The measurement results of bacterial VOCs depend greatly on the analytical methods adopted for fingerprinting, especially the gas sampling strategy. Although the charcoal-based matrix is a sufficiently suitable adsorbent for a wide range of compounds27, one of the major drawbacks of using the charcoal adsorbent used in this study was the loss of volatiles, which could be caused by the solvent dilution factor, lesser affinity with polar compounds, and overload of the sorbent matrix27,28,24. These might explain the detection of less putative VOC-markers in this study as compared to other reported study that used different adsorbents34 and technique such as thermal desorption28,35. However, the charcoal adsorbent has been proven to perform sufficiently well to detect the most predominant bacterial volatiles that were commonly reported by other researchers, such as alcohols and ketones.
In this study, alcohols were the most commonly detected VOCs in Campylobacter spp., produced mainly in the early stationary and stationary growth phase (Table 2). The detected alcohol-based VOCs included the fatty alcohols 3,7,11-trimethyl-3-dodecanol and 1-heptadecanol, as well as the non-cyclic alkene alcohol 1,8-nonadien-3-ol (Figure 2). The probable pathways for bacterial synthesis of fatty alcohol and long-chain alcohols are through hydrogenation of methyl esters of fatty acids36, as well as by the β- or α-oxidation of fatty acid by-products37. For instance, the production of fatty alcohols in E. coli begins with a thioesterase-mediated conversion of a fatty acyl-ACP (acyl carrier protein) to a free fatty acid. The free fatty acid is then converted to a fatty acyl-CoA by a fatty acyl-CoA synthase. The resulting fatty acyl-CoA can subsequently be metabolised via the β- oxidation route or reduced to its corresponding fatty alcohol by the generation of its corresponding fatty aldehyde in a NADPH-dependent fatty acyl-CoA reductase-catalysed process38. Additionally, 1,8-nonadien-3-ol has been reported to be emitted by Pseudomonas putida as an antimicrobial volatile against plant pathogens39, and commercial fermentation starters during food fermentation40. On the other hand, 1-heptadecanol was also found to be one of the antifungal volatiles emitted by Pseudomonas spp. isolated from canola and soybean41.
The two most predominantly isolated volatile ketones emitted by Campylobacter spp. in the current study were isophorone and 1s,4R,7R,11R-1,3,4,7-tetramethyltricyclo [5.3.1.0(4,11)] undec-2-en-8-one (Figure 2 and Table 2). The ketones were found to be emitted by the thermophilic Campylobacter (C. jejuni, C. coli and C. lari) in both Bolton broth and Brain Heart Infusion culture (Table 2). However, it is noteworthy to point out that ketones were detected generally after 12 hours of incubation at 24 and 48 hours, corresponding to the early phase of stationary and stationary growth phase of Campylobacter spp. in vitro (Table 2). Similarly, Reese et al.35 also reported on the observation of detection of ketones with long carbon chains in the stationary phase and beyond. Isophorone is an unsaturated cyclic ketone that has a peppermint-like smell, and it evaporates faster than water42. It can be naturally found in cranberries42, honey43 and as a beetle pheromone44. Also, it has been already reported by Schulz and Dickschat45 as a volatile emitted by the cyanobacteria Oscillatoria perornata. Whereas 1s,4R,7R,11R-1,3,4,7-tetramethyltricyclo [5.3.1.0(4,11)] undec-2-en-8-one is sesquiterpene ketones occurs naturally in fungi and plants46 and it has an important antibiotic role as a bacterial secondary metabolite47. 1s,4R,7R,11R-1,3,4,7-tetramethyltricyclo [5.3.1.0(4,11)] undec-2-en-8-one has been also reported as one the volatiles of lignin isolated from oil palm empty fruit48.
Generally, the biosynthesis pathway of ketones, particularly methyl ketones, involve the decarboxylation of fatty acids49. However, isophorone and 1s,4R,7R,11R-1,3,4,7-tetramethyltricyclo [5.3.1.0(4,11)] undec-2-en-8-one are terpenoids with a molecule of oxygen as the functional group of a ketone. Hence, the bacterial metabolic routes of terpenoids, unlike methyl ketones, are synthesised from the five-carbon building blocks, isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP). These molecules are likely to be originated from pyruvate and glyceraldehyde-3-phosphate in the methylerythritol-4-phosphate (MEP) and the Mevalonate (MVA) pathways50.
The presence of alcohols and ketones in the VOC profile of C. jejuni is in accordance with other studies that observed the prevalence of these compounds in the cultures of C. jejuni and in matrices contaminated by the bacterium. Probert et al.32 also noticed the presence of these chemicals in stool of patients contaminated with C. jejuni. Alcohols and ketones were detected as discriminant VOCs of chicken faeces contaminated with C. jejuni and C. coli33. Alcohols and ketones were also abundant in the bacterial samples of C. jejuni (ATCC 33560) cultivated in BHI broth after 20 hours of incubation34.
The VOC profiling of other clinically important Campylobacter species, such as C. coli, C. fetus subsp. fetus and C. lari, was a pioneering step towards the characterization of the volatilome of these pathogens. Isophorone and 3,5-bis(1,1-dimethylethyl)-phenol were discriminant volatiles emitted by C. coli., 3,7,11-trimethyl-3-dodecanol and 1s,4R,7R,11R-1,3,4,7-tetramethyltricyclo [5.3.1.0(4,11)] undec-2-en-8-one were identified as biomarkers of C. lari. However, the profile of C. fetus consisted of only not-identified compounds, that despite of their unconfirmed identity, these compounds were important to demonstrate the distinction of C. fetus among the other species. Nonetheless, future research should be able to widen the identification of the volatiles of these foodborne bacteria by employing more sensitive VOC-analytical methods.
The PCA analysis demonstrated clear distinguishable markers based on the VOCs profile emitted by the various strains of Campylobacter spp. included in this work; as well as VOCs profiles that is growth phase dependent (Figure 4 and Figure 5). PCA has been used by numerous researchers to identify similarity and variation in the multivariable dataset, such as Milanowski and co-workers51 that has demonstrated discrimination of salivary bacteria (Hafnia alvei, Pseudomonas luteola and Staphylococcus warneri) based on their volatiles’ profiles; and discrimination of F. tularensis subsp. novicida, Bacillus anthracis Sterne and Bacillus anthracis Ames in the study by Reese and co-workers35.
The study of the variation in the VOCs emitted by the various clinically important Campylobacter spp. including Campylobacter jejuni subsp. jejuni in vitro via active sampling with an activated charcoal sorbent, revealed the culture- and growth phase-associated volatilome profiles of thermophilic and non-thermophilic Campylobacter spp. The findings will provide an insight into the potential roles of bacterial VOCs in their growth and survival in various niches in environment and its host. The findings may also provide a baseline to development of VOC-based detection method of the fastidious Campylobacter spp. Nevertheless, more in-depth studies are required to identify the unknown VOCs detected in this study. Also, it will be meaningful to investigate other influencing factors, such as coculturing of Campylobacter spp. with other bacteria population to fully understand the mechanisms and role of bacterial VOCs.