Discrepancy in MALDI-TOF MS identification of uncommon Gram-negative bacteria from lower respiratory secretions in patients with cystic fibrosis.

Early identification of microbial organisms from respiratory secretions of patients with cystic fibrosis (CF) is important to guide therapeutic decisions. The objective was to compare the accuracy of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) relative to the conventional phenotypic method in identifying common bacterial isolates, including nonfermenting Gram-negative bacteria, in a cohort of patients with CF. A total of 123 isolates from 50 patients with CF representing 14 bacterial species from respiratory specimens were identified using MALDI-TOF MS in parallel with conventional phenotypic methods. Discrepancies were confirmed by 16S ribosomal RNA (rRNA) gene sequencing in five Gram-negative isolates. The MALDI-TOF MS managed to identify 122/123 (99.2%) bacterial isolates to the genus level and 118/123 (95.9%) were identified to the species level. The MALDI-TOF MS results were 100% consistent to the species level with conventional phenotypic identification for isolates of Staphylococcus aureus, Pseudomonas aeruginosa, Haemophilus influenzae, Streptococcus pyogenes, Achromobacter xylosoxidans, Stenotrophomonas maltophilia, and other uncommon organisms such as Chryseobacterium gleum and Enterobacter cloacae. The 5/123 (4.6%) isolates misidentified were all Gram-negative bacteria. The isolation of E. cloacae and Haemophilus paraphrohaemolyticus may extend the potentially pathogenic list of organisms isolated from patients with CF. Although the technique provides an early identification and antimicrobial therapy approach in patients with CF, limitation in the diagnosis of uncommon Gram-negative bacteria may exist

Genetic relatedness and host specificity of Pseudomonas aeruginosa isolates from cystic fibrosis and non-cystic fibrosis patients

Background: Pseudomonas aeruginosa is one of the primary pathogens isolated more frequently in cystic fibrosis (CF) and it exhibits innate resistance to a wide range of antibiotics. Purpose: We sought to determine whether the highly prevalent genotypes of P. aeruginosa are specifically linked to CF patients and have any related multidrug antibiotic resistance. Isolates from hospitalized non-CF patients and from environmental sources were also genotypically analyzed. Methods: Collections of P. aeruginosa from lower respiratory secretions (n=45) were genotyped using pulsed-field gel electrophoresis (PFGE). Phenotypic screening for antibiotic susceptibility was performed for the common antimicrobial agents by E-test and automated Phoenix method. Results: P. aeruginosa isolates from CF (n=32), hospitalized non-CF patients (n=13), and environment sources (n=5) were analyzed. The population structure of P. aeruginosa is highly diverse and population-specific. All PFGE results of P. aeruginosa isolates fall among four major clusters. Cluster 1 contained 16 P. aeruginosa isolates from CF patients and two from environmental sources; cluster 2 contained 11 P. aeruginosa isolates from CF and one each from non-CF and environmental sources; cluster 3 contained 12 P. aeruginosa isolates from hospitalized non-CF patients and two P. aeruginosa isolates from one CF patient and one environmental source; and cluster 4 consisted of three isolates from CF patients and one from the environment. The majority of multidrug-resistant P. aeruginosa isolates were in clusters 3 and 4. P. aeruginosa isolates from CF patients were resistant to ciprofloxacin (34.4%) followed by resistance to amikacin and gentamicin (each 28%), whereas the majority of isolates from non-CF patients were resistant to meropenem (69%) and were grouped in cluster 3. Conclusion: PFGE of P. aeruginosa isolates from CF patients shows a high degree of similarity, suggesting specific adaptation of these clones to CF-affected lungs. The hospitalized non-CF cluster has a different clonal origin, indicating specific clustering in a specific location, suggesting hospital-acquired P. aeruginosa infections.The Qatar National Research Fund (UREP 14-026-3-010)

Comparative genomic analysis of clinical Candida glabrata isolates identifies multiple polymorphic loci that can improve existing multilocus sequence typing strategy

Candida glabrata is the second leading cause of candidemia in many countries and is one of the most concerning yeast species of nosocomial importance due to its increasing rate of antifungal drug resistance and emerging multidrug-resistant isolates. Application of multilocus sequence typing (MLST) to clinical C. glabrata isolates revealed an association of certain sequence types (STs) with drug resistance and mortality. The current C. glabrata MLST scheme is based on single nucleotide polymorphisms (SNPs) at six loci and is therefore relatively laborious and costly. Furthermore, only a few high-quality C. glabrata reference genomes are available, limiting rapid analysis of clinical isolates by whole genome sequencing. In this study we provide long-read based assemblies for seven additional clinical strains belonging to three different STs and use this information to simplify the C. glabrata MLST scheme. Specifically, a comparison of these genomes identified highly polymorphic loci (HPL) defined by frequent insertions and deletions (indels), two of which proved to be highly resolutive for ST. When challenged with 53 additional isolates, a combination of TRP1 (a component of the current MLST scheme) with either of the two HPL fully recapitulated ST identification. Therefore, our comparative genomic analysis identified a new typing approach combining SNPs and indels and based on only two loci, thus significantly simplifying ST identification in C. glabrata. Because typing tools are instrumental in addressing numerous clinical and biological questions, our new MLST scheme can be used for high throughput typing of C. glabrata in clinical and research settings.We thank Dibyendu Kumar (Rutgers University) for help with C. glabrata PacBio sequencing. This work was supported by NIH 5R01AI109025 to D.S.P. TG group acknowledges support from the Spanish Ministry of Science and Innovation for grant PGC2018-099921-B-I00, cofounded by European Regional Development Fund (ERDF); from the Catalan Research Agency (AGAUR) SGR423; from the European Union's Horizon 2020 research and innovation programme (ERC-2016-724173); from the Gordon and Betty Moore Foundation (Grant GBMF9742) and from the Instituto de Salud Carlos III (INB Grant PT17/0009/0023 – ISCIII-SGEFI/ERDF).Peer ReviewedPostprint (published version

Genomic Understanding of an Infectious Brain Disease from the Desert

Rhinocladiella mackenziei accounts for the majority of fungal brain infections in the Middle East, and is restricted to the arid climate zone between Saudi Arabia and Pakistan. Neurotropic dissemination caused by this fungus has been reported in immunocompromised, but also immunocompetent individuals. If untreated, the infection is fatal. Outside of humans, the environmental niche of R. mackenziei is unknown, and the fungus has been only cultured from brain biopsies. In this paper, we describe the whole-genome resequencing of two R. mackenziei strains from patients in Saudi Arabia and Qatar. We assessed intraspecies variation and genetic signatures to uncover the genomic basis of the pathogenesis, and potential niche adaptations. We found that the duplicated genes (paralogs) are more susceptible to accumulating significant mutations. Comparative genomics with other filamentous ascomycetes revealed a diverse arsenal of genes likely engaged in pathogenicity, such as the degradation of aromatic compounds and iron acquisition. In addition, intracellular accumulation of trehalose and choline suggests possible adaptations to the conditions of an arid climate region. Specifically, protein family contractions were found, including short-chain dehydrogenase/reductase SDR, the cytochrome P450 (CYP) (E-class), and the G-protein b WD-40 repeat. Gene composition and metabolic potential indicate extremotolerance and hydrocarbon assimilation, suggesting a possible environmental habitat of oil-polluted desert soilinfo:eu-repo/semantics/publishedVersio

Azole-Resistance in Aspergillus terreus and Related Species: An Emerging Problem or a Rare Phenomenon?

Raquel Sabino was not included as an author in the published article. It was corrected a posteriori.Erratum in - Corrigendum: Azole-Resistance in Aspergillus terreus and Related Species: An Emerging Problem or a Rare Phenomenon? [Front Microbiol. 2018] Front Microbiol. 2019 Jan 14;9:3245. doi: 10.3389/fmicb.2018.03245. eCollection 2018.Disponível em: https://www.frontiersin.org/articles/10.3389/fmicb.2018.03245/fullFree PMC Article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5882871/ | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6340063/Objectives: Invasive mold infections associated with Aspergillus species are a significant cause of mortality in immunocompromised patients. The most frequently occurring aetiological pathogens are members of the Aspergillus section Fumigati followed by members of the section Terrei. The frequency of Aspergillus terreus and related (cryptic) species in clinical specimens, as well as the percentage of azole-resistant strains remains to be studied. Methods: A global set (n = 498) of A. terreus and phenotypically related isolates was molecularly identified (beta-tubulin), tested for antifungal susceptibility against posaconazole, voriconazole, and itraconazole, and resistant phenotypes were correlated with point mutations in the cyp51A gene. Results: The majority of isolates was identified as A. terreus (86.8%), followed by A. citrinoterreus (8.4%), A. hortai (2.6%), A. alabamensis (1.6%), A. neoafricanus (0.2%), and A. floccosus (0.2%). One isolate failed to match a known Aspergillus sp., but was found most closely related to A. alabamensis. According to EUCAST clinical breakpoints azole resistance was detected in 5.4% of all tested isolates, 6.2% of A. terreus sensu stricto (s.s.) were posaconazole-resistant. Posaconazole resistance differed geographically and ranged from 0% in the Czech Republic, Greece, and Turkey to 13.7% in Germany. In contrast, azole resistance among cryptic species was rare 2 out of 66 isolates and was observed only in one A. citrinoterreus and one A. alabamensis isolate. The most affected amino acid position of the Cyp51A gene correlating with the posaconazole resistant phenotype was M217, which was found in the variation M217T and M217V. Conclusions:Aspergillus terreus was most prevalent, followed by A. citrinoterreus. Posaconazole was the most potent drug against A. terreus, but 5.4% of A. terreus sensu stricto showed resistance against this azole. In Austria, Germany, and the United Kingdom posaconazole-resistance in all A. terreus isolates was higher than 10%, resistance against voriconazole was rare and absent for itraconazole.This work was supported by ECMM, ISHAM, and EFISG and in part by an unrestricted research grant through the Investigator Initiated Studies Programof Astellas, MSD, and Pfizer. This study was fundet by the Christian Doppler Laboratory for invasive fungal infections.info:eu-repo/semantics/publishedVersio

Resistance of Asian Cryptococcus neoformans Serotype A Is Confined to Few Microsatellite Genotypes

Contains fulltext : 109375.pdf (publisher's version ) (Open Access)BACKGROUND: Cryptococcus neoformans is a pathogenic yeast that causes cryptococcosis, a life threatening disease. The prevalence of cryptococcosis in Asia has been rising after the onset of the AIDS epidemic and estimates indicate more than 120 cases per 1,000 HIV-infected individuals per year. Almost all cryptococcal disease cases in both immunocompromised and immunocompetent patients in Asia are caused by C. neoformans var. grubii. Epidemiological studies on C. neoformans in pan-Asia have not been reported. The present work studies the genetic diversity of the fungus by microsatellite typing and susceptibility analysis of approximately 500 isolates from seven Asian countries. METHODOLOGY/PRINCIPAL FINDINGS: Genetic diversity of Asian isolates of C. neoformans was determined using microsatellite analysis with nine microsatellite markers. The analysis revealed eight microsatellite complexes (MCs) which showed different distributions among geographically defined populations. A correlation between MCs and HIV-status was observed. Microsatellite complex 2 was mainly associated with isolates from HIV-negative patients, whereas MC8 was associated with those from HIV-positive patients. Most isolates were susceptible to amphotericin B, itraconazole, voriconazole, posaconazole, and isavuconazole, but 17 (3.4%) and 10 (2%) were found to be resistant to 5-flucytosine and fluconazole, respectively. Importantly, five Indonesian isolates (approximately 12.5% from all Indonesian isolates investigated and 1% from the total studied isolates) were resistant to both antifungals. The majority of 5-flucytosine resistant isolates belonged to MC17. CONCLUSIONS: The findings showed a different distribution of genotypes of C. neoformans var. grubii isolates from various countries in Asia, as well as a correlation of the microsatellite genotypes with the original source of the strains and resistance to 5-flucytosine

Notes for genera: basal clades of Fungi (including Aphelidiomycota, Basidiobolomycota, Blastocladiomycota, Calcarisporiellomycota, Caulochytriomycota, Chytridiomycota, Entomophthoromycota, Glomeromycota, Kickxellomycota, Monoblepharomycota, Mortierellomycota, Mucoromycota, Neocallimastigomycota, Olpidiomycota, Rozellomycota and Zoopagomycota)

Compared to the higher fungi (Dikarya), taxonomic and evolutionary studies on the basal clades of fungi are fewer in number. Thus, the generic boundaries and higher ranks in the basal clades of fungi are poorly known. Recent DNA based taxonomic studies have provided reliable and accurate information. It is therefore necessary to compile all available information since basal clades genera lack updated checklists or outlines. Recently, Tedersoo et al. (MycoKeys 13:1--20, 2016) accepted Aphelidiomycota and Rozellomycota in Fungal clade. Thus, we regard both these phyla as members in Kingdom Fungi. We accept 16 phyla in basal clades viz. Aphelidiomycota, Basidiobolomycota, Blastocladiomycota, Calcarisporiellomycota, Caulochytriomycota, Chytridiomycota, Entomophthoromycota, Glomeromycota, Kickxellomycota, Monoblepharomycota, Mortierellomycota, Mucoromycota, Neocallimastigomycota, Olpidiomycota, Rozellomycota and Zoopagomycota. Thus, 611 genera in 153 families, 43 orders and 18 classes are provided with details of classification, synonyms, life modes, distribution, recent literature and genomic data. Moreover, Catenariaceae Couch is proposed to be conserved, Cladochytriales Mozl.-Standr. is emended and the family Nephridiophagaceae is introduced

Reduced Multidrug Susceptibility Profile Is a Common Feature of Opportunistic Fusarium Species: Fusarium Multi-Drug Resistant Pattern

The resistance among various opportunistic Fusarium species to different antifungal agents has emerged as a cause of public health problems worldwide. Considering the significance of multi-drug resistant (MDR), this paper emphasizes the problems associated with MDR and the need to understand its clinical significance to combat microbial infections. The search platform PubMed/MEDLINE and a review of 32 cases revealed a common multidrug-resistant profile exists, and clinically relevant members of Fusarium are intrinsically resistant to most currently used antifungals. Dissemination occurs in patients with prolonged neutropenia, immune deficiency, and especially hematological malignancies. Amphotericin B displayed the lowest minimum inhibitory concentrarions (MICs) followed by voriconazole, and posaconazole. Itraconazole and fluconazole showed high MIC values, displaying in vitro resistance. Echinocandins showed the highest MIC values. Seven out of ten (70%) patients with neutropenia died, including those with fungemia that progressed to skin lesions. Clinical Fusarium isolates displayed a common MDR profile and high MIC values for the most available antifungal agents with species- and strain-specific differences in antifungal susceptibility. Species identification of Fusarium infections is important. While the use of natamycin resulted in a favorable outcome in keratitis, AmB and VRC are the most used agents for the treatment of fusariosis in clinical settings

Serum Cytokine Profile in Patients with Candidemia versus Bacteremia

Bloodstream Candida infections constitute a major threat for hospitalized patients in intensive care units and immunocompromised hosts. Certain serum cytokines play a decisive role in anti-microbial host defense. Cytokines may act as discriminatory biomarkers that can significantly increase in candidemia compared to bacteremia patients. The concentration of secreted cytokine/chemokines was determined using a multiplexed cytometric bead array run on a cell analyzer. The cytokines tested during the study were interleukin (IL)-1β, IL-6, IL-17A, IL-10, IFN-γ, IL-4, IL-2, IL-8, IL-12p70 and the tumor necrosis factor (TNF)-α. The cytokines of 51 candidemia patients were characterized and compared to the cytokine levels of 20 bacteremia patients. Levels were significantly elevated in patients with bloodstream infections compared to healthy controls. Cytokines comprising IL-2, IL-17A, IL-6 and IL-10 were significantly elevated in the patients with bloodstream Candida infection as compared to the patients having bloodstream bacterial infections. The levels were found to be promising as a potential diagnostic marker for bloodstream Candida infections