Search

In elderly patients, several morbidities or medical treatments predisposing for fungal infections occur at a higher frequency, leading to high mortality and morbidity in this vulnerable patient group. Often, this is linked to an innately azole-resistant yeast species such as Candida glabrata or C. krusei. Additionally, host age per se and the wearing of dentures have been determined to influence the mix of colonizing species and, consequently, the species distribution of invasive fungal infections. Since both old age and the wearing of dentures are two tightly connected parameters, it is still unclear which of them is the main contributor. Here, we performed a cross-sectional study on a cohort (N = 274) derived from three groups of healthy elderly, diseased elderly, and healthy young controls. With increasing host age, the frequency of oral colonization by a non-albicans&nbsp, Candida species, mainly by C. glabrata, also increased, and the wearing of dentures predisposed for colonization by C. glabrata irrespectively of host age. Physically diseased hosts, on the other hand, were more frequently orally colonized by C. albicans than by other yeasts. For both C. albicans and C. glabrata, isolates from the oral cavity did not generally display an elevated biofilm formation capacity. In conclusion, intrinsically azole-drug-resistant, non-albicans&nbsp, Candida yeasts are more frequent in the oral cavities of the elderly, and fungal cells not contained in biofilms may predispose for subsequent systemic infection with these organisms. This warrants further exploration of diagnostic procedures, e.g., before undergoing elective abdominal surgery or when using indwelling devices on this patient group.

Background Extended-spectrum β-lactamases (ESBLs) are enzymes that can render their hosts resistant to various β-lactam antibiotics. CTX-M-type enzymes are the most prevalent ESBLs and the main cause of resistance to third-generation cephalosporins in Enterobacteriaceae. The number of described CTX-M types is continuously rising, currently comprising over 240 variants. During routine screening we identified a novel blaCTX-M gene. Objectives To characterize a novel blaCTX-M variant harboured by a multidrug-resistant Escherichia coli isolate of sequence type ST354. Methods Antibiotic susceptibilities were determined using broth microdilution. Genome and plasmid sequences were reconstructed using short- and long-read sequencing. The novel blaCTX-M locus was analysed using long-read and Sanger sequencing. Plasmid polymorphisms were determined in silico on a single plasmid molecule level. Results The novel blaCTX-M-243 allele was discovered alongside a nearly identical blaCTX-M-104-containing gene array on a 219 kbp IncHI2A plasmid. CTX-M-243 differed from CTX-M-104 by only one amino acid substitution (N109S). Ultra-deep (2300-fold coverage) long-read sequencing revealed dynamic scaling of the blaCTX-M genetic contexts from one to five copies. Further antibiotic resistance genes such as blaTEM-1 also exhibited sequence heterogeneity but were stable in copy number. Conclusions We identified the novel ESBL gene blaCTX-M-243 and illustrate a dynamic system of varying blaCTX-M copy numbers. Our results highlight the constant emergence of new CTX-M family enzymes and demonstrate a potential evolutionary platform to generate novel ESBL variants and possibly other antibiotic resistance genes.

This study was performed as a head-to-head comparison of the performance characteristics of (1) two SARS-CoV-2-specific rapid antigen assays with real-time PCR as gold standard as well as (2) a fully automated high-throughput transcription-mediated amplification (TMA) assay and real-time PCR in a latent class analysis-based test comparison without a gold standard with several hundred samples in a low prevalence “real world” setting. Recorded sensitivity and specificity of the NADAL and the LumiraDx antigen assays and the Hologic Aptima SARS-CoV-2 TMA assay were 0.1429 (0.0194, 0.5835), 0.7644 (0.7016, 0.8174), and 0.7157 (0, 1) as well as 0.4545 (0.2022, 0.7326), 0.9954 (0.9817, 0.9988), and 0.9997 (not estimable), respectively. Agreement kappa between the positive results of the two antigen-based assays was 0.060 (0.002, 0.167) and 0.659 (0.492, 0.825) for TMA and real-time PCR. Samples with low viral load as indicated by cycle threshold (Ct) values &gt, 30 were generally missed by both antigen assays, while 1:10 pooling suggested higher sensitivity of TMA compared to real-time PCR. In conclusion, both sensitivity and specificity speak in favor of the use of the LumiraDx rather than the NADAL antigen assay, while TMA results are comparably as accurate as PCR, when applied in a low prevalence setting.

Candida parapsilosis 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 C. parapsilosis 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 C. parapsilosis 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. This work was funded in part by grants or scholarships from the ZabaldUz program (Universidad del País Vasco/Euskal Herriko Unibertsitatea) to IDlP, the Consejería de Educación, Universidades e Investigación (GIC15/78 IT-990-16) of Gobierno Vasco-Eusko Jaurlaritza to GQ, the Ministerio de Economía y Competitividad (grants SAF2013-47570-P and SAF2017-86188-P, the latter co-financed by FEDER) of the Spanish government to P.G. and G.Q., and the FP7-PEOPLE-2013-ITN—Marie-Curie Action: “Initial Training Networks”: Molecular Mechanisms of Human Fungal Pathogen Host Interaction, ImResFun, MC-ITN-606786, to O.B. and U.G.

SUMMARYBackgroundThe global pandemic of COVID-19 (coronavirus disease 2019) is caused by the novel coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), with different prevalence rates across countries and regions. Dynamic testing strategies are mandatory to establish efficient mitigation strategies against the disease; to be cost effective, they should adapt to regional prevalences. Seroprevalence surveys that detect individuals who have mounted an immune response against COVID-19 will help to determine the total number of infections within a community and improve the epidemiological calculations of attack and case fatality rates of the virus. They will also inform about the percentage of a population that might be immune against re-infections.MethodsWe developed a sensitive and specific cell-based assay to detect conformational SARS-CoV-2 spike (SARS-2-S) S1 antibodies in human serum, and have cross-evaluated this assay against two FDA-approved SARS-CoV-2 antibody assays. We performed pseudovirus neutralization assays to determine whether sera that were rated antibody-positive in our assay were able to specifically neutralize SARS-2-S. We pooled up to 24 sera and assessed the group testing performance of our cell-based assay. Group testing was further optimized by Monte Carlo like simulations and prospectively evaluated.FindingsHighly significant correlations could be established between our cell-based assay and commercial antibody tests for SARS-CoV-2. SARS-2-S S1 antibody-positive sera neutralized SARS-2-S but not SARS-S, and were sensitively and specifically detected in pools of 24 samples. Monte Carlo like simulations demonstrated that a simple two-step pooling scheme with fixed pool sizes performed at least equally as well as Dorfman’s optimal testing across a wide range of antibody prevalences.InterpretationWe demonstrate that a cell-based assay for SARS-2-S S1 antibodies qualifies for group testing of neutralizing anti-SARS-2-S antibodies. The assay can be combined with an easily implemented algorithm which greatly expands the screening capacity to detect anti-SARS-2-S antibodies across a wide range of antibody prevalences. It will thus improve population serological testing in many countries.FundingThis work was supported by the Bundesministerium für Bildung und Forschung within the network project RAPID (risk assessment in pre-pandemic respiratory infectious diseases [grant number 01KI1723D, S.P.]).

Candida yeasts causing human infections are spread across the yeast phylum with Candida glabrata being related to Saccharomyces cerevisiae, Candida krusei grouping to Pichia spp., and Candida albicans, Candida parapsilosis and Candida tropicalis belonging to the CTG-clade. The latter lineage contains yeasts with an altered genetic code translating CUG codons as serine using a serine-tRNA with a mutated anticodon. It has been suggested that the CTG-clade CUG codons are mistranslated to a small extent as leucine due to mischarging of the serine-tRNA(CAG). The mistranslation was suggested to result in variable surface proteins explaining fast host adaptation and pathogenicity. Here, we re-assessed this potential mistranslation by high-resolution mass spectrometry-based proteogenomics of multiple CTG-clade yeasts, various C. albicans strains, isolated from colonized and from infected human body sites, and C. albicans grown in yeast and hyphal forms. Our in vivo data do not support CUG codon mistranslation by leucine. Instead, (i) CUG codons are mistranslated only to the extent of ribosomal mistranslation with no preference for specific amino acids, (ii) CUG codons are as unambiguous (or ambiguous) as the related CUU leucine and UCC serine codons, (iii) tRNA anticodon loop variation across the CTG-clade yeasts does not result in any difference of the mistranslation level, and (iv) CUG codon unambiguity is independent of C. albicans’ strain pathogenicity or growth form.

Enterococci are commensals of the intestinal tract of many animals and humans. Of the various known and still unnamed new enterococcal species, only isolates of Enterococcus faecium and Enterococcus faecalis have received increased medical and public health attention. According to textbook knowledge, the majority of infections are caused by E. faecalis. In recent decades, the number of enterococcal infections has increased, with the increase being exclusively associated with a rising number of nosocomial E. faecium infections. This increase has been accompanied by the dissemination of certain hospital-acquired strain variants and an alarming progress in the development of antibiotic resistance namely vancomycin resistance. With this review we focus on a description of the specific situation of vancomycin resistance among clinical E. faecium isolates in Germany over the past 30 years. The present review describes three VRE episodes in Germany, each of which is framed by the beginning and end of the respective decade. The first episode is specified by the first appearance of VRE in 1990 and a country-wide spread of specific vanA-type VRE strains (ST117/CT24) until the late 1990s. The second decade was initially marked by regional clusters and VRE outbreaks in hospitals in South-Western Germany in 2004 and 2005, mainly caused by vanA-type VRE of ST203. Against the background of a certain "basic level" of VRE prevalence throughout Germany, an early shift from the vanA genotype to the vanB genotype in clinical isolates already occurred at the end of the 2000s without much notice. With the beginning of the third decade in 2010, VRE rates in Germany have permanently increased, first in some federal states and soon after country-wide. Besides an increase in VRE prevalence, this decade was marked by a sharp increase in vanB-type resistance and a dominance of a few, novel strain variants like ST192 and later on ST117 (CT71, CT469) and ST80 (CT1065). The largest VRE outbreak, which involved about 2,900 patients and lasted over three years, was caused by a novel and until that time, unknown strain type of ST80/CT1013 (vanB). Across all periods, VRE outbreaks were mainly oligoclonal and strain types varied over space (hospital wards) and time. The spread of VRE strains obviously respects political borders; for instance, both vancomycin-variable enterococci which were highly prevalent in Denmark and ST796 VRE which successfully disseminated in Australia and Switzerland, were still completely absent among German hospital patients, until to date.

SUMMARY The incidence of azole resistance in Aspergillus species has increased over the past years, most importantly for Aspergillus fumigatus . 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 A. fumigatus 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 cyp51 genes for interpretation of DNA sequencing data, especially in the absence of cultured isolates.

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.

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.

Hintergrund: Systemische Infektionen durch Candidahefen stellen ein erhebliches medizinisches Problem nach gastrointestinal-chirurgischen Eingriffen dar. Das Benchmarking-Pilotprojekt erhebt aktuelle epidemiologische Daten zur Bedeutung der Candidamie in deutschen viszeralchirurgischen Akutkrankenhausern. Material und Methoden: Zur Ermittlung von Benchmarking-Inzidenzen der Candidamie wurden Daten von 47 704 gastrointestinal-chirurgischen Eingriffen, die im Jahr 2010 in deutschen Akutkrankenhausern durchgefuhrt worden waren, ausgewertet. Ergebnisse und Schlussfolgerung: In einem hohen Anteil von 20,3 % der Falle wurden Candida spp. als Ursache kulturell positiver Blutkulturen gefunden. Bezogen auf die Gesamtzahl gastrointestinal-chirurgischer Eingriffe, wurde eine Benchmarking-Inzidenz der Candidamie von 0,15 % (95 %-KI: 0,10–0,21 %) ermittelt. Fur nach dem Eingriff intensivpflichtige Patienten wurde eine Inzidenz von 0,89 % (95 %-KI: 0,57–1,38 %) gefunden, die sich auf 3,13 % (95 %-KI: 2,09–4,66 %) erhohte, wenn bei Patienten eine Blutkulturdiagnostik durchgefuhrt wurde. Krankenhausspezifische Inzidenzzahlen konnen von den Teilnehmern beim Nationalen Referenzzentrum fur Systemische Mykosen Web-basiert abgefragt werden, um Masnahmen der Infektionsvermeidung einzuleiten (www.nrz-mykosen.de/gastrointestinalchirurgie).

Understanding the pathogenesis of an infectious disease is critical for developing new methods to prevent infection and diagnose or cure disease. Adherence of microorganisms to host tissue is a prerequisite for tissue invasion and infection. Fungal cell wall adhesins involved in adherence to host tissue or abiotic medical devices are critical for colonization leading to invasion and damage of host tissue. Here, with a main focus on pathogenic Candida species, we summarize recent progress made in the field of adhesins in human fungal pathogens and underscore the importance of these proteins in establishment of fungal diseases.

Two novel isolates of Candida glabrata exhibiting reduced sensitivity to amphotericin B (MIC, 8 μg ml −1 ) were found to be ERG2 mutants, wherein Δ 8 -sterol intermediates comprised >90% of the total cellular sterol fraction. Both harbored an alteration at Thr 121 in ERG2; the corresponding residue (Thr 119 ) in Saccharomyces cerevisiae is essential for sterol Δ8-Δ7 isomerization. This constitutes the first report of C. glabrata harboring mutations in ERG2 and exhibiting reduced sensitivity to amphotericin B.

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.

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.

MALDI-ToF mass spectrometry offers fast and reliable species identification for bacteria and yeasts under clinical routine conditions. Here, we produced mass spectra for identification of clinically important species of the Pseudallescheria/Scedosporium complex using the recently suggested new nomenclature and use this example to discuss to what extent the principle of DNA barcoding might be transferred to mass spectrometry.

Summary Despite PCR per se being a powerful and sensitive technique, regarding the detection of fungi in patients’ blood, no consensus for a standardised PCR protocol yet exists. To complement other ongoing or accomplished studies which tackle this problem, the German Reference Center for Systemic Mycoses conducted an interlaboratory comparison starting with blood samples spiked with fungal cell elements. Altogether, six laboratories using in-house PCR-protocols from Germany and Austria participated in the trial. Blood samples were spiked with vital cells of Candida albicans or Aspergillus fumigatus. Candida was used in the yeast form, whereas Aspergillus cells were either spiked as conidia or as very young germlings, also known as smoo cells. Spiked blood samples contained between 10 and 10 000 cells ml−1. Depending on the techniques used for fungal cell disruption and DNA-amplification, detection quality was variable between laboratories, but also differed within single laboratories in different trials particularly for samples spiked with less than 100 cells ml−1. Altogether, at least regarding the detection of A. fumigatus, two of six laboratories showed constant reliable test results also with low fungal cell number spiked samples. Protocols used by these labs do not differ substantially from others. However, as particularities, one protocol included a conventional phenol chloroform extraction during the DNA preparation process and the other included a real time PCR-protocol based on FRET probes. Other laboratory comparisons on the basis of clinical samples should follow to further evaluate the procedures. The difficulties and problems of such trials in general are discussed.

Summary Mucormycosis is associated with high morbidity and mortality and is perceived as an emerging fungal infection. However, contemporary paediatric data are limited. We present a series of paediatric cases of mucormycosis reported from Germany and Austria collected within a voluntary epidemiological survey through standardised, anonymized case report forms. Twelve cases were reported between January 2004 and December 2008 (six men; mean age: 12.6 years, range: 0.1–17 years). Mucormycosis was proven in nine, and probable in three cases. Isolates included Lichtheimia (syn. Absidia pro parte, Mycocladus) (five), Rhizopus (three) and Mucor (one) species. Infection was limited to soft tissue in three cases, the lung in two cases, and an infected thrombus in one case; rhinocerebral disease was found in three cases, and pulmonarymediastinal, pulmonary-cerebral and soft tissue-cerebral involvement in one case each. All three patients with isolated soft tissue infection were cured, whereas seven of the remaining patients died (one patient without follow-up). The overall mortality rate was 67%. While these data cannot provide conclusive data on incidence and disease burden of mucormycosis in paediatric patients, they reflect the continuing threat of these infections to immunocompromised patients and the need for improved diagnosis and management.

Sterol analysis identified four Candida albicans erg3 mutants in which ergosta 7,22-dienol, indicative of perturbations in sterol Δ 5,6 -desaturase (Erg3p) activity, comprised >5% of the total sterol fraction. The erg3 mutants (CA12, CA488, CA490, and CA1008) were all resistant to fluconazole, voriconazole, itraconazole, ketoconazole, and clotrimazole under standard CLSI assay conditions (MIC values, ≥256, 16, 16, 8, and 1 μg ml −1 , respectively). Importantly, CA12 and CA1008 retained an azole-resistant phenotype even when assayed in the presence of FK506, a multidrug efflux inhibitor. Conversely, CA488, CA490, and three comparator isolates (CA6, CA14, and CA177, in which ergosterol comprised >80% of the total sterol fraction and ergosta 7,22-dienol was undetectable) all displayed azole-sensitive phenotypes under efflux-inhibited assay conditions. Owing to their ergosterol content, CA6, CA14, and CA177 were highly sensitive to amphotericin B (MIC values, −1 ); CA1008, in which ergosterol comprised −1 ). CA1008 harbored multiple amino acid substitutions in Erg3p but only a single conserved polymorphism (E266D) in sterol 14α-demethylase (Erg11p). CA12 harbored one substitution (W332R) in Erg3p and no residue changes in Erg11p. CA488 and CA490 were found to harbor multiple residue changes in both Erg3p and Erg11p. The results suggest that missense mutations in ERG3 might arise in C. albicans more frequently than currently supposed and that the clinical significance of erg3 mutants, including those in which additional mechanisms also contribute to resistance, should not be discounted.

A clinical isolate of Candida albicans was identified as an erg5 (encoding sterol C22 desaturase) mutant in which ergosterol was not detectable and ergosta 5,7-dienol comprised >80% of the total sterol fraction. The mutant isolate (CA108) was resistant to fluconazole, voriconazole, itraconazole, ketoconazole, and clotrimazole (MIC values, 64, 8, 2, 1, and 2 μg ml −1 , respectively); azole resistance could not be fully explained by the activity of multidrug resistance pumps. When susceptibility tests were performed in the presence of a multidrug efflux inhibitor (tacrolimus; FK506), CA108 remained resistant to azole concentrations higher than suggested clinical breakpoints for C. albicans (efflux-inhibited MIC values, 16 and 4 μg ml −1 for fluconazole and voriconazole, respectively). Gene sequencing revealed that CA108 was an erg11 erg5 double mutant harboring a single amino acid substitution (A114S) in sterol 14α-demethylase (Erg11p) and sequence repetition (10 duplicated amino acids), which nullified C22 desaturase (Erg5p) function. Owing to a lack of ergosterol, CA108 was also resistant to amphotericin B (MIC, 2 μg ml −1 ). This constitutes the first report of a C. albicans erg5 mutant isolated from the clinic.

The glycosylphosphatidylinositol-modified protein Rhd3/Pga29 of the human pathogen Candida albicans belongs to a family of cell wall proteins that are widespread among Candida species but are not found in other fungi. Pga29 is covalently linked to the beta-1,3-glucan framework of the cell wall via beta-1,6-glucan. It is a small and abundant O-glycosylated protein and requires the protein-O-mannosyl transferase Pmt1 for glycosylation. Furthermore, Pga29 is strongly expressed in yeast cells but is downregulated in hyphae. Removal of the PGA29 gene in C. albicans leads to a significant reduction of cell wall mannan; however, Pga29 does not seem to have a major role in maintaining cell wall integrity. In addition, adhesion capacity and hyphae formation appear normal in pga29 deletion mutants. Importantly, the pga29 deletion mutant is less virulent, and infection of reconstituted human epithelium with the pga29 mutant results in a diminished induction of proinflammatory cytokines, such as GM-CSF, TNF, IL-6 and IL-8. We propose that the reduced virulence of the pga29 mutant is a consequence of altered surface properties, resulting in altered fungal recognition.

The prevalence of tetracycline resistance, tetracycline MICs andtet(O) gene localization were investigated in 83Campylobacterisolates from patients suffering from acute gastroenteritis in Germany. Combined biochemical and molecular markers identified 74 isolates (89 %) asCampylobacterjejuni, including seven atypical isolates that failed to hydrolyse hippurate, and nine isolates (11 %) asCampylobactercoli. Tetracycline resistance was detected in six out of nineCampylobacter coliisolates (67 %) and 13 out of 74C. jejuniisolates (18 %). Low-level tetracycline resistance was observed forC. coli(MIC 16 μg ml−1for all strains), whereasC. jejunishowed high-level resistance (MIC >256 μg ml−1for all strains). Both low- and high-level tetracycline resistance was associated with the presence of thetet(O) gene. InC. jejuni,tet(O) was plasmid-encoded in 54 % of tetracycline-resistant isolates, whereas inC. coli,tet(O) appeared to be located on the chromosome.

Cryptococcal meningitis infections cause high mortality rates among HIV-infected patients in Sub-Saharan Africa. The high incidences of cryptococcal infections may be attributed to common environmental sources which, if identified, could lead to institution of appropriate control strategies. To determine the genotypes of Cryptococcus gattii/C. neoformans- species complex from Nairobi, Kenya, 123 clinical and environmental isolates were characterised. Typing was done using orotidine monophosphate pyrophosphorylase (URA5) gene restriction fragment length polymorphism (URA5-RFLP). The majority of the isolates [105/123; 85.4%] were C. neoformans genotype (AFLPI/VNI) and 1.6% AFLP1A/VNB/VNII, whereas (13%) were C. gattii (AFLP4/VGI). This is the first report on the genotypes of C. gattii/C. neoformans species complex from clinical and environmental sources in Nairobi, Kenya and the isolation of C. gattii genotype AFLP4/VGI from the environment in Kenya.

Azole antifungal drug resistance in Aspergillus fumigatus is an emerging problem in several parts of the world. Here we investigated the distribution of such strains in soils from Germany. At a general positivity rate of 12%, most prevalently, we found strains with the TR 34 /L98H and TR 46 /Y121F/T289A alleles, dispersed along a corridor across northern Germany. Comparison of the distributions of resistance alleles and genotypes between environment and clinical samples suggests the presence of local clinical clusters.

Although new molecular diagnostic tools have been introduced, and improved antifungal agents such as new generation triazoles and the echinocandins are now available, the mortality rate of systemic candidosis in high risk patients is still intolerably high. The emerging resistance to antifungal drugs of Candida albicans and the increasing significance of intrinsically resistant non- albicans Candida species in human infections are a major concern. Unfortunately, comprehensive epidemiological data for Germany and Europe are fragmentary. The completion of two genome projects have made the diploid genome sequence of C. albicans (May 2002) and the haploid genome sequence of Candida glabrata (July 2004) available to the scientific community. Postgenomic approaches, including transcriptional and proteomic methodologies for the study of gene functions have shed light on features such as virulence factors, morphogenetic plasticity and signal transduction. Understanding the molecular basis of pathogenic mechanisms is an obvious prerequisite for any significant progress in the diagnosis and treatment of Candida infections and in unravelling the interactive processes between fungal pathogen and host. During the Interdisciplinary Forum on Candidosis (IFOCAN) 2005, held at the Institute of Medical Microbiology of the University of Gottingen, Germany from 23 to 25 September 2005, internationally recognized Candida scientists and renowned clinicians with expertise in medical mycology exchanged their views on the current status, essential future developments, and opportunities in research, diagnosis and therapy of Candida infections. The meeting was a collaborative effort of the German Society for Hygiene and Microbiology (DGHM), section Eukaryotic Pathogens, members of the Priority Programme on Human Pathogenic Fungi (SPP 1160) of the Deutsche Forschungsgemeinschaft (DFG), and the German National Reference Centre for Systemic Mycoses. The German Reference Centre for Systemic Mycoses has established a network of 40 laboratories distributed across Germany to study the epidemiology of invasive Candida infections and the current resistance situation (Margarete …

Objectives. Enterocyte invasion of Campylobacter jejuni 81-176 has been reported to depend upon the virulence plasmid pVir. The objective of this study was to determine the prevalence of pVir in clinical C. jejuni isolates, to investigate DNA homologies between C. jejuni plasmids and the significance of plasmids for C. jejuni invasiveness. Methods. DNA homologies between C. jejuni plasmids were studied by southern blot hybridization. C. jejuni invasion into human intestinal Caco-2 cells was assessed in a gentamicin exclusion assay. Results. Twenty-nine percent of C. jejuni isolated from patients with bloody or watery diarrhoea harboured plasmids of various sizes. One plasmid (7%) was a pVir homologue whereas, the majority of the plasmids (53%) belonged to a subgroup distinct from pVir. The plasmids of this novel subgroup share extensive DNA sequence homology with each other, including homologues to so-called invasion-promoting genes. However, conjugative transfer of these plasmids clearly did not increase invasiveness of plasmidless recipient C. jejuni strains. Conclusion. This study indicates that only a small proportion of C. jejuni strains carry the virulence factor pVir and that at least one other distinctive group of plasmids in C. jejuni exists, which does not seem to be associated with invasiveness.

Candida glabrata is an important cause of systemic candidiasis in humans. This paper reports a systematic analysis of the putative glycosylphosphatidylinositol-modified (GPI) proteins of C. glabrata, a large part of which are covalently bound to the cell wall glucan network and the remainder of which are retained in the plasma membrane, and of cell wall proteins (CWPs) which are covalently bound in a mild-alkali-sensitive manner. In silico genomic analysis revealed 106 putative GPI proteins. Fifty-one of these GPI proteins could be categorized as adhesive proteins, potentially implicated in fungus-host interactions or biofilm formation during the development of fungal infections. Eleven proteins belonged to well-known GPI protein families of glycoside hydrolases, probably involved in cell wall expansion and remodelling during growth. Other identified GPI proteins included phospholipases, aspartic proteases, homologues of ScEcm33p and ScKre1p, and structural CWPs. Interestingly, the GPI algorithm predicted three orthologues of an abundant CWP in S. cerevisiae, Cwp1p, which is absent in Candida albicans. To evaluate the in silico predictions, isolated cell walls were extracted using HF-pyridine, which specifically cleaves phosphodiester bonds, to release GPI-CWPs. Immunological analysis of the extract using one-dimensional SDS-PAGE and anti-ScCwp1p antiserum indicated the presence of a Cwp1p homologue in C. glabrata cell walls. Further analysis by two-dimensional gel electrophoresis and electrospray ionization tandem mass spectrometry (ESI-MS/MS) confirmed the presence of two of the predicted Cwp1p proteins, Cwp1.1p and Cwp1.2p. Crh1p, a putative 1,3-beta-glucan remodelling enzyme, was also identified. In silico genomic analysis further revealed five putative Pir proteins (Pir1-5p) and five members of the Bgl2 glycoside hydrolase family 17, belonging to a class of putative CWPs that can be extracted with NaOH. Immunological analysis of mild-alkali-extracted CWPs showed the presence of a ScPir2p homologue. Together, these experimental data and in silico predictions represent the first systematic analysis of the C. glabrata cell wall proteome.

Invasive aspergillosis (IA) is a serious life-threatening complication in immunocompromised children. The commonest risk groups are children with acquired immunodeficiency syndrome, leukaemia, corticosteroid and other immunosuppressive therapy, chronic granulomatous disease and severe combined immunodeficiency as well as neonates. The clinical manifestations are heterogeneous and many organ systems can be involved. Diagnosis based on the clinical presentation alone is cumbersome. Innovative and sensitive laboratory test systems which detect fungal antigens or DNA in clinical specimens have been recently developed. Specific Aspergillus antibody detection using recombinant antigen technique has also been introduced. Although each individual technique has drawbacks, the combined use of culture with antigen and antibody ELISA as well as PCR should result in an earlier and more definitive diagnosis of IA in children presenting with clinical and/or radiological signs of aspergillosis. In high risk children these methods are valuable for serial screening and early detection of Aspergillus infection. The implementation of accurate diagnostic criteria and standardised diagnostic flow charts in children at risk will lead to a better outcome of IA in the future. Conclusion: definite, well-timed early diagnosis and sufficient therapy is elementary for a successful outcome of invasive aspergillosis in immunocompromised children. To date, the diagnosis of invasive aspergillosis remains a combination of clinical presentation, radiology and microbiological tests.

Aspergillus fumigatus– Virulenz und Opportunismus? The Interdisciplinary Forum on Aspergillosis, organized by the Deutsche Gesellschaft fur Hygiene und Mikrobiologie (DGHM) section, Eucaryotic Pathogens, and the Deutschsprachige Mykologische Gesellschaft (DmykG), was held at the Georg-August-University of Gottingen, Germany, 7–8 April 2000.

In fungi, the cell wall plays a major role in host–pathogen interactions. Despite this, little is known about the molecular basis of cell wall assembly in Candida glabrata, which has emerged as the second most common cause of systemic candidosis. A C. glabrata gene family, CgGAS1–3, that shares significant homologies with both the GAS1 gene of Saccharomyces cerevisiae, which is necessary for cell wall assembly, and the pH-regulated genes PHR1 and PHR2 of Candida albicans, which are involved in cell wall assembly and required for virulence, has been cloned. Among the members of this family, CgGAS1–3 display a unique expression pattern. Both CgGAS1 and CgGAS2 are constitutively expressed. In contrast, CgGAS3 transcript was not detectable under any of the assayed conditions. The C. glabrata actin gene, CgACT1, has also been cloned to be used as a meaningful loading control in Northern blots. CgGAS1 and CgGAS2 were deleted by two different methodological approaches. A rapid PCR-based strategy by which gene disruption was achieved with short regions of homology (50 bp) was applied successfully to C. glabrata. ΔCggas1 or ΔCggas2 cells demonstrated similar aberrant morphologies, displaying an altered bud morphology and forming floccose aggregates. These phenotypes suggest a role for CgGAS1 and CgGAS2 in cell wall biosynthesis. Further evidence for this hypothesis was obtained by successful functional complementation of a gas1 null mutation in S. cerevisiae with the C. glabrata CgGAS1 or CgGAS2 gene.