Effects of biological and methodological factors on volatile organic compound patterns during cultural growth of Mycobacterium avium ssp. paratuberculosis

The following three MAP strains (A, B, C) were included in the project:

• (A)  
  MAP 44133 (type-II, reference strain, bovine origin, DSMZ GmbH, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany);
• (B)  
  MAP 04 A 0386 (type-III, field isolate from sheep);
• (C)  
  MAP 12 MA 1245 (type-II, field isolate from cattle).

Field strains were isolates from tissue or feces, and were isolated and cultivated according to standard protocols recommended by the National Reference Laboratory for Paratuberculosis.

Two independent studies were performed consecutively (figure 1).

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Study I focused on evaluating the influence of culture media on VOC profiles. The following five media (1–5) were included:

• (1)  
  HEYM: Herrold's Egg Yolk Medium containing Mycobactin J and amphotericin, nalidixic acid and vancomycin (Becton Dickinson, Sparks, USA);
• (2)  
  Sto: Stonebrink Medium with Polymyxin B, Amphotericin B, carbenicillin, trimethoprim, and pyruvate and Mycobactin J (Bioservice Waldenburg GmbH, Germany);
• (3)  
  LJ: Löwenstein–Jensen containing Polymyxin B, Amphotericin B, carbenicillin, and glycerol and Mycobactin J (Bioservice Waldenburg GmbH, Germany);
• (4)  
  Du: Dubos Oleic Agar with Mycobactin J (produced according to accredited instructions from the National Reference Laboratory for MAP);
• (5)  
  MB: Middlebrook 7H10 Medium with Mycobactin J containing oleic acid, albumin, dextrose, catalase and polymyxin B, amphotericin B, carbenicillin and trimethoprim (produced according to accredited instructions from the National Reference Laboratory for MAP).

At least eight vials per medium were used for each of the three MAP strains (A, B, C). In total, 123 vials inoculated with bacteria (about 24 of each medium) were compared to 18 control vials (2–5 of each medium).

For inoculation, three loops of cultured bacteria were added to 4 ml of phosphate buffered saline containing disodium, potassium dihydrogen orthophosphate, and sodium chloride (PBS). The bacterial suspension was thoroughly vortexed and diluted to an optical density of 0.312  ±  0.039, measured via a spectral photometer (Dr Lange Cadas 30, Dr Bruno Lange GmbH, Düsseldorf, Germany), which resulted in a bacterial count of 7.44  ±  13 * 10⁸ cfu ml⁻¹. Dilutions of 10⁻², 10⁻⁴ and 10⁻⁶ were then prepared from the original bacterial suspension. Next, 100 µl of the bacterial suspensions of each dilution were inoculated onto each of six slants of each medium. The vials were sealed with Silicone/Teflon septa (Si/PTFE, PAS Technology Deutschland GmbH, Magdala, Germany). Cultures were incubated at 37 °C in a horizontal position for two weeks and then a further three weeks in an upright position. They were then kept at room temperature (21  ±  1 °C) until sampling (see section 2.2).

Study II aimed to assess the time-dependent formation of VOCs during cultivation. The three MAP strains (A, B, C) were cultivated on HEYM only. In total, 71 inoculated vials and 28 control vials were included in this study.

We added 2 ml of original bacterial suspension containing PBS originating from study I to 8 ml of Middlebrook 7H9 liquid medium containing oleic acid, albumin, dextrose, catalase and polymyxin B, amphotericin B, carbenicillin and trimethoprim (MB-Bouillon, produced according to accredited instructions by the National Reference Laboratory for MAP). This suspension was incubated for 54 d at 37 °C in an incubator shaker (70 rotations per min) in the presence of sterile glass beads. The bacterial suspension was thoroughly vortexed and diluted to an optical density of 0.316  ±  0.015, which resulted in a bacterial count of 3.36  ±  0.14 * 10⁵ cfu ml⁻¹. Dilutions of 10⁻², 10⁻⁴ and 10⁻⁶ were then prepared from the original bacterial suspension. Next, 100 µl of the bacterial suspensions of each dilution were inoculated onto each of 18 slants of HEYM. The vials were sealed with silicone/teflon septa and incubated at 37 °C in a horizontal position for one week and then further in an upright position. Sampling (see section 2.2) was performed after two, four, and six weeks of incubation. Afterwards, all vials were kept in incubation for 11 weeks in total to be able to assess any growth of MAP.

Vials of each medium inoculated with 100 µl PBS instead of bacteria served as control.

Bacterial growth in all vials was visually assessed at regular intervals until the end of the study (11 weeks in total) as shown in figure 1, and was scored from 0.5 to 5 points.

The headspace above MAP cultures and pure media control slants was pre-concentrated by means of needle trap microextraction (NTME), as described by Bergmann et al [16] and Fischer et al [17]. The triple-bed needle trap devices (NTDs, Shinwa Ltd, Japan) were packed with divinylbenzene (DVB, 80/100 mesh, 1 cm), Carbopack X (60/80 mesh, 1 cm), and Carboxen 1000 (60/80 mesh, 1 cm). The NTDs were conditioned in a heating device (PAS Technology Deutschland GmbH, Magdala, Germany) at 250 °C for at least 12 h under permanent nitrogen flow (1.5 bar) before first use, and re-conditioned at 250 °C for 30 min before being applied for pre-concentration of the samples. All vials were warmed up in a dry block heating bath (Unitek, Germany) at 37 °C for 20 min immediately before sampling. Needles were pierced through the septum (figure 2) and 20 ml of headspace was conducted through the needle by inflating and releasing a 1 ml disposable syringe (Transcoject GmbH, Neumünster, Germany). Each NTD was sealed using a Teflon cap (Shinwa LTD, Japan/PAS Technology Deutschland GmbH, Magdala, Germany) before and immediately after collecting the sample.

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VOC analyses were performed by means of GC-MS. VOCs desorbed from NTDs were separated by GC (Agilent 7890A) and detected by mass selective detector (Agilent 5975C inert XL MSD). This principle has been described previously [10, 16, 18]. VOCs were initially identified via a mass spectral library search (NIST 2005 Gatesburg, PA, USA). Subsequent identification and quantification was established by analysis of pure reference substances (the origin of the chemicals can be found in table S1 (stacks.iop.org/JBR/10/037103/mmedia)) and comparison of GC retention times and mass spectra of all selected marker substances.

Using a liquid calibration unit (LCU, Ionicon Analytik GmbH, Innsbruck, Austria), humidified standards of pure references in different concentration levels were produced for NTME calibration. By measuring 10 blank samples and integrating signal areas of baseline for each marker, the substance limit of detection (LOD, signal-to-noise ratio 3:1) and limit of quantification (LOQ, signal-to-noise ratio 10:1) were calculated. VOC concentrations below LOD were set to zero. Table S2 provides confirmed substances through retention time, mass spectra and quantitative parameters such as LOD and LOQ.

NTME GC-MS measurements resulted in more than 100 individual substances detected in the headspace of vials and quantified by analysis and calibration of pure reference substances (section 2.3). The total concentrations of the volatiles in the headspace of the vials were considered. To differentiate between VOCs originating from the material or the media, the ones arising or being consumed from MAP cultures, and the ones existing in the surrounding air of the laboratory, we compared results from above cultures, above pure media slants and the laboratory room air. VOCs serving as potential marker substances for MAP growth were selected according to the following criteria. (i) The VOC profile was defined after 6 weeks of cultural incubation, based on the assumption that the bacteria underwent exponential growth throughout this period. (ii) The concentration of VOCs above MAP, disregarding strains and bacterial count, needed to be significantly higher than in the surrounding laboratory room air and needed to differ significantly from VOC emissions above pure media.

All 18 control vials and 123 inoculated vials from study I along with 28 control vials and 71 inoculated vials from study II were included in statistical data analyses. IBM SPSS Statistics 19 (version 19.0, IBM Corporation, NY, USA), Microsoft Excel 2010 (Microsoft Corporation, USA) and STATGRAPHICS Centurion XVI.I (version 16.1.18, Statistical Graphics Corporation) were used. Numerical data are presented as medians and percentiles (25–75%). To identify significant differences between two groups of unpaired data, the Mann–Whitney U-test (exact test) was applied. Multifactorial analysis of variance (ANOVA) was used to identify significant influences of methodological effects (different bacterial counts, different duration of incubation) and biological factors (inter-strain variability) on concentrations of selected substances. Values of p  <  0.05 were considered statistically significant. For visualization, a three-colour heatmap with normalized values was used. To evaluate the influence of different culture media, a principal component analysis was applied and the scatterplot depicts variations in VOC emissions based on the primary ingredients of the media.

Different culture media were associated with remarkable differences in VOC emissions (figure S1) and with significant differences in VOC concentrations.

Substances from all chemical classes were measurable above all five culture media. Some VOCs, like cyclohexane and 3-methylfuran, were emitted from all five culture media, but showed significantly different concentrations. Others were either highly concentrated above egg-containing media (figures 3 and S1) and minimally concentrated above synthetic media, like 3-methyl-1-butanol, or vice versa (e.g. benzene). In contrast, several VOCs were emitted in measurable amounts above some media yet showed concentration values below LOD above other media, for example 2-propylfuran and benzaldehyde. The ingredients of the culture media are listed in table S3. Figure 3 illustrates that the concentration and composition of VOC emissions depended on the ingredients of the media and that score two from the principal component analysis defined a VOC-profile linked to all egg-containing media. Eight VOCs showed significant differences between inoculated and control vials on HEYM, Sto, and LJ, and all inoculated vials had significantly higher or lower VOC concentrations than all individual control vials. The concentrations in MAP-inoculated slopes were lower for 1-propanol, benzaldehyde, and hexanal, and higher for 2-methyl-1-butanol, 3-methyl-1-butanol, 2-hydroxy-benzoate, methyl-acetate, and 3-pentanone.

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According to the criteria for potential marker substances (section 2.4), 43 VOCs assessed above MAP cultures could be identified as potential biomarkers. These marker substances belonged to the classes of alcohols, aldehydes, esters, ketones, nitrogen compounds, aliphatic hydrocarbons, and aromatic hydrocarbons. Table 1 shows absolute concentrations of selected VOCs. Three alcohols (1-propanol, 2-methyl-1-butanol, and 3-methyl-1-butanol), two esters (2-hydroxy-benzoate and methyl acetate), two ketones (2,3-butanedione and 3-octanone), and eight hydrocarbons (2,4-dimethyl-1-heptene, 2,4-dimethylheptane, 4-methyloctane, cyclohexane, ethylbenzene, heptane, octane, and styrene) were not detectable above control vials at all, but were highly concentrated above MAP cultures. In contrast, four VOCs (1-octen-3-ol, 2-methyl-2-butenal, heptanal, and 2-methyl-butanenitrile) had no detectable concentration above bacteria after six weeks of incubation, but showed significantly higher concentrations in control vials. Nine volatiles (1-octen-3-ol, 2-heptanone, all nitrogen containing compounds, and six aldehydes) were less concentrated above bacteria than in the headspace above pure media.

Multifactorial ANOVA (table 2) revealed no significant differences between the three MAP strains for 35 of the 43 VOCs, but eight volatiles showed significant differences between different MAP strains. This applied for the reference and the C-type field strain, which showed slightly lower values for some VOCs, for example 2,4-dimethylheptane (figure 4). The lower bacterial density tubes in particular presented higher variation in their VOC concentrations. Figure 4 also illustrates that VOCs above MAP showed significant differences in their concentration compared to control vials, regardless of strain.

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Due to the different dilutions of the original bacterial suspension, an exponentially increasing bacterial count prevailed in inoculated vials which led to significantly different growth: the higher the original bacterial count, the better the bacterial growth. The original bacterial count ensured significant differences for 36 of the 43 VOCs, except for 2-methyl-1-butanol, ethanol, isopropyl-alcohol, 2-methyl-butanenitrile, 2,4-dimethylheptane, and styrene (table 2). The multifactorial ANOVA for the period of incubation resulted in significant concentration differences for 41 of the 43 VOCs, except 1-propanol and 2,4-methyl-1-heptene (table 2). After two weeks of incubation, visually apparent growth was already seen in vials with high bacterial count, while most vials with low bacterial count did not show apparent growth after four or even six weeks of incubation. After two weeks of incubation, VOC concentrations of different bacterial counts were all similar to each other (figure 5(a)), even though vials with high bacterial density showed visible bacterial growth. On the other hand, after four weeks of incubation there were several vials without visual bacterial growth, but significant emissions of 34 VOCs (figure 5(b)).

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After two weeks of incubation, VOC concentrations of MAP resembled control vials, while most VOC concentrations changed significantly after four weeks of incubation. Three different patterns of MAP-related VOCs were obvious (figure 6). (i) Concentrations of 34 VOCs above MAP were directly related to increasing bacterial density, either by initial inoculum or by bacterial growth over time. Some of these substances decreased after reaching a peak, for example 2-hydroxy benzoate, and hexane (figure 6(a)). (ii) Other VOCs kept increasing without reaching a peak, for example 2-methyl-1-butanol and 2-butanone (figure 6(b)). (iii) In contrast, concentrations of nine VOCs decreased significantly with increasing bacterial density up to a concentration lower than LOD (figure 6(c)). A remarkable pattern was shown by 2-heptanone, which presented decreasing concentrations with increasing initial bacterial count, but increasing concentrations within the dilution with increasing duration of incubation. Also, 2-heptanone and six aldehydes (except 2-methylpropanal) represented a special instance of this pattern. VOC concentrations were significantly higher in vials with low bacterial density than controls and decreased further with increasing bacterial density to lower values than controls (figure 6(c), 2-methyl-2-butenal, figure 5(b)).

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Remarkable time-dependent changes in the VOC concentrations in the headspace above control slants were also noted. With increasing duration of incubation, all VOCs presenting concentrations higher than LOD increased (figures 5(b) and 6).

As the bacterial density of MAP increased, either by initial inoculum or by bacterial growth over time, the concentrations of the volatiles emitted could be assigned to three different patterns (figure 6). We speculate that VOCs with decreasing concentrations originated from the media and were consumed by replicating MAP. Volatiles with increasing concentrations most likely originated from MAP and could be intermediates or metabolites of several pathways or could function as signaling substances. VOCs which reached a peak had similar origin but either the metabolic pathways stopped due to reduced replication, or synthesis and degradation of substances leveled out, because other metabolic pathways took over. Metabolic regulation always depends on ambient conditions such as available surface area on the medium, nutrient supply, competitors and messenger substances. High levels of emitted aldehydes above vials without visible bacterial growth pointed to the importance of these substances in metabolism of MAP. These could be messenger substances or metabolites in the signaling system for environmental adaptation, comparable with proteins responding to stress like heat, hypoxia or nutrient starvation [19, 20].

The literature contains some studies examining parts of metabolic pathways of Mycobacteria. The cell wall of Mycobacteria presents many specific molecules. As described by Dhimann et al and Appelmelk et al [21, 22], lipoarabinomannan (LAM) suppresses the host's macrophage functions. The core of the molecule is linked to α-arabinofuranan and D-galactofuran, and is mainly built during the logarithmic phase of bacterial growth. Furans can be generated during LAM synthesis. Mycolic acids, a formation of long-chain fatty acids, ketones and alcohols [23], are a special feature of the cell wall of Mycobacteria. During synthesis of mycolic acids, esters [24, 25] may also be generated. Due to the structure of mycolic acids, branched hydrocarbons may be generated via cleavage. However, the origin of VOCs is mostly unknown and is subject to speculation.

As described by many authors, Mycobacteria must adapt metabolically to in vitro conditions [26]. Russell et al [27] described primary carbon source changes from host-derived lipids in vivo to glucose and glycerol in vitro. This may explain differences in VOC emissions above different media. Future studies should focus on elucidating the origin of VOCs in metabolic pathways and the effects of different culture conditions.

For diagnostic purposes, VOCs above MAP need to differ significantly from VOCs above pure media slants. More than 100 different substances were detectable over MAP cultures. After applying the selection criteria, only 43 VOCs remained more concentrated than the surrounding laboratory room air and those differing significantly between inoculated and pure media slants. The concentration above MAP strains could be either higher or lower than those above control slants. Overall, we included 43 volatiles in the VOC profile. Our results agree with those from an earlier study by Trefz et al [10]. Both projects resulted in about 40 volatiles belonging to the same chemical classes. About half of these were included in both VOC profiles and showed the same concentration pattern. The variation could be due to numerically larger experimental groups or different culture media and incubation duration. In vitro VOC profiles of M. tuberculosis described by Syhre et al [28] differed from those defined in this project for MAP. To confirm the suitability of VOC analysis for diagnostic applications, more detailed studies comparing many other Mycobacterium species are necessary.

To display a diagnostic benefit, VOCs need to be independent of biological (inter-strain variability) and methodological factors (culture media, bacterial count, duration of incubation).

4.3.1. Biological factors

4.3.2. Methodological factors