specimen samples. The Sepsityper workflow worked well on samples derived from BacT/Alert blood culture bottles inoculated with synovial fluid, giving concordant results to identification from solid media. Host remnant material in the inoculum, such as blood or pus, had no detrimental effect on identification score values of the BioTyper system after processing with the Sepsityper workflow, and neither had the initial viscosity of the synovial sample.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0243790PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7732097PMC
January 2021

Remote near infrared identification of pathogens with multiplexed nanosensors.

Nat Commun 2020 11 25;11(1):5995. Epub 2020 Nov 25.

Institute of Physical Chemistry, Göttingen University, Göttingen, Germany.

Infectious diseases are worldwide a major cause of morbidity and mortality. Fast and specific detection of pathogens such as bacteria is needed to combat these diseases. Optimal methods would be non-invasive and without extensive sample-taking/processing. Here, we developed a set of near infrared (NIR) fluorescent nanosensors and used them for remote fingerprinting of clinically important bacteria. The nanosensors are based on single-walled carbon nanotubes (SWCNTs) that fluoresce in the NIR optical tissue transparency window, which offers ultra-low background and high tissue penetration. They are chemically tailored to detect released metabolites as well as specific virulence factors (lipopolysaccharides, siderophores, DNases, proteases) and integrated into functional hydrogel arrays with 9 different sensors. These hydrogels are exposed to clinical isolates of 6 important bacteria (Staphylococcus aureus, Escherichia coli,…) and remote (≥25 cm) NIR imaging allows to identify and distinguish bacteria. Sensors are also spectrally encoded (900 nm, 1000 nm, 1250 nm) to differentiate the two major pathogens P. aeruginosa as well as S. aureus and penetrate tissue (>5 mm). This type of multiplexing with NIR fluorescent nanosensors enables remote detection and differentiation of important pathogens and the potential for smart surfaces.
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http://dx.doi.org/10.1038/s41467-020-19718-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7689463PMC
November 2020

Phenotypic Variability in a Coinfection With Three Independent Lineages.

Front Microbiol 2020 27;11:1994. Epub 2020 Aug 27.

Institute for Medical Microbiology, University Medical Center Göttingen, Göttingen, Germany.

The human pathogenic yeast has gained significant importance over the past decades as one of the principal causes of fungal bloodstream infections. Isolates of are known to be able to switch between several different colony morphologies in vitro, which are correlated with different cell shapes, altered cell surface properties, and thus different capacities to form biofilms on indwelling medical devices. In a set of six clinical specimens from a single surgery patient yielding stable smooth- as well as crepe-morphology isolates, we investigated the differences between five of them on a phenotypic and genomic level. In contrast to the initial assumption that they were switched forms of a clonal strain, karyotyping and genome sequencing showed that the patient was colonized by at least three distinct linages. Statistical analysis placed these groups distantly across the population of . Interestingly the single blood culture isolate was of smooth morphology and matched with an isolate from the patient's nose of similar morphology. Strong variation between the isolates was seen in adhesin-encoding genes, where repeat regions showed significant variation in length and repeat-numbers, most strikingly in of the smooth isolates. Although no differences in drug susceptibility were evident, the high phylogenetic distance separating the individual strains highlights the need for testing of multiple colonies in routine practice. The absence of biofilm formation in the blood stream isolate indicates a lack of respective adhesins in the cell wall, in turn pointing toward lack of adhesion as a positively contributing factor for dissemination.
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http://dx.doi.org/10.3389/fmicb.2020.01994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7481391PMC
August 2020

Variation Among Biosynthetic Gene Clusters, Secondary Metabolite Profiles, and Cards of Virulence Across Species.

Genetics 2020 Oct 17;216(2):481-497. Epub 2020 Aug 17.

Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235

is a major human pathogen. In contrast, and the recently described , the two species most closely related to , are not known to be pathogenic. Some of the genetic determinants of virulence (or "cards of virulence") that A. possesses are secondary metabolites that impair the host immune system, protect from host immune cell attacks, or acquire key nutrients. To examine whether secondary metabolism-associated cards of virulence vary between these species, we conducted extensive genomic and secondary metabolite profiling analyses of multiple , one , and multiple strains. We identified two cards of virulence (gliotoxin and fumitremorgin) shared by all three species and three cards of virulence (trypacidin, pseurotin, and fumagillin) that are variable. For example, we found that all species and strains examined biosynthesized gliotoxin, which is known to contribute to virulence, consistent with the conservation of the gliotoxin biosynthetic gene cluster (BGC) across genomes. For other secondary metabolites, such as fumitremorgin, a modulator of host biology, we found that all species produced the metabolite but that there was strain heterogeneity in its production within species. Finally, species differed in their biosynthesis of fumagillin and pseurotin, both contributors to host tissue damage during invasive aspergillosis. biosynthesized fumagillin and pseurotin, while biosynthesized fumagillin and biosynthesized neither. These biochemical differences were reflected in sequence divergence of the intertwined fumagillin/pseurotin BGCs across genomes. These results delineate the similarities and differences in secondary metabolism-associated cards of virulence between a major fungal pathogen and its nonpathogenic closest relatives, shedding light onto the genetic and phenotypic changes associated with the evolution of fungal pathogenicity.
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http://dx.doi.org/10.1534/genetics.120.303549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536862PMC
October 2020

Molecular Typing of Candida glabrata.

Mycopathologia 2020 Oct 15;185(5):755-764. Epub 2019 Oct 15.

Institute for Medical Microbiology, University Medical Center Göttingen, Kreuzbergring 57, 37075, Göttingen, Germany.

The yeast Candida glabrata has emerged, second only to Candida albicans, to be one of the most frequently isolated fungi in clinical specimen from human. Its frequent resistance towards azole antifungal drugs and the high capacity to form biofilms on indwelling catheters of individual isolates render it an often difficult to treat pathogen. Hence, there is a notably increasing scientific and clinical interest in this species. This has led to the development of a variety of molecular tools for genetic modification, strain collections, and last but not least different approaches to analyse the population structure among isolates of different geographical and clinical contexts. Often, these are used to study correlations (or the absence thereof) with different pathogenicity, virulence, or drug resistance traits. Three molecular methods have been used to type within the C. glabrata population on a genetic level by multiple studies: multi-locus sequence typing, microsatellite length polymorphisms, and clustering of whole-genome sequencing data, and these are subject of this review.
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http://dx.doi.org/10.1007/s11046-019-00388-xDOI Listing
October 2020

Proteotyping of as Alternate Typing Method to Ribotyping Is Able to Distinguish the Ribotypes RT027 and RT176 From Other Ribotypes.

Front Microbiol 2019 10;10:2087. Epub 2019 Sep 10.

Institut für Medizinische Mikrobiologie, Universitätsmedizin Göttingen, Göttingen, Germany.

, a Gram-positive spore-forming bacterium, is the leading cause of nosocomial diarrhea worldwide and therefore a substantial burden to the healthcare system. During the past decade, hypervirulent PCR-ribotypes (RT) e.g., RT027 or RT176 emerged rapidly all over the world, associated with both, increased severity and mortality rates. It is thus of great importance to identify epidemic strains such as RT027 and RT176 as fast as possible. While commonly used diagnostic methods, e.g., multilocus sequence typing (MLST) or PCR-ribotyping, are time-consuming, proteotyping offers a fast, inexpensive, and reliable alternative solution. In this study, we established a MALDI-TOF-based typing scheme for A total of 109 ribotyped strains representative for five MLST clades were analyzed by MALDI-TOF. MLST, based on whole genome sequences, and PCR-ribotyping were used as reference methods. Isoforms of MS-detectable biomarkers, typically ribosomal proteins, were related with the deduced amino acid sequences and added to the proteotyping scheme. In total, we were able to associate nine biomarkers with their encoding genes and include them in our proteotyping scheme. The discriminatory capacity of the proteotyping scheme was mainly based on isoforms of L28-M (2 main isoforms), L35-M (4 main isoforms), and S20-M (2 main isoforms) giving rise to at least 16 proteotyping-derived types. In our test population, five of these 16 proteotyping-derived types were detected. These five proteotyping-derived types did not correspond exactly to the included five MLST-based clades, nevertheless the subtyping depth of both methods was equivalent. Most importantly, proteotyping-derived clade B contained only isolates of the hypervirulent RT027 and RT176. Proteotyping is a stable and easy-to-perform intraspecies typing method and a promising alternative to currently used molecular techniques. It is possible to distinguish the group of RT027 and RT176 isolates from non-RT027/non-RT176 isolates using proteotyping, providing a valuable diagnostic tool.
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http://dx.doi.org/10.3389/fmicb.2019.02087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6747054PMC
September 2019

Identification of a distinct subset of disease-associated gain-of-function missense mutations in the STAT1 coiled-coil domain as system mutants.

Mol Immunol 2019 10 20;114:30-40. Epub 2019 Jul 20.

Department of Psychosomatic Medicine and Psychotherapy, University Medical Center Göttingen, Göttingen, Germany. Electronic address:

Heterozygous gain-of-function (GOF) mutations in the cytokine-regulated transcription factor STAT1 (signal transducer and activator of transcription 1) lead to chronic mucocutaneous candidiasis (CMC). However, the molecular basis of these pathogenic missense mutations is largely unknown. In this study, we characterized in more detail the CMC-associated GOF substitution mutation of arginine-to-tryptophan at position 274 (R274W) and, in addition, the adjacent glutamine-to-alanine mutation at position 275 (Q275A). Both mutants displayed elevated tyrosine phosphorylation levels, prolonged nuclear accumulation, and increased transcriptional responses to interferon-γ (IFNγ) stimulation. No difference was observed between wild-type (WT) and mutant STAT1 in DNA sequence-specificity or dissociation kinetics from high-affinity DNA-binding elements known as gamma-activated sites (GAS). Furthermore, all variants exhibited similar cooperative DNA binding. Unexpectedly, in vitro dephosphorylation rates using the recombinant STAT1-inactivating Tc45 phosphatase in both the absence and presence of double-stranded GAS elements were similar in all STAT1 variants. Likewise, the rate of tyrosine phosphorylation by Janus kinase 2 (JAK2) was unaltered as compared to the WT molecule, excluding that the phenotype of these mutants is caused by either defective Tc45-catalyzed dephosphorylation or JAK2-induced hyper-activation. Interestingly, within 10 min of IFNγ exposure, the majority of R274W and Q275A molecules had entered the nucleus, whereas the wild-type protein remained predominantly cytosolic. Thus, the exchange of critical residues located at the binding interface in the antiparallel dimer conformer led to a premature accumulation of phospho-STAT1 in the nuclear compartment. In summary, our data show that the hyper-activity of the GOF mutations results, at least in part, from the premature nuclear import of the tyrosine-phosphorylated molecules and not from alterations in their phosphorylation or dephosphorylation rates.
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http://dx.doi.org/10.1016/j.molimm.2019.07.008DOI Listing
October 2019

Differentiation of Subspecies by Proteotyping.

Eur J Microbiol Immunol (Bp) 2019 Jun 20;9(2):62-71. Epub 2019 May 20.

Institut für Medizinische Mikrobiologie, Universitätsmedizin Göttingen, Göttingen, Germany.

is a causative agent of intestinal illness and, occasionally, severe systemic infections and meningitis. currently comprises three subspecies: subspecies subspecies , and subspecies and are primarily associated with mammals whereas is associated with reptiles. To offer an alternative to laborious sequence-based techniques such as multilocus sequence typing (MLST) and polymerase chain reaction (PCR)-ribotyping for this species, the purpose of the study was to develop a typing scheme based on proteotyping. In total, 41 representative strains were analyzed by intact cell mass spectrometry and compared to MLST results. Biomarkers detected in the mass spectrum of subsp. reference strain LMG 6442 (NCTC 10842) as well as corresponding isoforms were associated with the respective amino acid sequences and added to the proteotyping scheme. In combination, the 9 identified biomarkers allow the differentiation of subspecies strains from and subspecies strains. Biomarkers to distinguish between and were not found. The results of the study show the potential of proteotyping to differentiate different subspecies, but also the limitations of the method.
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http://dx.doi.org/10.1556/1886.2019.00006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6563684PMC
June 2019

Proteotyping as alternate typing method to differentiate Campylobacter coli clades.

Sci Rep 2019 03 12;9(1):4244. Epub 2019 Mar 12.

Institut für Medizinische Mikrobiologie, Universitätsmedizin Göttingen, Kreuzbergring 57, 37075, Göttingen, Germany.

Besides Campylobacter jejuni, Campylobacter coli is the most common bacterial cause of gastroenteritis worldwide. C. coli is subdivided into three clades, which are associated with sample source. Clade 1 isolates are associated with acute diarrhea in humans whereas clade 2 and 3 isolates are more commonly obtained from environmental waters. The phylogenetic classification of an isolate is commonly done using laborious multilocus sequence typing (MLST). The aim of this study was to establish a proteotyping scheme using MALDI-TOF MS to offer an alternative to sequence-based methods. A total of 97 clade-representative C. coli isolates were analyzed by MALDI-TOF-based intact cell mass spectrometry (ICMS) and evaluated to establish a C. coli proteotyping scheme. MLST was used as reference method. Different isoforms of the detectable biomarkers, resulting in biomarker mass shifts, were associated with their amino acid sequences and included into the C. coli proteotyping scheme. In total, we identified 16 biomarkers to differentiate C. coli into the three clades and three additional sub-clades of clade 1. In this study, proteotyping has been successfully adapted to C. coli. The established C. coli clades and sub-clades can be discriminated using this method. Especially the clinically relevant clade 1 isolates can be differentiated clearly.
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http://dx.doi.org/10.1038/s41598-019-40842-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6414644PMC
March 2019

Genome Comparisons of Serial Clinical Isolates Reveal Patterns of Genetic Variation in Infecting Clonal Populations.

Front Microbiol 2019 12;10:112. Epub 2019 Feb 12.

Bioinformatics and Genomics Programme, Centre for Genomic Regulation, Barcelona Institute of Science and Technology, Barcelona, Spain.

is an opportunistic fungal pathogen that currently ranks as the second most common cause of candidiasis. Although the mechanisms underlying virulence and drug resistance in are now starting to be elucidated, we still lack a good understanding of how this yeast adapts during the course of an infection. Outstanding questions are whether the observed genomic plasticity of plays a role during infection, or what levels of genetic variation exist within an infecting clonal population. To shed light onto the genomic variation within infecting populations, we compared the genomes of 11 pairs and one trio of serial clinical isolates, each obtained from a single patient. Our results provide a catalog of genetic variations existing within clonal infecting isolates, and reveal an enrichment of non-synonymous changes in genes encoding cell-wall proteins. Genetic variation and the presence of non-synonymous mutations and copy number variations accumulated within the host, suggest that clonal populations entail a non-negligible level of genetic variation that may reflect selection processes that occur within the human body. As we show here, these genomic changes can underlie phenotypic differences in traits that are relevant for infection.
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http://dx.doi.org/10.3389/fmicb.2019.00112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379656PMC
February 2019

High diversity of Candida glabrata in a tertiary hospital-Mwanza, Tanzania.

Med Mycol 2019 Oct;57(7):914-917

Institute of Medical Microbiology, University Medical Center Gottingen, Germany.

Candida glabrata is a genetically diverse human pathogenic yeast, whose subpopulations have been documented to vary geographically. Here, we report MLST genotypes and antifungal drug susceptibility of C. glabrata isolates from Africa. Among 47 mostly urogenital isolates, we found 13 sequence types, amounting to a 27% genetic population difference. More than half of the isolates were of novel sequence types. ST18 was most predominant and had reduced susceptibility to fluconazole. There was clear segregation of STs between urine and vaginal specimen. In Tanzania, the C. glabrata population is genetically diverse, and divergent from those seen in other countries.
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http://dx.doi.org/10.1093/mmy/myy151DOI Listing
October 2019

Rapid and Sensitive Detection of Azole-Resistant Aspergillus fumigatus by Tandem Repeat Loop-Mediated Isothermal Amplification.

J Mol Diagn 2019 03 4;21(2):286-295. Epub 2018 Dec 4.

Centre for Bio-Inspired Technology, Institute of Biomedical Engineering, Department of Electrical and Electronic Engineering.

Invasive fungal infections caused by multiazole-resistant Aspergillus fumigatus are associated with increasing rates of mortality in susceptible patients. Current methods of diagnosing infections caused by multiazole-resistant A. fumigatus are, however, not well suited for use in clinical point-of-care testing or in the field. Loop-mediated isothermal amplification (LAMP) is a widely used method of nucleic acid amplification with rapid and easy-to-use features, making it suitable for use in different resource settings. Here, we developed a LAMP assay to detect a 34 bp tandem repeat, named TR34-LAMP. TR34 is a high-prevalence allele that, in conjunction with the L98H single-nucleotide polymorphism, is associated with the occurrence of multiazole resistance in A. fumigatus in the environment and in patients. This process was validated with both synthetic double-stranded DNA and genomic DNA prepared from azole-resistant isolates of A. fumigatus. Use of our assay resulted in rapid and specific identification of the TR34 allele with high sensitivity, detecting down to 10 genomic copies per reaction within 25 minutes. Fluorescent and colorimetric detections were used for the analysis of 11 clinical isolates as cross validation. These results show that the TR34-LAMP assay has the potential to accelerate the screening of clinical and environmental A. fumigatus to provide a rapid and accurate diagnosis of azole resistance, which current methods struggle to achieve.
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http://dx.doi.org/10.1016/j.jmoldx.2018.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6419584PMC
March 2019

Disseminated cryptococcosis in a HIV-negative patient: Case report of a newly diagnosed hypertensive adult presenting with hemiparesis.

Med Mycol Case Rep 2018 Dec 12;22:4-7. Epub 2018 Jul 12.

Department of Microbiology and Immunology, Weill Bugando School of Medicine, Catholic University of Health and Allied Sciences, Mwanza, Tanzania.

We report a case of disseminated cryptococcosis in a 42-year old immunocompetent female. Prior to admission at Bugando Medical Center, the patient was attended at three hospitals for hypertension and clinically diagnosed malaria. Following diagnosis of disseminated at our center, she was successfully treated with fluconazole but remained with visual loss. Blood cultures should be considered in the management of any adult presenting with fever to enable early detection of the least expected differentials like in this case.
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http://dx.doi.org/10.1016/j.mmcr.2018.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235752PMC
December 2018

Virulence and susceptibility patterns of clinical Candida spp. isolates from a tertiary hospital, Tanzania.

Med Mycol 2018 Oct 31. Epub 2018 Oct 31.

Department of Microbiology and immunology, Weill Bugando School of Medicine, Catholic University of Heath and Allied Sciences Mwanza, Tanzania.

Despite the increased burden of human immunodeficiency virus (HIV) and other comobidities in developing countries, information regarding antifungal susceptibility patterns of Candida spp. and their virulence potential are still limited. Here, we report the virulence and antifungal susceptibility patterns of Candida spp. from varieties spectrum of candidiasis in a tertiary hospital, Tanzania. The study was conducted from March to December 2017. Candida spp. from clinical samples were characterized. Antifungal susceptibility patterns based on EUCAST guidelines and virulence activities (phospholipase, protease, hemolysin, and coagulase activity) were determined. A total of 399 Candida spp. isolates were obtained, of these, 278, 51 and 47 were C. albicans, C. tropicalis, and C. glabrata, respectively. Phospholipase 193/268, protease 32/51 and coagulase 25/47 were the most frequently detected virulence activities in C. albicans, C. tropicalis, and C. glabrata, respectively. Protease and phospholipase were frequently detected virulence activities from C. albicans from blood and esophageal brushes. The median zone diameter of protease activities was significantly larger among C. tropicalis than C. albicans. C. albicans, and C. tropicalis isolates were 100% sensitive to caspofungin. The proportions of C. albicans isolate resistant to fluconazole, voriconazole and posaconazole were 3.1, 3.6%, and 1.8%, respectively. In conclusion, the majority of Candida spp. isolates were sensitive to fluconazole. There are different phenotypes of C. albicans, C. glabrata and C. tropicalis based on susceptibility and virulence activities patterns, necessitating further molecular characterizations to place them in global perspective. Routine antifungal susceptibility testing to guide clinical therapy should be encouraged in developing countries.
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http://dx.doi.org/10.1093/mmy/myy107DOI Listing
October 2018

Comparison of Two Molecular Assays for Detection and Characterization of Triazole Resistance and Mutations in Clinical Isolates and Primary Clinical Samples of Immunocompromised Patients.

Front Microbiol 2018 27;9:555. Epub 2018 Mar 27.

Department of Hematology and Oncology, University Hospital Mannheim, Heidelberg University, Mannheim, Germany.

In hematological patients, the incidence of invasive aspergillosis (IA) caused by azole resistant (ARAf) is rising. As the diagnosis of IA is rarely based on positive culture in this group of patients, molecular detection of resistance mutations directly from clinical samples is crucial. In addition to the in-house azole resistance ARAf polymerase chain reaction (PCR) assays detecting the frequent mutation combinations TR34/L98H, TR46/Y121F/T289A, and M220 in the () gene by subsequent DNA sequence analysis, we investigated in parallel the commercially available AsperGenius® real time PCR system in detecting the alterations TR34/L98H and Y121F/T289A directly from 52 clinical samples (15 biopsies, 22 bronchoalveolar lavage (BAL), 15 cerebrospinal fluid (CSF) samples) and ARAf isolates ( = 3) of immunocompromised patients. We analyzed DNA aliquots and compared both methods concerning amplification and detection of DNA and alterations. As positive control for the feasibility of our novel Y121F and T289A PCR assays, we used two isolates with the TR46/Y121F/T289A mutation combination isolated from hematological patients with known C alterations and a lung biopsy sample of a patient with acute myeloid leukemia (AML). The rate of positive ARAf PCR results plus successful sequencing using the ARAf PCR assays was 61% in biopsies, 29% in CSF, 67% in BAL samples and 100% in isolates. In comparison the amount of positive PCRs using the AsperGenius® assays was 47% in biopsies, 42% in CSF, 59% in BAL samples and 100% in isolates. Altogether 17 alterations were detected using our ARAf PCRs plus DNA sequencing and therefrom 10 alterations also by the AsperGenius® system. The comparative evaluation of our data revealed that our conventional PCR assays are more sensitive in detecting ARAf in BAL and biopsy samples, whereby differences were not significant. The advantage of the AsperGenius® system is the time saving aspect. We consider non-culture based molecular detection of triazole resistance to be of high epidemiological and clinical relevance in patients with hematological malignancies.
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http://dx.doi.org/10.3389/fmicb.2018.00555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5890139PMC
March 2018

Comparative evaluation of different gradient diffusion tests for detection of azole resistance in Aspergillus fumigatus.

Diagn Microbiol Infect Dis 2018 May 9;91(1):52-54. Epub 2018 Jan 9.

Institute for Medical Microbiology, University Medical Center Göttingen, Kreuzbergring 57, 37075 Göttingen, Germany.

Gradient diffusion assays Etest and MIC Test Strip for itraconazole, posaconazole and voriconazole as well as isavuconazole (MIC Test Strip) were evaluated for Aspergillus fumigatus against EUCAST broth microdilution. Both assays demonstrated generally good performance; however, posaconazole MIC Test Strip showed low agreement with broth microdilution due to MIC overestimation.
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http://dx.doi.org/10.1016/j.diagmicrobio.2018.01.003DOI Listing
May 2018

Processing of Ece1p Is Critical for Candidalysin Maturation and Fungal Virulence.

mBio 2018 01 23;9(1). Epub 2018 Jan 23.

Mucosal and Salivary Biology Division, Dental Institute, King's College London, London, United Kingdom.

is an opportunistic fungal pathogen responsible for superficial and life-threatening infections in humans. During mucosal infection, undergoes a morphological transition from yeast to invasive filamentous hyphae that secrete candidalysin, a 31-amino-acid peptide toxin required for virulence. Candidalysin damages epithelial cell plasma membranes and stimulates the activating protein 1 (AP-1) transcription factor c-Fos (via p38-mitogen-activated protein kinase [MAPK]), and the MAPK phosphatase MKP1 (via extracellular signal-regulated kinases 1 and 2 [ERK1/2]-MAPK), which trigger and regulate proinflammatory cytokine responses, respectively. The candidalysin toxin resides as a discrete cryptic sequence within a larger 271-amino-acid parental preproprotein, Ece1p. Here, we demonstrate that kexin-like proteinases, but not secreted aspartyl proteinases, initiate a two-step posttranslational processing of Ece1p to produce candidalysin. Kex2p-mediated proteolysis of Ece1p after Arg61 and Arg93, but not after other processing sites within Ece1p, is required to generate immature candidalysin from Ece1p, followed by Kex1p-mediated removal of a carboxyl arginine residue to generate mature candidalysin. strains harboring mutations of Arg61 and/or Arg93 did not secrete candidalysin, were unable to induce epithelial damage and inflammatory responses , and showed attenuated virulence in a murine model of oropharyngeal candidiasis. These observations identify enzymatic processing of Ece1p by kexin-like proteinases as crucial steps required for candidalysin production and fungal pathogenicity. is an opportunistic fungal pathogen that causes mucosal infection in millions of individuals worldwide. Successful infection requires the secretion of candidalysin, the first cytolytic peptide toxin identified in any human fungal pathogen. Candidalysin is derived from its parent protein Ece1p. Here, we identify two key amino acids within Ece1p vital for processing and production of candidalysin. Mutations of these residues render incapable of causing epithelial damage and markedly reduce mucosal infection Importantly, candidalysin production requires two individual enzymatic events. The first involves processing of Ece1p by Kex2p, yielding immature candidalysin, which is then further processed by Kex1p to produce the mature toxin. These observations identify important steps for pathogenicity at mucosal surfaces.
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http://dx.doi.org/10.1128/mBio.02178-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5784256PMC
January 2018

Patterns of Genomic Variation in the Opportunistic Pathogen Candida glabrata Suggest the Existence of Mating and a Secondary Association with Humans.

Curr Biol 2018 01 14;28(1):15-27.e7. Epub 2017 Dec 14.

Bioinformatics and Genomics Programme, Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Dr. Aiguader 88, 08003 Barcelona, Spain; Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain; Institució Catalana de Recerca i Estudis Avançats (ICREA), Passeig Lluís Companys 23, 08010 Barcelona, Spain. Electronic address:

Candida glabrata is an opportunistic fungal pathogen that ranks as the second most common cause of systemic candidiasis. Despite its genus name, this yeast is more closely related to the model yeast Saccharomyces cerevisiae than to other Candida pathogens, and hence its ability to infect humans is thought to have emerged independently. Moreover, C. glabrata has all the necessary genes to undergo a sexual cycle but is considered an asexual organism due to the lack of direct evidence of sexual reproduction. To reconstruct the recent evolution of this pathogen and find footprints of sexual reproduction, we assessed genomic and phenotypic variation across 33 globally distributed C. glabrata isolates. We cataloged extensive copy-number variation, which particularly affects genes encoding cell-wall-associated proteins, including adhesins. The observed level of genetic variation in C. glabrata is significantly higher than that found in Candida albicans. This variation is structured into seven deeply divergent clades, which show recent geographical dispersion and large within-clade genomic and phenotypic differences. We show compelling evidence of recent admixture between differentiated lineages and of purifying selection on mating genes, which provides the first evidence for the existence of an active sexual cycle in this yeast. Altogether, our data point to a recent global spread of previously genetically isolated populations and suggest that humans are only a secondary niche for this yeast.
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http://dx.doi.org/10.1016/j.cub.2017.11.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5772174PMC
January 2018

Molecular Tools for the Detection and Deduction of Azole Antifungal Drug Resistance Phenotypes in Aspergillus Species.

Clin Microbiol Rev 2017 10;30(4):1065-1091

Institute for Medical Microbiology, University Medical Center Göttingen, Göttingen, Germany

The incidence of azole resistance in species has increased over the past years, most importantly for . This is partially attributable to the global spread of only a few resistance alleles through the environment. Secondary resistance is a significant clinical concern, as invasive aspergillosis with drug-susceptible strains is already difficult to treat, and exclusion of azole-based antifungals from prophylaxis or first-line treatment of invasive aspergillosis in high-risk patients would dramatically limit drug choices, thus increasing mortality rates for immunocompromised patients. Management options for invasive aspergillosis caused by azole-resistant strains were recently reevaluated by an international expert panel, which concluded that drug resistance testing of cultured isolates is highly indicated when antifungal therapy is intended. In geographical regions with a high environmental prevalence of azole-resistant strains, initial therapy should be guided by such analyses. More environmental and clinical screening studies are therefore needed to generate the local epidemiologic data if such measures are to be implemented on a sound basis. Here we propose a first workflow for evaluating isolates from screening studies, and we compile the MIC values correlating with individual amino acid substitutions in the products of genes for interpretation of DNA sequencing data, especially in the absence of cultured isolates.
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http://dx.doi.org/10.1128/CMR.00095-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608879PMC
October 2017

Oral candidiasis among African human immunodeficiency virus-infected individuals: 10 years of systematic review and meta-analysis from sub-Saharan Africa.

J Oral Microbiol 2017 21;9(1):1317579. Epub 2017 Jun 21.

Department of Microbiology and Immunology, Weill Bugando School of Medicine, Catholic University of Heath and Allied Sciences, Mwanza, Tanzania.

Oral candidiasis (OC) is the most common opportunistic fungal infection among immunocompromised individuals. This systematic review and meta-analysis reports on the contribution of non- species in causing OC among human immunodeficiency virus (HIV)-infected individuals in sub-Saharan Africa between 2005 and 2015. Thirteen original research articles on oral infection/colonization among HIV-infected African populations were reviewed. The prevalence of OC ranged from 7.6% to 75.3%. Pseudomembranous candidiasis was found to range from 12.1% to 66.7%. The prevalence of non- species causing OC was 33.5% [95% confidence interval (CI) 30.9-36.39%]. Of 458 non- species detected, . (23.8%; 109/458) was the most common, followed by . (22%; 101/458) and . (10.7%; 49/458). The overall fluconazole resistance was 39.3% (95% CI 34.4-44.1%). was significantly more resistant than non-albicans species to fluconazole (44.7% vs 21.9%;  < 0.001). One-quarter of the cases of OC among HIV-infected individuals in sub-Saharan Africa were due to non- species. isolates were more resistant than the non- species to fluconazole and voriconazole. Strengthening the capacity for fungal diagnosis and antifungal susceptibility testing in sub-Saharan Africa is mandatory in order to track the azole resistance trend.
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http://dx.doi.org/10.1080/20002297.2017.1317579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5508360PMC
June 2017

Progressive Dispersion of Azole Resistance in Aspergillus fumigatus: Fatal Invasive Aspergillosis in a Patient with Acute Myeloid Leukemia Infected with an A. fumigatus Strain with a TR Y121F M172I T289A Allele.

Antimicrob Agents Chemother 2017 08 25;61(8). Epub 2017 Jul 25.

Laboratory of Mycology Research, Center for Antimicrobial Resistance and Microbial Genomics, McGovern Medical School, Houston, Texas, USA

Patients with hematologic malignancies as well as allogeneic hematopoietic stem cell transplantation (HSCT) patients are at high risk for invasive aspergillosis. Here, we report a culture- and autopsy-proven fatal invasive aspergillosis in an allogeneic HSTC patient which he developed despite posaconazole prophylaxis. The agent was determined to be an azole-resistant strain bearing the mutation combination TR Y121F M172I T289A. At increasing frequency, the azole resistance of is being reported globally, limiting treatment options and complicating regimens.
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http://dx.doi.org/10.1128/AAC.00270-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5527573PMC
August 2017

Prevalence of pregnancy-relevant infections in a rural setting of Ghana.

BMC Pregnancy Childbirth 2017 Jun 6;17(1):172. Epub 2017 Jun 6.

Institute for Medical Microbiology, University Medical Center Göttingen, Kreuzbergring 57, D-37075, Göttingen, Germany.

Background: Although infectious diseases still account for a high burden of morbidity and mortality in sub-Saharan Africa, simultaneous investigations on multiple infections affecting maternal and child health are missing.

Methods: We conducted a cross-sectional, single-centre pilot study in a rural area of Ghana to assess the infectiological profile during pregnancy. Screening of 180 expectant mothers was done by vaginal swabs and serology to detect the most common pregnancy-relevant infections. They were also interviewed for potential risk factors, outcome of previous pregnancies, and socio-economic aspects.

Results: We found a high prevalence of infections caused by hepatitis B virus (16.7% HBs antigen positive). In contrast, infections caused by hepatitis C virus (1.1% anti-HCV) and HIV (0.6%) were rare. Maternal malaria was frequent (10.6%), despite increasing acceptance of intermittent preventive treatment during pregnancy (IPTp). Group B streptococci were present in 10.6% of all pregnant women. Absence of antibodies against varicella zoster virus in 43.2%, Toxoplasma gondii in 26.8%, parvovirus B19 in 20.0%, and rubella virus in 15.7% makes a significant proportion of pregnant women susceptible for acquiring primary infections. Whereas all study participants had specific IgG antibodies against human cytomegalovirus, infections with Listeria, Brucella, or Neisseria gonorrhoeae as well as active syphilis were absent.

Conclusions: Our pilot study in a rural community in Ghana indicates an urgent need for action in dealing at least with high-prevalent pregnancy-relevant infections, such as hepatitis B, malaria and those caused by group B streptococci. In addition, the resulting prevalence rates of various other infections may offer guidance for health officials to prioritize possible future intervention schemes.
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http://dx.doi.org/10.1186/s12884-017-1351-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460405PMC
June 2017

Oral findings and dental behaviour before and after liver transplantation - a single-centre cross-sectional study.

Int Dent J 2017 Aug 10;67(4):244-251. Epub 2017 Mar 10.

Department of Cariology, Endodontology and Periodontology, University of Leipzig, Leipzig, Germany.

Objective: The aim of this single-centre, cross-sectional study was to evaluate dental, periodontal and mycological findings, as well as oral behaviour, in patients before (pre-LTx) and after (post-LTx) liver transplantation.

Methods: A total of 47 patients pre-LTx and 119 patients post-LTx were asked to participate. Oral health behaviour was assessed using a standardised questionnaire. Oral examinations included dental [decayed, missing and filled teeth (DMFT) index] and periodontal [papillary bleeding index (PBI), periodontal probing depth (PPD) and clinical attachment loss (CAL)] findings. For Candida screening, swabs from the oral mucosa were cultured. Statistical analysis was performed using the Student's t-test or the Mann-Whitney U-test, depending on whether or not the data followed a normal distribution; Fisher's exact test was also performed. The significance level was α = 5%.

Results: A total of 110 patients were included (pre-LTx, n = 35; post-LTx, n = 75). Different patients were investigated in the post-LTx and pre-LTx groups. Lack of use of supplemental oral-hygiene aids was noted. Between-group comparisons failed to find significant overall differences in DMFT and periodontal status. The post-LTx group showed fewer decayed teeth (P = 0.03). A total of 86% of patients pre-LTx and 84% of patients post-LTx were found to need dental treatment, and 60% of patients pre-LTx and 55% of patients post-LTx showed a need for periodontal treatment. The prevalence of Candida albicans was high; however, there were no statistically significant differences between the groups in regard to fungal infection.

Conclusion: Improved dental care pre- and post-transplant, including screening for fungal infections, is recommended to avoid systemic infections in LTx patients. Increased attention to oral health care, and interdisciplinary collaboration to provide guidance, is needed to improve the oral health of patients before and after LTx.
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http://dx.doi.org/10.1111/idj.12290DOI Listing
August 2017

Epidemiology of invasive aspergillosis and azole resistance in patients with acute leukaemia: the SEPIA Study.

Int J Antimicrob Agents 2017 Feb 12;49(2):218-223. Epub 2016 Dec 12.

Department I of Internal Medicine, University Hospital of Cologne, Cologne, Germany; German Centre for Infection Research, Partner Site Bonn-Cologne, Cologne, Germany. Electronic address:

Invasive aspergillosis (IA) is a serious hazard to high-risk haematological patients. There are increasing reports of azole-resistant Aspergillus spp. This study assessed the epidemiology of IA and azole-resistant Aspergillus spp. in patients with acute leukaemia in Germany. A prospective multicentre cohort study was performed in German haematology/oncology centres. The incidence of probable and proven aspergillosis according to the revised EORTC/MSG criteria was assessed for all patients with acute leukaemia [acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL)]. Cases were documented into a web-based case report form, and centres provided data on standards regarding prophylactic and diagnostic measures. Clinical isolates were screened centrally for azole resistance and, if applicable, underlying resistance mechanisms were analysed. Between September 2011 and December 2013, 179 cases of IA [6 proven (3.4%) and 173 probable (96.6%)] were diagnosed in 3067 patients with acute leukaemia. The incidence of IA was 6.4% among 2440 AML patients and 3.8% among 627 ALL patients. Mortality at Day 84 was 33.8% (49/145) and attributable mortality was 26.9% (39/145). At Day 84, 53 patients (29.6%) showed a complete response, 25 (14.0%) a partial response and 17 (9.5%) a deterioration or failure. A total of 77 clinical Aspergillus fumigatus isolates were collected during the study period. Two episodes of azole-resistant IA (1.1%) were caused by a TR/L98H mutation in the cyp51A gene. With only two cases of IA due to azole-resistant A. fumigatus, a change of antifungal treatment practices in Germany does not appear warranted currently.
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http://dx.doi.org/10.1016/j.ijantimicag.2016.10.019DOI Listing
February 2017

Diversity and Antifungal Drug Susceptibility of Cryptococcus Isolates in Thailand.

Med Mycol 2017 Aug;55(6):680-685

Interdisciplinary Program, Medical Microbiology, Graduate School, Chulalongkorn University, Bangkok, Thailand.

Yeasts of the Cryptococcus species complex are the causative agent of cryptococcosis, especially in human immunodeficiency virus (HIV) positive individuals. Cerebral or disseminated cryptococcosis has a very high mortality rate worldwide, including in Thailand. Additionally, an increasing rate of antifungal drug resistant cryptococcal isolates has been reported in several neighboring countries, complicating therapeutic approaches. To understand the situation of this infection in Thailand, we retrospectively investigated the molecular epidemiology and antifungal drug resistance in a collection of 74 clinical, 52 environmental and two veterinary isolates using the URA5-RFLP for typing and the EUCAST guideline for susceptibility testing. Where no EUCAST breakpoints (AMB and 5FC) were available, CLSI epidemiologic cutoff values were used for interpretation. Cryptococcal molecular type diversity showed most isolates were C. grubii, molecular type VNI. One clinical isolate was C. deuterogattii (mol. type VGII) and another C. grubii (mol. type VNII). One strain from environment was classified as C. grubii (mol. type VNII). No resistant strains were detected in this retrospective study for either of the antimycotics tested; however, monitoring of the epidemiology of Cryptococcus species in infected patients in Thailand needs to be continued to detect emergence of resistance.
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http://dx.doi.org/10.1093/mmy/myw130DOI Listing
August 2017

Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics (MSPP).

J Vis Exp 2016 10 30(116). Epub 2016 Oct 30.

Institut für Medizinische Mikrobiologie, Universitätsmedizin Göttingen.

MALDI-TOF MS offers the possibility to differentiate some bacteria not only at the species and subspecies level but even below, at the strain level. Allelic isoforms of the detectable biomarker ions result in isolate-specific mass shifts. Mass spectrometry-based phyloproteomics (MSPP) is a novel technique that combines the mass spectrometric detectable biomarker masses in a scheme that allows deduction of phyloproteomic relations from isolate specific mass shifts compared to a genome sequenced reference strain. The deduced amino acid sequences are then used to calculate MSPP-based dendrograms. Here we describe the workflow of MSPP by typing a Campylobacter jejuni ssp. doylei isolate collection of seven strains. All seven strains were of human origin and multilocus sequence typing (MLST) demonstrated their genetic diversity. MSPP-typing resulted in seven different MSPP sequence types, sufficiently reflecting their phylogenetic relations. The C. jejuni ssp. doylei MSPP scheme includes 14 different biomarker ions, mostly ribosomal proteins in the mass range of 2 to 11 kDa. MSPP can in principle, be adapted to other mass spectrometric platforms with an extended mass range. Therefore, this technique has the potential to become a useful tool for strain level microbial typing.
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http://dx.doi.org/10.3791/54165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5226127PMC
October 2016

Fungal Species Identification by MALDI-ToF Mass Spectrometry.

Authors:
Oliver Bader

Methods Mol Biol 2017 ;1508:323-337

Institute for Medical Microbiology, University Medical Center Göttingen, Kreuzbergring 57, Göttingen, D-37075, Germany.

MALDI-TOF MS has become the standard method for routine identification of most microbial organisms in clinical laboratories and has largely replaced biochemical assays. Classification relies on extensive well curated databases, ideally covering the full spectrum of microorganisms encountered in the specimens at hands. The protocols for harvesting cells and procuring material suitable for downstream MALDI-TOF MS analyses vary in specific details between the different groups of organisms, e.g., gram-positive or -negative bacteria, mycobacteria, or fungi. With respect to fungi, methods further vary between yeasts and moulds; and even among different mould genera if they do not lyse in a similar fashion. Purification of microbial materials from clinical specimen allows the direct identification of bacteria; however this is not yet fully adapted to fungi. In this chapter, I look into the differences between the underlying methods for yeast and moulds, and for production of samples suitable for MALDI-TOF MS species identification from cultures and different clinical materials.
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http://dx.doi.org/10.1007/978-1-4939-6515-1_19DOI Listing
January 2018

Prevalence of azole-resistant Aspergillus fumigatus in the environment of Thailand.

Med Mycol 2017 Jun;55(4):429-435

Institute for Medical Microbiology, University Medical Center Göttingen, Kreuzbergring 57, 37075 Göttingen, Germany.

Occurrence of azole-resistant Aspergillus fumigatus (ARAF) in the environment is an emerging problem worldwide, likely impacting on patient treatment. Several resistance mutations are thought to have initially arisen through triazole-based fungicide use in agriculture and subsequently being propagated in a similar manner. Here we investigated the prevalence of ARAF in the environment of Thailand and characterized their susceptibility profiles toward clinically used azole compounds along with underlying resistance mutations. Three hundred and eight soil samples were collected and analyzed, out of which 3.25% (n = 10) were positive for ARAF. All isolates obtained were resistant to itraconazole (MIC ≥ 8 μg/ml), two showed additional increased MIC values toward posaconazole (MIC = 0.5 μg/ml), and one other toward voriconazole (MIC = 2 μg/ml). Sequencing of the respective cyp51A genes revealed that eight of the isolates carried the TR34/L98H allele and those two with elevated MIC values to posaconazole the G54R substitution. Although a clear correlation between the use of triazole-based fungicides and isolation of ARAF strains from agricultural lands could not be established for Thailand, but this study clearly demonstrates the spread of globally observed ARAF strains to the environment of South East Asia.
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http://dx.doi.org/10.1093/mmy/myw090DOI Listing
June 2017

Erratum to: Identification of Stachybotrys spp. by MALDI-TOF mass spectrometry.

Anal Bioanal Chem 2016 11 9;408(27):7879. Epub 2016 Sep 9.

Chair of Food Safety, Department of Veterinary Sciences, LMU Munich, Schoenleutnerstr. 8, 85764, Oberschleissheim, Germany.

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http://dx.doi.org/10.1007/s00216-016-9921-1DOI Listing
November 2016