Due to excellent separation capacity for complex mixtures of chemicals, comprehensive two-dimensional gas chromatography (GC × GC) is being utilized with increasing frequency for metabolomics ...analyses. This review describes recent advances in GC × GC method development for metabolomics, organismal sampling techniques compatible with GC × GC, metabolomic discoveries made using GC × GC, and recommendations and best practices for collecting and reporting GC × GC metabolomics data.
•The excellent separation capacity of GC × GC is well suited for metabolomics.•Methods are being developed to optimize metabolome sampling and analysis.•GC × GC metabolome analyses are moving toward identifying mechanisms.•Data reporting recommendations are provided to optimize data sharing via new metabolomics databases.•Best practices for GC × GC metabolomics analyses are described.
The study of chemical bioactivity in the rhizosphere has recently broadened to include microbial metabolites, and their roles in niche construction and competition via growth promotion, growth ...inhibition, and toxicity. Several prior studies have identified bacteria that produce volatile organic compounds (VOCs) with antifungal activities, indicating their potential use as biocontrol organisms to suppress phytopathogenic fungi and reduce agricultural losses. We sought to expand the roster of soil bacteria with known antifungal VOCs by testing bacterial isolates from wild and cultivated cranberry bog soils for VOCs that inhibit the growth of four common fungal and oomycete plant pathogens, and
Trichoderma
sp. Twenty one of the screened isolates inhibited the growth of at least one fungus by the production of VOCs, and isolates of
Chromobacterium vaccinii
had broad antifungal VOC activity, with growth inhibition over 90% for some fungi. Fungi exposed to
C. vaccinii
VOCs had extensive morphological abnormalities such as swollen hyphal cells, vacuolar depositions, and cell wall alterations. Quorum-insensitive
cviR
−
mutants of
C. vaccinii
were significantly less fungistatic, indicating a role for quorum regulation in the production of antifungal VOCs. We collected and characterized VOCs from co-cultivation assays of
Phoma
sp. exposed to wild-type
C. vaccinii
MWU328, and its
cviR
−
mutant using stir bar sorptive extraction and comprehensive two-dimensional gas chromatography—time-of-flight mass spectrometry (SBSE-GC × GC-TOFMS). We detected 53 VOCs that differ significantly in abundance between microbial cultures and media controls, including four candidate quorum-regulated fungistatic VOCs produced by
C. vaccinii
. Importantly, the metabolomes of the bacterial-fungal co-cultures were not the sum of the monoculture VOCs, an emergent property of their VOC-mediated interactions. These data suggest semiochemical feedback loops between microbes that have co-evolved for sensing and responding to exogenous VOCs.
Valley fever (coccidioidomycosis) is an endemic fungal pneumonia of the North and South American deserts. The causative agents of Valley fever are the dimorphic fungi
and
, which grow as mycelia in ...the environment and as spherules within the lungs of vulnerable hosts. Current diagnostics for Valley fever are severely lacking due to poor sensitivity and invasiveness, contributing to a 23-day median time to diagnosis, and therefore, new diagnostic tools are needed. We are working toward the development of a breath-based diagnostic for coccidioidomycosis, and in this initial study, we characterized the volatile metabolomes (or volatilomes) of
cultures of
Using solid-phase microextraction (SPME) and comprehensive two-dimensional gas chromatography coupled to time of flight mass spectrometry (GC×GC-TOFMS), we characterized the volatile organic compounds (VOCs) produced by six strains of each species during mycelial or spherule growth. We detected a total of 353 VOCs that were at least 2-fold more abundant in a
culture than in medium controls and found that the volatile metabolome of
is more dependent on the growth phase (spherules versus mycelia) than on the species. The volatile profiles of
and
have strong similarities, indicating that a single suite of Valley fever breath biomarkers can be developed to detect both species.
Coccidioidomycosis, or Valley fever, causes up to 30% of community-acquired pneumonias in highly populated areas of the U.S. desert southwest where the disease is endemic. The infection is difficult to diagnose by standard serological and histopathological methods, which delays appropriate treatment. Therefore, we are working toward the development of breath-based diagnostics for Valley fever. In this study, we characterized the volatile metabolomes (or volatilomes) of six strains each of
and
, the dimorphic fungal species that cause Valley fever. By analyzing the volatilomes during the two modes of growth of the fungus-mycelia and spherules-we observed that the life cycle plays a significant role in the volatiles produced by
In contrast, we observed no significant differences in the
versus
volatilomes. These data suggest that life cycle, rather than species, should guide the selection of putative biomarkers for a Valley fever breath test.
Missing data is a significant issue in metabolomics that is often neglected when conducting data preprocessing, particularly when it comes to imputation. This can have serious implications for ...downstream statistical analyses and lead to misleading or uninterpretable inferences. In this study, we aim to identify the primary types of missingness that affect untargeted metabolomics data and compare strategies for imputation using two real-world comprehensive two-dimensional gas chromatography (GC × GC) data sets. We also present these goals in the context of experimental replication whereby imputation is conducted in a within-replicate-based fashionthe first description and evaluation of this strategyand introduce an R package MetabImpute to carry out these analyses. Our results conclude that, in these two GC × GC data sets, missingness was most likely of the missing at-random (MAR) and missing not-at-random (MNAR) types as opposed to missing completely at-random (MCAR). Gibbs sampler imputation and Random Forest gave the best results when imputing MAR and MNAR compared against single-value imputation (zero, minimum, mean, median, and half-minimum) and other more sophisticated approaches (Bayesian principal component analysis and quantile regression imputation for left-censored data). When samples are replicated, within-replicate imputation approaches led to an increase in the reproducibility of peak quantification compared to imputation that ignores replication, suggesting that imputing with respect to replication may preserve potentially important features in downstream analyses for biomarker discovery.
New Breath Diagnostics for Fungal Disease Diefenderfer, Jenna; Bean, Heather D.; Higgins Keppler, Emily A.
Current clinical microbiology reports,
06/2024, Letnik:
11, Številka:
2
Journal Article
Recenzirano
Odprti dostop
Purpose of Review
Diagnosis of fungal disease etiology is often difficult, compounded by inaccurate or delayed diagnostic methods. Breath-based biomarkers are being investigated as a novel target for ...clinical diagnostics. This review aims to summarize recent advancements, identify gaps, and discuss future research directions for breath-based fungal diagnostics.
Recent Findings
Studies conducted in vitro, in animal models, and in human breath show fungi produce a large and diverse volatile metabolome. Recent studies on
Aspergillus
,
Candida
,
Rhizopus
,
Coccidioides
,
Trichoderma
,
Fusarium
, and
Alternaria
demonstrate the feasibility of identifying infectious etiology using fungal volatile profiles. However, the majority of data on fungal volatiles come from in vitro analyses, which have limited translatability to in vivo infections; thus, future studies should focus on in vivo volatile profiles to develop breath tests for diagnosing infections and monitoring antifungal therapy.
Summary
This review describes recent studies that examine volatile organic compounds (VOCs) as biomarkers to detect and differentiate pathogenic fungi, highlighting the feasibility of breath-based diagnostics for fungal disease.
Coccidioides immitis and Coccidioides posadasii are soil-dwelling fungi of arid regions in North and South America that are responsible for Valley fever (coccidioidomycosis). Forty percent of ...patients with Valley fever exhibit symptoms ranging from mild, self-limiting respiratory infections to severe, life-threatening pneumonia that requires treatment. Misdiagnosis as bacterial pneumonia commonly occurs in symptomatic Valley fever cases, resulting in inappropriate treatment with antibiotics, increased medical costs, and delay in diagnosis. In this proof-of-concept study, we explored the feasibility of developing breath-based diagnostics for Valley fever using a murine lung infection model. To investigate potential volatile biomarkers of Valley fever that arise from host−pathogen interactions, we infected C57BL/6J mice with C. immitis RS (n = 6), C. posadasii Silveira (n = 6), or phosphate-buffered saline (n = 4) via intranasal inoculation. We measured fungal dissemination and collected bronchoalveolar lavage fluid (BALF) for cytokine profiling and for untargeted volatile metabolomics via solid-phase microextraction (SPME) and two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC×GC-TOFMS). We identified 36 volatile organic compounds (VOCs) that were significantly correlated (p < 0.05) with cytokine abundance. These 36 VOCs clustered mice by their cytokine production and were also able to separate mice with moderate-to-high cytokine production by infection strain. The data presented here show that Coccidioides and/or the host produce volatile metabolites that may yield biomarkers for a Valley fever breath test that can detect coccidioidal infection and provide clinically relevant information on primary pulmonary disease severity.
Abstract only
Chromobacterium
spp. have been found to grow in a variety of environments and have an extensive metabolic flexibility. Due to this flexibility, studies have shown that
Chromobacterium
...spp. produce an array of properties that can positively impact medicine, industry, and agriculture.
C. vaccinii
, isolated from cranberry bog soil, has been found to inhibit the growth of multiple pathogenic plant fungi through the production of volatile compounds. Fungal inhibition has been observed when the fungi is co‐cultured with
C. vaccinii
in separate petri dishes but with shared gas exchange. The same fungal inhibition is not observed when the fungus is exposed to
C. vaccinii
metabolites produced in liquid media. Based on this observation, we hypothesize that the volatiles produced in liquid media are not the same as the volatiles produced in agar. Experiments are being conducted to study the influence of growth conditions on
C. vaccinii
volatile metabolites.
C. vaccinii
will be cultured in liquid and semi‐solid media, the volatiles will be collected using thin film solid phase microextraction (TF‐SPME) and analyzed with two‐dimensional gas chromatography – time‐of‐flight mass spectroscopy (GC×GC‐TOFMS). The goal is to determine the differences between the volatiles produced in the different types of media. These results will lead to a better understanding of this natural occurring soil bacteria.
Support or Funding Information
NSF LEAP Scholars Program
Abstract
Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM
Objectives
Valley fever (coccidioidomycosis) is an endemic pneumonia of the North and South American deserts, and is responsible ...for up to 30% of community-acquired pneumonia in endemic and highly populated areas of the US desert southwest. The causative agents of Valley fever are the dimorphic fungi Coccidioides immitis and C. posadasii, which grow as mycelia in the environment and spherules within the lungs of vulnerable hosts. The current diagnostics for Valley fever are severely lacking due to poor sensitivity and invasiveness, contributing to a 23-day median time-to-diagnosis. There is a critical need for novel diagnostics for detecting and identifying Valley fever lung infections. Our long-term goal is to develop a breath-based diagnostic for coccidioidomycosis lung infections. Our current objective is to identify and validate volatile biomarkers of C. immitis and C. posadasii infections via metabolomics analyses of in vitro cultures, mouse model lung infections, and lung specimens from humans with Valley fever. Thus far we have characterized the volatile organic compounds (VOCs) produced by C. immitis and C. posadasii in vitro and evaluated the relationship of the volatile metabolomes to lifecycle, and we have investigated the VOC profiles of bronchoalveolar lavage fluid (BALF) samples from mouse model lung infections of Valley fever.
Methods
For in vitro analyses, six strains each of C. immitis and C. posadasii were cultured in triplicate to induce mycelial or spherule formation. For mouse model infections, female C57BL/6 mice were infected by intranasal inoculation with C. immitis RS (n = 6), C. posadasii Silveira (n = 6), or vehicle control (n = 4), and BALF fluid was collected 10 days post-infection. The in vitro spent media and BALF sample VOCs were analyzed by headspace solid-phase microextraction and comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (SPME-GC × GC-TOFMS). The BALF samples were analyzed for cytokines using a mouse magnetic 26-Plex ProcartaPlexTM panel and the mouse spleen and brain were quantified for fungal dissemination. Data analysis: Hierarchical clustering analysis (HCA), principal component analysis (PCA), and Kendall correlation were performed on volatile and cytokine data.
Results
We detected a total of 353 VOCs that were at least two-fold more abundant in Coccidioides cultures versus medium controls and found the volatile metabolome of Coccidioides is more dependent on lifecycle (mycelia vs. spherule) than species (Fig. 1). The BALF samples indicate that lung infection VOCs are correlated to cytokine production (Fig. 2) and classify mice based on their individual level of infection. We did not observe any separation between the C. immitis and C. posadasii infected mice by their BALF VOCs via PCA; however, separation of these classes was observed by PCA of the cytokines.
Conclusions
Our pilot data indicate that Coccidioides spp. and the host produce volatile metabolites that may yield biomarkers for a Valley fever breath test. We have collected BALF and sputum from human patients with community-acquired pneumonia, and the next steps will be to determine which Valley fever biomarkers can differentiate between bacterial and fungal etiologies of disease.
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