Initial study of three different pathogenic microorganisms by gas chromatography-mass spectrometry

Introduction

Infectious diseases are the main reason for morbidity and mortality in developing countries, especially among children¹. Staphylococcus aureus is a common inhabitant of the upper respiratory tract in children, and the causative agent for many infections. It is believed that people under 20 are more likely to have these bacteria. There is a greater possibility that S. aureus exists in the respiratory tract of infants aged 3 months or younger than in people of other ages². Moreover, S. aureus is colonized in the nasopharynx in 10–35% of children, and in almost 35% of the adult population³.

Escherichia coli is one of the most significant pathogens affecting preterm infants⁴. Some studies in developing countries have suggested that gram-negative rods (such as E. coli) are the major causes of infection in premature infants (0–6 days)^5–7. Furthermore, infections caused by E. coli are one of the most important causes of death in the early neonatal period⁵. Candida albicans is an opportunistic pathogen and an agent of nosocomial infection⁸.

Generally, the causative agents of respiratory tract infections are diagnosed in late phases of the disease⁷. Such infections need broad-spectrum antibiotic therapy, the consequences of which are a reduction in the pathogen load, but not eradication. Moreover, such therapies increase the probability of drug-resistant infections spreading⁹. Accurate and rapid detection of pathogens is a critical step for adequate treatment of infection¹⁰. and a non-invasive diagnostic method that has a high degree of accuracy needs to be developed¹¹.

It has been shown that bacteria produce organic gases. Different types of microorganisms have a distinct metabolism, and they produce various types of volatile organic compounds (VOCs)^12–14. Attempts have been made to identify the VOCs of pathogenic organisms^15–20. There are several sophisticated methods available that have been used for recognizing VOCs; these include gas chromatography-mass spectrometry (GC-MS)²¹, selected ion flow tube mass spectrometry (SIFT-MS)²², electronic noses (eNoses)²³, and ion-molecule reaction mass spectrometry (IMRMS)²⁴. Previous studies suggest that GC-MS is the most appropriate and reliable technique for the isolation and identification of VOCs^25–27.

The current study aimed to identify the volatile organic compounds (VOCs) produced by three respiratory tract pathogens, including Staphylococcus aureus, Escherichia coli and Candida albicans, to determine if these could be used as biomarkers.

Materials and methods

Results

The VOCs produced by S. aureus, E. coli and C. albicans were assessed under six different conditions (using two types of media and taking measurements at three time points). The Xcalibur raw files for these three pathogens are available at https://doi.org/10.6084/m9.figshare.5178004.v1³².

One chromatogram of the six chromatograms obtained is displayed in Figure 1, showing the chromatogram obtained 4 hours after culture in TSB medium, for each pathogen. The five other chromatograms are also available, as Supplementary File S1, Supplementary File S2, Supplementary File S3, Supplementary File S4 and Supplementary File S5.

Figure 1. Three chromatograms, for samples taken 4 hours after culture in TSB media.

EC: E. coli, SA: S. aureus and CA: C. albicans. The other chromatograms are available in the Supplementary material.

The processed GC-MS data obtained in the current study is available in a total of 18 tables as supplementary GC-MS data. It shows the details of the VOCs detected for each of the three pathogens, each analyzed under different conditions (using two types of media and taking measurements at three time points, as explained above).

For a better overview the detected VOCs are shown in three tables (at the 2 hour time point in Table 1, at the 4 hour time point in Table 2 and at the 24 hour time point in Table 3), alongside the percentage of the total area that the average peak of the detected VOC covered. In other words it is proportional to amount of the compound that is present.

Some VOCs were common among organisms and were generated by two or three organisms at an approximately equal rate, including 1,2-benzenedicarboxylic acid, 1,9-decadiene, 2,5-(1,1-dimethylethyl)-phenol, 2,6-bis(1,1-dimethylethyl)-4-methyl-phenol, 3-propionyl oxy pentadecane and anisol (Table 1). Some common VOCs were produced at a greater rate between one organism and another. It can be concluded that these VOCs could also be more important in the organism that produces greater quantities. 1-penten-3-ol was produced from E . coli in TSB medium after 2 hours (0.02%); under identical conditions, more of it was produced by S. aureus (5.14%) than by E. coli. Furthermore, indole was produced from E. coli after 2 hours of culture in two types of medium (82.61% for MB and 90.97% for TSB) and was also produced by S. aureus after 2 hours in TSB medium, although at a much lower rate (0.48%) (Table 1).

Uncommon VOCs of E. coli detected 2 hours after culture included 1-(1,5-dimethyl)-4-hexyl-4-methyl-benzene, 2,3-pentandione, 2,6-dibutyl-2,5-cyclohexadiene-1,4-dione, benzophenone, bisabolene, copaene, decanol, dodecanol, indole, limonene, muurola-4,5-diene, neryl acetate, phenyl ethyl pyrrole and tetradecane (Table 1).

Uncommon VOCs of S. aureus detected 2 hours after culture included 1,2-butadiene, 1-penten-3-ol, 2,5-dimethyl pyrazine, 2-ethyl hexanol, allyl butyl hydroquinone, benzene acetaldehyde, butyl cyclohexyl acetate, caryophyllene, cedrol, cyclohexene, 4-ethenyl-, decene, dimethyl octenal, heptadecane, humulen, longifolene, methone, nonadecanone and tetrabutyl cyclohexyl acetate (Table 1).

Uncommon VOCs of C. albicans detected 2 hours after culture included 1,3-butadiene, 1,5-decadiene, 2-hexan-1-ol, 3-methyl-1,5-heptadiene, butyraldehyde, cadinene, carbamic acid, dodecenol, eicosane, ethyl butanoate, longifolene, ocimene, octyl acetate, tetradecanol and β-santaloland (Table 1).

Uncommon VOCs of E. coli identified 4 hours after culture included 1,9-decadiene, 2-acetyl-1-pyrroline, 2-heptanone and indole (Table 2).

Uncommon VOCs of S. aureus identified 4 hours after culture included 1,2-butadiene, 1,3-heptadiene, 1-decyne, 1-methoxy-2-propanol, 2,3-pentandione, 2-ethyl hexanol, 2-methyl-2-undecanethiol, butyraldehyde, cis-dihydro-α-terpinyl acetate, cyclohexene,4-ethenyl- and levomenthol (Table 2).

Uncommon VOCs of C. albicans identified 4 hours after culture included (e)-2-hexyl ester- butanoic acid, (z)-2-octene-1-ol, (z)-4-decan-1-ol, 1,2-benzenedicarboxulic acid, 1,3-butadiene, 1,5-decadiene, 2-(phenyl methylene)-octanal, 2,5-(1,1-dimethylethyl)-phenol, 2,5-dimethyl pyrazine, 2-ethenyl-6-methyl-pyrazine, 2-hexan-1-ol, 4-t-butyl-2-(1-methyl-2-nitroethyl) cyclohexane, 5.5-dodecadinyl-1, 12-diol, 6-methyl-5-hepten-2-one, benzaldehyde, cadinene, carbamic acid, cedrol, , dimethyl octenal, dimethyl ethyl cyclohexanol, dodecenal, dodecenol, eicosane, longifolene, longifolol, methyl isopropyl hexenal, naphthalenol, octacosane, octyl acetate, phthalic acid butyl ester, tetradecanol, tridecanol, zingiberene and sesquiphellandrene (Table 2).

Uncommon VOCs of E. coli identified 24 hours after culture included 1,9-decadiene, 2-acetyl-1-pyrroline, 2-heptanone, 2-methyl tetradecane and indole (Table 3).

Uncommon VOCs of S. aureus identified in 24 hours after culture were included; 1,2-butadiene, 1,3-heptadiene, 1-decyne, 1-methoxy-2-propanol, 2,3-pentandione, 2,5-dimethyl pyrazine, 2-decenal, 2h-tetrazole-5-carboxylicacid, 2-phenyl, 3-methyl-1,5-heptadiene, caryophyllene, cis-dihydro-α-terpinyl acetate, cyclohexene,4-ethenyl-, ethyl butanoate, levomenthol and thiophene (Table 3).

Uncommon VOCs of C. albicans identified 24 hours after culture included (z)-2-octene-1-ol, (z)-4-decan-1-ol, 1,2-benzenedicarboxylic acid, 1,5-decadiene, 2-(phenyl methylene)-octanal, 2,5-dimethyl pyrazine, 2-methyl-1-propanol, 2-methyl-2-undecanethiol, 2-octyl-1-ol, 2-octyne, 3-methy-4-pentene-3-ol, 3-methyl-1-pentene, 4-t-butyl-2-(1-methyl-2-nitroethyl) cyclohexane, carbamic acid, cedrol, , ethyl acetoacetate, longifolol, octacosane and zingiberene (Table 3).

Discussion

As previous studies have shown, organisms are able to produce either common or specific VOCs^33–35. In the current study, GC-MS was used to detect VOCs generated by three pathogenic organisms in the human respiratory tract. The VOCs of E. coli, S. aureus and C. albicans were analyzed at three different time points, using two different types of media (Figure 1).

Results of the current study suggest that VOCs exclusively produced by E. coli are 1-(1,5-dimethyl)-4-hexyl-4-methyl-benzene, 2,6-dibutyl-2,5-cyclohexadiene-1,4-dione, benzophenone, bisabolene, copaene, decanol, dodecanol, indole, limonene, muurola-4,5-diene, nerylacetate, phenyl ethyl pyrrole, sesquiphellandrene, tetradecane, 2-acetyl-1-pyrroline and 2-methyl tetradecane. The most important compound among these is Indole, because it is generated at the three time points and also it was the most produced VOC by E. coli (at least 82%). Other studies have confirmed this finding^28,29,35. E. coli produced tryptophanase and this enzyme degrades tryptophan to indole and the other compounds³⁶. In future studies, it is advisable to measure the amount of indole in the exhaled air of infected patients with E. coli and compare it with the current results. This is because in the patient’s lungs the level of tryptophan is not the same as culture medium. It is also suggested that the amount of released indole from this bacterium should be evaluated under at in-vitro conditions and with using the simplest culture medium (relative to TSB and MB). In this way, we will have a more detailed thought of the importance of the Indole production by E. coli.

The current study has shown that the specific VOCs produced by S. aureus are 1,2-butadiene, 1-penten-3-ol, 2,5-dimethyl pyrazine, 2-ethyl hexanol, allyl butyl hydroquinone, benzene acetaldehyde, butylcyclohexyl acetate, caryophyllene, cyclohexene, 4-ethenyl-, decene, heptadecane, humulen, longifolene, methone, nonadecanone, tetrabutylcyclohexyl acetate, 1,3-heptadiene, 1-decyne, 1-methoxy-2-propanol, 2,3-pentandione, cis-dihydro-α-terpinyl acetate, levomenthol, 2-decenal, ethyl butanoate and thiophene. Moreover, 1,2-butadiene, 2,5-dimethyl pyrazine, 2-ethyl hexanol, caryophyllene, cyclohexene, 4-ethenyl, 1,3-heptadiene, 1-decyne, 1-methoxy-2-propanol, 2,3-pentandione, cis-dihydro-α-terpinyl acetate, and levomenthol were detected under more than one of the six conditions that were tested, so they are significant. Another important point is that the percentage of the total area that the average peaks for 2,3-pentandione, cis-dihydro-α-terpinyl acetate, 1-decyne, 1,3-heptadiene, 2,5-dimethyl pyrazine, ethyl butanoate and cyclohexene,4-ethenyl covered were at least 15%; thus, they are remarkable VOCs for S. aureus. Some of the VOCs produced by S. aureus in the current study have been reported in other studies^34,37 but some of them have not^28,33. The origin of all produced VOCs is not exactly known. However it is believed some released VOCs by this bacterium is because of the ability to degrade amino acids in its growth environment¹¹.

This study suggested that the specific VOCs produced by C. albicans include 1,3-butadiene, 1,5-decadiene, 2-hexan-1-ol, cadinene, carbamic acid, dodecenol, eicosane, longifolene, ocimene, octyl acetate, tetradecanol, β-sesquiphellandrene, (z)-2-octene-1-ol, (z)-4-decan-1-ol, 2-(phenyl methylene)-octanal, 4-t-butyl-2-(1-methyl-2-nitroethyl) cyclohexane, longifolol, 6-methyl-5-hepten-2-one, dodecenal, methyl isopropyl hexenal, tridecanol, 2-methyl-2-undecanethiol, 2-octyl-1-ol, 2-octyne, 3-methy-4-pentene-3-ol, 2-methyl-1-propanol and 3-methyl-1-pentene. also, 1,3-butadiene, 1,5-decadiene, 2-hexan-1-ol, cadinene, carbamic acid, dodecenol, eicosane, longifolene, octyl acetate, tetradecanol, β-sesquiphellandrene, (z)-2-octene-1-ol, (z)-4-decan-1-ol, 2-(phenyl methylene)-octanal, 4-t-butyl-2-(1-methyl-2-nitroethyl) cyclohexane, longifolol, octyl acetate, β-sesquiphellandreneand 2-methyl-2-undecanethiol were detected under more than one of the six conditions that were tested, so they are significant. Furthermore, 1,3-butadiene, carbamic acid, longifolol, β-santalol, 2-methyl-1-propanol, 2-methyl-2-undecanethiol and 4-t-butyl-2-(1-methyl-2-nitroethyl) cyclohexane were produced in greater quantities . Several studies have analyzed the VOCs of C. albicans and have noted that most of these identified compounds are alcohols^38–40. That is because if favorable growth conditions are available for this bacterium (a sufficient level of oxygen, aromatic amino acids, and an alkaline pH) will produce large amounts of alcohol that results from its metabolism⁴¹.

It is suggested that the findings of future studies on the exhaust air of respiratory infections patients with these three pathogens should be compared with the identified VOCs in this study. Although there may be some differences between the results of in-vitro and in-vivo studies there seems to be significant similarities over the dominant detected VOCs.

Finding a non-invasive and rapid method for diagnosis of infectious agents is a subject of interest, so it has been investigated in several studies^33,42–45. The current study showed that using SPME fiber and GC-MS for extraction and detection of VOCs allowed detection of more specific VOCs for the three pathogenic respiratory tract organisms, E. coli, S. aureus and C. albicans, which could be used as biomarkers for their identification. It is essential that more comprehensive studies be conducted to create a more complete profile of VOCs for these organisms, and so that the methods can be developed further.

Data availability

The Xcalibur raw files for the three studied pathogens are available at https://doi.org/10.6084/m9.figshare.5178004.v1³².