Colocaciones frente a combinaciones léxicas especializadas

Pseudomonas aeruginosa (strain ATCC 27853) and a clinical isolate ofA. fumiga- tus (AZN 8196 clinical isolate), were selected for this study.

Pseudomonas aeruginosa was inoculated into 50 mL Brain Heart Infusion (BHI) broth (Mediaproducts BV, The Netherlands) in an initial concentration of approximately 5 x 106 _{colony forming units (CFU)/mL.}

Aspergillus fumigatus was stored in 10% glycerol broth at -80◦C, and revived by subculturing on Sabouraud dextrose agar (SAD) supplemented with 0.02% chloramphenicol, for 2 x 7 days at 37 ◦C. Using a swab, the conidia were har- vested and suspended into 5 mL of BHI broth plus 0.1% Tween 80 (Boom B.V., Meppel, The Netherlands) until an average concentration of 2.6 x 106 _CFU/mL was reached, as assessed by measuring the transmission using a spectrophotometer at 530 nm [9]. This suspension was further diluted (10x) into 50 mL BHI broth to a final concentration of 2.6 x 105 _CFU/mL.

Co-cultures (cultures with both A. fumigatus and P. aeruginosa) were ob- tained by preparing a culture of A. fumigatus (2.6 x 105 _{CFU/mL) as described} above. Since P. aeruginosa was expected to overgrow A. fumigatus if both pathogens are inoculated simultaneously, P. aeruginosa (5 x 106 _{CFU/mL) was manually} added fifteen hours after the inoculation of A. fumigatus.

4.2.2

Growth curves

The growth of A. fumigatus in mono- and co-culture was assessed by measuring galactomannan levels in the growth medium [10]. Samples were taken at 1 hour, 6, 12, 20, 24 and 48 hours after inoculation. Determination of galactomannan concentrations in the medium was performed by the Platelia Aspergillus Enzyme Immuno Assay (EIA) (Platelia Aspergillus; Sanofi Diagnostics, Marnes-La Co- quette, France) according to the manufacturers instructions. Values of EIA were expressed as galactomannan index (GI) plotted over time.

The growth of P. aeruginosa in mono-culture was monitored by counting the colony forming units (CFU) according to standard procedures. In brief, samples for CFU counting were taken at 30 minutes after inoculation of the bacteria, then subsequently every hour from 1 to 8 hours, every 2 hours from 8 to 16 hours, every 8 hours from 16 to 32 hours and finally at 48 hours. Tenfold serial dilutions of these samples were made in saline (0.9% NaCl), 10 L was subsequently plated in triplicate on Mueller Hinton agar plates (Oxoid, Landsmeer, the Netherlands). The colonies on the plates were counted after approximately 12 hours, and the CFU/mL was calculated.

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Chapter 4. VOC biomarker combinations of mono- and co-cultures

In the co-cultures, P. aeruginosa growth was also monitored by CFU counting. The counting was performed as described above and samples were taken every hour from 1 to 9 hours, every 2 hours from 9 to 13 hours, every 4 hours from 13 to 25 hours and at 33 hours after adding P. aeruginosa to A. fumigatus.

4.2.3

Headspace sampling

For sampling the headspace of the cultures, a setup was used as described earlier [11]. Briefly, all cultures were placed in an environmental chamber at 37 ◦C. Cultures were constantly shaken on an orbital shaker (Sanyo orbital shaker, Osaka, Japan) at 100 revolutions per minute (rpm) in silicon coated 250 mL round-bottom Erlenmeyer flasks. Each flask was closed using a glass stopper. This stopper contained two Teflon open-close valves acting as the inlet and outlet. Bacterial filters were placed on the inlet and outlet of the flasks to prevent contamination. The headspaces of the cultures were constantly flushed with 3.5 L/h of catalysed compressed air.

To sample 3.5 litre of headspace, a glass tube filled with Tenax TA® (Shi- madzu, Kyoto, Japan), was connected to the outlet of the glass stopper for 60 minutes. Sterile BHI broth, kept under the same environmental conditions as the cultures, was used as a control experiment. The headspaces of the BHI broth medium and the mono-cultures were sampled at 16, 24 and 48 hours after in- oculation. In the co-culture, samples were taken at 16, 24 and 48 hours after inoculation of the first pathogen, A. fumigatus. Each experiment was performed independently 6 times using 2 technical replicates (n=12).

4.2.4

Headspace analysis

The headspace samples were analysed using thermal desorption (TD20) coupled to QP2010 Ultra GC-MS (Shimadzu, Kyoto, Japan). After headspace sampling, the Tenax tubes were flushed with 6 L/h nitrogen for 60 seconds, in a custom-made setup, to dry the samples. Thereafter, the tubes were transported to a thermal desorption unit where the tubes were heated to 250◦_{C for 8 minutes with a flow} of 60 mL/min for desorption of all trapped compounds. A cold trap at 10◦C cap- tured the VOCs, and was subsequently heated to 250◦C to release the molecules through a heated transfer line (split 1:20) at 260◦C onto a CP-Sil 19 CF capillary column (25 m, 0.25 mm inner diameter, 1.2 m film thickness, Agilent Technologies Netherlands BV, Amstelveen, The Netherlands) at 1.02 mL/min column flow. The oven temperature profile started at 40◦C for 4 minutes, then increased to 250◦C with a 5◦C/min heating rate, and finished with 14 minutes at 250 ◦C. Electron ionization (EI) at 70 eV was used to ionize the molecules, and a quadrupole mass

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4.2. Materials and Methods

spectrometer detected the fragment ions in the mass to charge (m/z ) range of 30- 500 Da. Compounds were putatively identified based on 80% minimal similarity of the MS spectra compared to the National Institute of Standards and Technology (NIST) libraries NIST08 and NIST08s.

4.2.5

Standard compounds

The assignment of the VOC biomarkers resulted from the statistical analysis was performed with standards with a purity of ≥ 96%. Dimethyl trisulfide, 2- furaldehyde, methyl thiolacetate, 1-undecene and 2-nonanone were purchased from Sigma Aldrich Chemie B.V., Zwijndrecht, the Netherlands, 2-octanone was pur- chased from Acros Organics (Fisher Scientific, Landsmeer, the Netherlands) and 8-nonen-2-one from AKos GmbH, Steinen, Germany. The standards were diluted 10.000 times in either methanol (2-nonanone, 2-octanone, methyl thiolacetate, dimethyl trisulfide and 8-nonen-2-one) or acetone (2-furaldehyde, 1-undecene) and 2 µL of this solution was injected onto the Tenax tube. To dry the samples, the tubes were flushed with 6 L/h nitrogen for 60 seconds. The retention times (RTs) and mass spectra of these pure standards were compared with those obtained in the experimental samples to confirm the correct identification of the compound.

4.2.6

Multivariate analysis

Multivariate analysis by Partial Least Squares Discriminant Analysis (PLS-DA) [12] was performed to identify VOC biomarkers for the presence of P. aeruginosa, A. fumigatus or both. A separate PLS-DA model was fitted for each sampling time point (16, 24 and 48h).

Partial Least Squares Discriminant Analysis makes linear combinations of the Total Ion Current (TIC) of every VOC to form Latent Variables (LVs); consider- ing compounds that were present in at least 50% of replicates per culture. This analysis reduces the complexity of the data considerably and simultaneously de- termines strong correlations between VOCs. These correlations are expressed in loadings and scores. Loadings indicate the importance of each VOC on every LV, while scores reflect the differences between the samples.

The scores and loadings were represented together in a two-dimensional biplot [13] from which the relevance of each VOC to distinguish between culture types can be interpreted in two dimensions.

To establish time-independent VOC biomarkers linked to the presence of one or both pathogens, a group of selected VOCs was composed for each culture type using all three models. The VOCs were ranked by analysing the loadings for their positive contribution in the direction of each culture type. These VOC rankings were multiplied over the three time points. The number of compounds to include

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Chapter 4. VOC biomarker combinations of mono- and co-cultures

was determined by comparing the rank products of all VOCs visualized in bar graphs. One rank product threshold was set for all three culture types based on a steep increase in rank product when including more VOCs. The VOC biomarkers were represented in a Venn diagram [14]. The power of these time-independent VOC biomarker combinations was determined using one PLS-based classifica- tion model including all sampling time points. As an additional validation, a PLS-DA model was made for each sampling time point using only these time- independent VOC biomarker combinations. Furthermore, to test whether these time-independent VOCs can successfully distinguish the presence of pathogens from the culture medium at all-time points, a four-class PLS-DA model has been constructed including the culture medium.

All PLS-DA models were fitted using double cross-validation [15]. To assess the model performance on a test set, six-fold cross validation was used. An underlying five-fold cross validation was used to determine the optimal number of LVs to include for classification. This means that a model was built using 80% of all samples, which was subsequently tested on the remaining 20% of the samples. This was repeated for increasing numbers of LVs and the number with the highest prediction accuracy (smallest amount of misclassified samples) was selected to fit the final model. The final estimation of prediction accuracy was given as the mean performance of the six cross-validation models. This resulted in a cross-table for each of the three models showing the percentage of samples with correctly classified culture type.

Multivariate data analysis was performed in Matlab R2014a (The MathWorks, Inc., Natick, Massachusetts, USA). PLS-DA was performed using PLS Toolbox 7.8.2 (Eigenvector Research, Inc., Wenatchee, Washington, USA).

4.3

Results