The aim of this work was to investigate whether the detection and perhaps identification of bacteria can be achieved by the determination of characteristic volatile metabolites released. This work should provide the basis for the application of breath-gas analysis in the early and non-invasive diagnosis of bacterial lung infections by monitoring the presence of the specific pathogen-derived markers in exhaled breath. Exhaled breath analysis is a relatively new field of research and still requires extensive basic research to evaluate the candidate compounds, which may serve as biomarkers, and to delineate their possible biochemical and cellular sources. Volatile compounds in exhaled breath may be of endogenous (i.e. derived from host cells), exogenous or microbial origin.
Hence it is crucial to investigate the contribution of microorganisms of the normal flora (originating from body compartments like the gut, upper airways, sinuses, nose or mouth) and of microorganisms expanded during infections to the VOCs found in human breath.
Numerous species which are found in normal flora of humans may also become pathogenic, e.g. when the immune system is weakened [2
]. In this work two different bacterial species [2
] were investigated with respect of the release of VOCs. In the past, such or similar investigations were performed applying GC-MS, however, mostly with only qualitative and not quantitative analysis of detected VOCs [6
] or for instance with indirect quantification without calibration of VOCs of interest [30
In our in vitro work we found that the patterns of VOC release from S. aureus and P. aeruginosa are only in part identical, and considerable differences were found concerning the dynamics of VOC production and especially the uptake of volatile metabolites. Thus, P. aeruginosa takes up or catabolizes (but never releases) aldehydes, in contrast to S. aureus, which releases high concentrations of aldehydes. Similarly, no acids were significantly released by P. aeruginosa in our study. Despite higher proliferation rate of P. aeruginosa the concentrations of released metabolites were lower from those secreted by S. aureus. A greater variety of volatile compounds was found in the headspace of P. aeruginosa as compared to S. aureus comprising diverse ketones, esters, sulfur containing compounds, hydrocarbons and additionally nitrogen containing compounds, which were not detectable in the headspace of S. aureus.
Zechman and co-workers have identified several identical compounds as reported here in the headspace of S. aureus
and P. aeruginosa
(e.g. acetoin and methylbutanal for S. aureus
, 1-undecene and ketones for P. aeruginosa
and DMDS and iso
-pentanol for both species) using aerobic conditions similar to us with application of liquid culture and tryptic soy broth as culture medium [6
]. However, they did only qualitative analyses at one incubation time point of 24
h. Besides similarities in our study to other works, also divergent results were obtained [6
]. In this respect, Scott-Thomas [26
] and Labows [30
] identified 2-aminoacetophenone as an important volatile metabolite of P. aeruginosa,
which allows discrimination of cystic fibrosis patients colonized with P. aeruginosa
from control groups (healthy subjects and CF patients colonized with other bacteria species) [26
]. This compound could not be detected in the headspace of P. aeruginosa
in our study since it is derived from tryptophan, whose concentration might be too low in the used soy based medium to form aminoacetophenone in detectable concentrations. Similarly, Allardyce et al. reported strong release of acetic acid and acetaldehyde from P. aeruginosa
], whereas acetaldehyde was clearly decreasing in the Pseudomonas
cultures in our study. Presumably, culture conditions (especially nutrient availability) and analytical methodologies may have a strong influence on the release of VOCs from bacteria cells, stressing the importance to standardize these factors.
Although it might be insufficient to reveal the full spectrum of potential volatile metabolites, a single growth medium (tryptic soy broth) was used for bacteria cultivation in our experiments. This medium is standard for bacteria culture ensuring fast proliferation of microorganisms. Standardization of culture conditions (e.g. proposed here application of the same medium for both species) will be a challenge for the future as bacteria differ in their requirements for nutrients and the composition of the medium in use may affect the nature of the compounds released.
The sampling of headspace gas was performed at several different time points to gain insight into the dynamics of microbial VOC production. This approach demonstrated varying VOC concentration profiles. Accurate diagnosis will require knowledge at what time after inoculation volatile metabolites show either maximum release or become steady in concentration. Although this study was limited to two species we observed specific VOC patterns for each bacterium, demonstrating the procedure developed is suitable to discriminate between pathogenic bacteria. An important issue which should be addressed in future studies is to gain insight into the VOC profiles of further clinically relevant microorganisms and to address the effect of the presence of additional pathogenic organisms in the samples as well as of the presence of host cells.
The metabolic origin of VOCs released is not completely elucidated but it is known that production of branched-chain aldehydes results from the catabolism of amino acid (Figure ) [19
]. Aldehydes then can be reduced to alcohols by alcohol dehydrogenases (e.g. 3-methylbutanal to 3-methyl-1-butanol) or oxidized to carboxylic acids by an aldehyde dehydrogenase (e.g. 3-methylbutanal to isovaleric acid) as observed for S. aureus
. Since all aforementioned compounds were found to be released by S. aureus
in our in vitro
study we presume that amino acid degradation rather than synthesis of fatty acids from alkanes is the underlying pattern of VOCs released by S. aureus
, especially since the culture medium used in our experiments consisted mainly of amino acids, peptides and glucose. This hypothesis is also supported by other published work, where a link between availability of branched amino acids (e.g. valine, isolecine) and production of branched alcohols and aldehydes was reported [6
Figure 2 Catabolism of leucine leads to the formation of 3-methylbutanal, 3-methyl-1-butanol and 3-methylbutyric acid (isovaleric acid), which were found to be significantly released byS. aureus. Modified after Marilley et al. 
Additionally, pyruvate or citrate are starting materials for the formation of short-chain flavor compounds such as acetoin, 2,3-butanedione, 1-butanol, 2-propanol, acetic acid, acetaldehyde and ethanol through glycolytic, lactate converting and non-glycolytic carbohydrates fermentations or fermentations of nitrogenous compounds [44
]. The catabolism of pyruvate (presented on Figure ) seems to play an important role in case of S. aureus
since the products of this metabolic pathway were found in the headspace of this bacterium in our study and also by other researchers, inter alia ethanol, acetaldehyde, acetic acid [11
] and acetoin [6
Figure 3 Simplified scheme of pyruvate metabolism via glycolytic fermentations and lactate converting fermentations, modified after Michal et al.. Exclusively, pathways which lead to the production of VOCs significantly released by S. aureus in this study (more ...)
Detailed investigation of the subspecies of the genus Staphylococcus
shows that acetoin is produced by the subspecies aureus
and not by the subspecies anaerobius
. On the other hand, Pseudomonads
are described as organisms with strictly respiratory metabolism mostly with oxygen and in some species nitrate as terminal electron acceptor [45
], hence the release of alcohols and acids from these microorganisms is not expected. Indeed, carboxylic acids were not observed to be released by P. aeruginosa
in our in vitro
study, but a very week production of 2-butanol and substantially stronger of ethanol and 3-methyl-1-butanol were found. These may be related to altered activity of aldehyde- and alcoholdehydrogenase as reported by Nosova et al. [46
] while the metabolism of amino acids [44
] rather than glycolysis of carbohydrates via Entner-Doudoroff pathway [1
] yields pyruvate as starting material under conditions applied in our study. Nevertheless, it seems that the most dominant metabolic process in P. aeruginosa
cultures is the catabolism of organic compounds such as aldehydes as carbon and energy sources. The versatile nutritional requirements of Pseudomonas
are commonly known and some of its subspecies utilize over 100 different compounds of diverse chemical classes what makes them particularly important organisms of bioremediation in environment (degradation of oil spills, pesticides and other xenobiotics) [1
]. In respect to this feature, the substantial uptake of aldehydes from culture medium was observed in experiments with P. aeruginosa
, while acetaldehyde, 3-methylbutanal, 2-methylpropanal, benzaldehyde and butanal were most strongest metabolized.
Our results confirm the production of sulfur-containing compounds, especially by P. aeruginosa
, extending the earlier works of other researchers [6
]. VSCs such as dimethylsulfide, dimethyldisulfide and dimethyltrisulfide originate from auto-oxidation of methanethiol [19
] that can be produced though metabolism of the sulfur-containing amino acids, e.g. via demethiolation [50
], transamination [51
] or recombination pathway [54
One of the most interesting observations in experiments with P. aeruginosa is the early and strong release of the nitrogen containing compounds pyrrole, 1-vinyl aziridine and 3-methylpyrrole with aberrant release patterns concerning the first two mentioned compounds compared to all other released metabolites. This finding is unique among tested bacteria species and particularly interesting from the point of view of early detection of P. aeruginosa infections.
Both investigated bacteria release in part the same compounds, mostly alcohols, esters and VSCs (Tables and ). As such, these compounds cannot be used for an unambiguous identification of the underlying pathogen. However, they can be used in exhaled breath analysis to monitor development of disease (e.g. emerging pneumonia), especially that some of them are released at as high concentration levels as several hundreds of ppbv (e.g. methanethiol, 3-methyl-1-butanol). Nevertheless, both bacteria S. aureus and P. aeruginosa normally do not coexist as the pathogens of pneumonia. In addition, our in vitro study clearly shows that both bacteria produce pathogen-specific metabolites allowing their identification by means of gas phase analysis. VOCs exclusively released by S. aureus comprise mostly low molecular weight analytes, while the compounds within the range of C3 - C5 have the biggest contribution, being 76% of all unique metabolites for this bacterium. Similarly, there is a set of metabolites exclusively released by P. aeruginosa. Several compounds show significantly increased concentrations already in the first few hours of bacterial growth. Among them, nitrogen-containing VOCs were released early after incubation of P. aeruginosa, but also ketones (besides methyl isobutyl ketone) and most of unsaturated hydrocarbons.
Compounds like acetone, isoprene, acetaldehyde and butane are normally present in human breath [55
] resulting in substantially high background level and therefore they are unsuitable as biomarkers. We propose a candidate compound should not be present in more than 5% of healthy non-smoking subjects, ideally. Volatile metabolites fulfilling our criteria are listed in Table . In this respect, particularly intriguing substances are nitrogen-containing metabolites such as 1-vinylaziridine and 3-methylpyrrole, which are increasing strongly during the first incubation phase of P. aeruginosa
and have a decreasing tendency afterwards.
Table 4 Maximum median concentrations [ppbv] with respective time of bacteria growth [h] as well as appearance in exhaled breath of healthy volunteers for selected metabolites which fulfill the criteria for biomarker ofStaphylococcus aureusandPseudomonas aeruginosa (more ...)
Very encouraging results were obtained also for α-unsaturated hydrocarbons, especially 1- undecene which was one of the most abundant VOCs produced by P. aeruginosa
. 1-Undecene was significantly released from the first time-point of the experiment (1.5
h) and was never found in exhaled breath of healthy non-smokers. Interesting is also 2-nonanone, which was significantly released immediately after inoculation of P. aeruginosa
, but never found in any exhaled breath sample. Similarly, acetoin and acetol meet all requirements for a perfect biomarker of S. aureus