Application of mass spectrometry in mycology Quiles Melero, Inmaculada; Peláez, Teresa; Rezusta López, Antonio ...
Enfermedades infecciosas y microbiologia clinica
34 Suppl 2
Journal Article
Peer reviewed
MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometry (MS) is becoming an essential tool in most microbiology laboratories. At present, by using a characteristic ...fungal profile obtained from whole cells or through simple extraction protocols, MALDI-TOF MS allows the identification of pathogenic fungi with a high performance potential. This methodology decreases the laboratory turnaround time, optimizing the detection of mycoses. This article describes the state-of-the-art of the use of MALDI-TOF MS for the detection of human clinical fungal pathogens in the laboratory and discusses the future applications of this technology, which will further improve routine mycological diagnosis.
The genus
Aspergillus
consists of a vast number of medically and environmentally relevant species.
Aspergillus
species classified in series
Versicolores
are ubiquitous in the environment and include ...the opportunistic pathogen
Aspergillus sydowii
, which is associated with onychomycosis and superficial skin infections. Despite frequent clinical reports of
A. sydowii
and related series
Versicolores
species, antifungal susceptibility data are scarce, hampering optimal treatment choices and subsequent patient outcomes. Here, we employed antifungal susceptibility testing (AFST) based on microbroth dilution on a set of 155 series
Versicolores
strains using the common antifungals amphotericin B, itraconazole, voriconazole, posaconazole, isavuconazole and micafungin with the addition of luliconazole and olorofim. All strains were identified using partial calmodulin gene sequencing, with 145 being
A. sydowii,
seven
A. creber
and three
A. versicolor
, using the latest taxonomic insights. Overall, tested antifungals were potent against the entire strain collection. In comparison to
A. fumigatus
, azole and amphotericin B MICs were slightly elevated for some strains. AFST with luliconazole and olorofim, here reported for the first time, displayed the highest in vitro activity, making these antifungals interesting alternative drugs but clinical studies are warranted for future therapeutic use.
We aimed to assess the percentage of azole resistance in Aspergillus fumigatus in Spain.
Thirty participating Spanish hospitals stored all morphologically identified A. fumigatus sensu lato clinical ...isolates—regardless their clinical significance—from 15 February to 14 May 2019. Isolates showing azole resistance according to the EUCAST 9.3.2 methodology were molecularly identified and the cyp51A gene was studied in A. fumigatus sensu stricto isolates.
Eight hundred and forty-seven isolates from 725 patients were collected in 29 hospitals (A. fumigatus sensu stricto (n = 828) and cryptic species (n = 19)). Isolates were mostly from the lower respiratory tract (94.0%; 797/847). Only cryptic species were amphotericin B resistant. Sixty-three (7.4%) out of the 847 isolates were resistant to ≥1 azole(s). Azole resistance was higher in cryptic species than in A. fumigatus sensu stricto (95%, 18/19 vs. 5.5%, 45/828); isavuconazole was associated to the lowest number of non-wild type isolates. The dominant mechanism of resistance was the presence of TR34-L98H substitutions (n = 24 out of 63). Out of the 725 patients, 48 (6.6%) carried either cryptic species (n = 14) or A. fumigatus sensu stricto (n = 34; 4.7%) resistant isolates. Aspergillus fumigatus sensu stricto harbouring either the TR34-L98H (n = 19) or TR46/Y121F/T289A (n = 1) mutations were detected in patients in hospitals located at 7/24 studied cities.
Of the patients, 6.6% carry azole-resistant A. fumigatus sensu lato isolates in Spain. TR34-L98H is the dominant cyp51A gene substitutions, although its presence is not widespread.
This report describes a 49-year-old male construction worker who acquired a Bacillus anthracis infection after working on a sheep farm. He experienced a severe respiratory infection, septic shock, ...and hemorrhagic meningoencephalitis with severe intracranial hypertension. After several weeks with multiple organ dysfunction syndrome, he responded favorably to antibiotic treatment. Three weeks into his hospitalization, an intracranial hemorrhage and cerebral edema led to an abrupt deterioration in his neurological status. A single dose of raxibacumab was added to his antimicrobial regimen on hospital day 27. His overall status, both clinical and radiographic, improved within a few days. He was discharged 2 months after admission and appears to have fully recovered.
An interlaboratory study using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to determine the identification of clinically important yeasts (n = 35) was ...performed at 11 clinical centers, one company, and one reference center using the Bruker Daltonics MALDI Biotyper system. The optimal cutoff for the MALDI-TOF MS score was investigated using receiver operating characteristic (ROC) curve analyses. The percentages of correct identifications were compared for different sample preparation methods and different databases. Logistic regression analysis was performed to analyze the association between the number of spectra in the database and the percentage of strains that were correctly identified. A total of 5,460 MALDI-TOF MS results were obtained. Using all results, the area under the ROC curve was 0.95 (95% confidence interval CI, 0.94 to 0.96). With a sensitivity of 0.84 and a specificity of 0.97, a cutoff value of 1.7 was considered optimal. The overall percentage of correct identifications (formic acid-ethanol extraction method, score ≥ 1.7) was 61.5% when the commercial Bruker Daltonics database (BDAL) was used, and it increased to 86.8% by using an extended BDAL supplemented with a Centraalbureau voor Schimmelcultures (CBS)-KNAW Fungal Biodiversity Centre in-house database (BDAL+CBS in-house). A greater number of main spectra (MSP) in the database was associated with a higher percentage of correct identifications (odds ratio OR, 1.10; 95% CI, 1.05 to 1.15; P < 0.01). The results from the direct transfer method ranged from 0% to 82.9% correct identifications, with the results of the top four centers ranging from 71.4% to 82.9% correct identifications. This study supports the use of a cutoff value of 1.7 for the identification of yeasts using MALDI-TOF MS. The inclusion of enough isolates of the same species in the database can enhance the proportion of correctly identified strains. Further optimization of the preparation methods, especially of the direct transfer method, may contribute to improved diagnosis of yeast-related infections.
•MB-aPDT causes photoinactivation of S. aureus and P. aeruginosa in planktonic cultures and biofilms.•GN enhances MB-aPDT-induced bacterial photinactivation of planktonic cultures.•GN enhances ...MB-aPDT-induced bacterial photinactivation of P. aeruginosa biofilms.•The combination of GN + MB-aPDT exerts no synergistic antibacterial effects in S. aureus biofilms.•GN combined with MB-aPDT caused no adverse effects in our experimental setting.
Antimicrobial photodynamic therapy (a-PDT), combined or not with antibiotics, constitutes a promising therapy for superficial infections caused by bacteria implicated in multidrug resistance processes.
We compared the efficacy of aPDT using the photosensitizer methylene blue (MB), combined or not with the antibiotic gentamicin (GN), against Staphylococcus aureus and Pseudomonas aeruginosa.
Different concentrations of MB (0.03–7000 μg/mL), with or without GN (1–20 μg/mL), were added to planktonic cultures or biofilms and the samples irradiated with a LED lamp (λ 625 nm, 7 mW/cm2, 18 J/cm2). The number of viable bacteria in the samples and in corresponding nonirradiated controls was quantified by counting colony-forming units to evaluate the individual effects of MB, GN, and irradiation.
MB-aPDT resulted in significant bacterial photoinactivation. The combination of GN and MB-aPDT exerted a synergistic bactericidal effect against planktonic cultures of S. aureus and P. aeruginosa. This combination did not significantly alter the photoinactivating effect of MB against S. aureus biofilms, but exerted a positive bactericidal effect against P. aeruginosa biofilms.
These results underscore the need for further clinical studies of this therapeutic combination for the management of difficult-to-treat skin and mucous infections, especially those caused by P. aeruginosa.
Abstract
Objectives
To determine the prevalence of penicillin susceptibility among MSSA causing bloodstream infections (BSIs) in 16 Spanish hospitals and to characterize the penicillin-susceptible ...MSSA (MSSA-PENS) isolates.
Methods
A total of 1011 Staphylococcus aureus isolates were collected from blood cultures in 16 Spanish hospitals during 2018–19 (6–12 months) and their susceptibility to 18 antimicrobials was determined. The MSSA-PENS isolates were selected and examined by PCR to determine the presence of the blaZ gene, other resistance genes and the genes lukF/lukS-PV, eta, etb and tst. The immune evasion cluster (IEC) type was also analysed. All the MSSA-PENS isolates were submitted to S. aureus protein A (spa) typing and the clonal complexes (CCs) were assigned according to their spa type.
Results
The prevalence of MSSA was 74.6% (754/1011) and 14.9% (151/1011) were MSSA-PENS-blaZnegative. MSSA-PENS-blaZnegative isolates (n = 151) were ascribed to 88 spa types and 11 CCs. The most frequent CCs were CC5 (35/151) and CC398 (25/151), with t002-CC5 and t571-CC398 being the most common lineages. Pan-susceptibility was identified in 117 of the 151 MSSA-PENS-blaZnegative isolates (77.5%). In the remaining isolates, erythromycin and clindamycin resistance was the most frequent resistance found, although tobramycin, ciprofloxacin, fusidic acid, mupirocin and/or tetracycline resistance was also detected. Thirty-eight MSSA-PENS-blaZnegative isolates were IEC negative and four isolates were Panton–Valentine leucocidin (‘PVL’) positive.
Conclusions
A high penicillin susceptibility rate was detected among MSSA, opening therapeutic opportunities for BSIs. The emergence of new successful MSSA-PENS clones could be responsible for these data. The detection among MSSA-PENS-blaZnegative isolates of the clonal lineage CC398 or the absence of an IEC raises questions about their possible animal origin, requiring further analysis.
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