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

Background: Diagnoses  of  respiratory  tract  infections  usually happen  in  the  late  phase  of  the  disease  and  usually  result  in  reduction  of  the  pathogen  load after broad-spectrum  antibiotic  therapy,  but  not  in eradication of the pathogen.  The  development  of a  non-invasive,  fast,  and  accurate  method  to  detect  pathogens  has  always  been  of  interest  to  researchers  and  clinicians  alike.  Previous studies have shown that bacteria produce organic gases.  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.
Methods: The  VOCs  produced  were identified by gas chromatography-mass spectrometry (GC-MS), with  prior  collection  of  microbial  volatile  compounds  using  solid  phase  microextraction  (SPME)  fiber.  The volatile compounds were collected by obtaining bacterial headspace samples.
Results: Results  showed  that  these  three  organisms  have  various  VOCs,  which  were  analyzed  under  different  conditions.  By ignoring common VOCs, some species-specific VOCs could be detected.  The most important VOC of E. coli was Indole, also some important VOCs produced by S. aureus  were 2,3-Pentandione,  cis-Dihydro-α-terpinyl  acetate,  1-Decyne,  1,3-Heptadiene-3-yne,  2,5-dimethyl  Pyrazine,  Ethyl  butanoate  and  Cyclohexene,4-ethenyl  furthermore,  most  of  identified  compounds  by  C.  albicans are  alcohols. Conclusions: The  detection  of  VOCs  produced  by  infectious  agents  maybe  the  key  to  make   a  rapid  and  precise  diagnosis  of  infection,  but  more  comprehensive  studies  must  be  conducted  in this  regard.

Introduction

Infectious diseases are the main reason for morbidity and mortality in developing countries, especially among children [1]. 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 ages [2]. Moreover, S. aureus is colonized in the nasopharynx in 10–35% of children, and in almost 35% of the adult population [3].

Escherichia coli is one of the most significant pathogens affecting preterm infants [4]. 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 period [5]. Candida albicans is an opportunistic pathogen and an agent of nosocomial infection [8].

Generally, the causative agents of respiratory tract infections are diagnosed in late phases of the disease [7]. 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 spreading [9]. Accurate and rapid detection of pathogens is a critical step for adequate treatment of infection [10]. and a non-invasive diagnostic method that has a high degree of accuracy needs to be developed [11].

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

Model organisms, medium and growth conditions

The bacterial strains used in this study were E. coli (ATCC 25922) and S. aureus (ATCC 25923), as gram-negative and gram-positive model organisms, and C. albicans (ATCC 10231) was used as a human pathogenic fungi model. These organisms model were obtained from the Microbiology Laboratory of Medicinal plants and Drugs Research Institute, Shahid Beheshti University. Monocultures of all strains were cultured 24 hours in nutrient agar, and then sub-cultured aerobically at 37°C in 30 ml of two different types of broth medium, Mueller Hinton broth (MB) and tryptic soy broth (TSB), in 100 ml sterilized glass bottles. For a more careful assessment of VOCs produced by each microorganism, the headspace was extracted from both media at three different time points: 2, 4 and 24 hours. To increase the possibility of VOC production, bottles containing cultured microorganism were shaken at 150 rpm during incubation time [28]. A suspension of microorganisms with approximately OD 600 ~0.5 in culture media was used during the headspace extraction [10], and the corresponding sterile broth mediums were used as the blank samples [29].

Headspace extraction

A solid phase microextraction (SPME) fiber holder (57330-U, Sigma-Aldrich) containing fiber coated with divinyl benzene/carboxen/poly dimethyl siloxane 50/30 µm (DVB/CAR/PDMS) (57328-U, Sigma-Aldrich) was used for absorption of volatile compounds from the headspace of pathogens. To provide conditions that increase the rate of VOC absorption, after incubation time, 2ml of NaCl 36% was added to each culture. Then the DVB/CAR/PDMS fiber was suspended from the top of the bottle containing the culture and placed on a magnetic stirrer hotplate at 70°C for 30 minutes [30]. After that, the fiber was placed at the injection site of GC-MS and all the absorbed VOCs entered the device. Eventually each VOC is represented as a chromatogram peak in the monitor that is connected to the GC-MS. For thermal desorption, the SPME fiber remained in the injector for 2 minutes before it was exposed to the headspace of the pathogen samples [31]. To avoid possible false discoveries each state was tested at least three times.

GC-MS

To study the bacterial VOCs, a Thermo-Finnigan Trace GC-MS system (Thermo Quest-Finnigan Co) equipped with a DB-5 column (60 m length, 0.25 mm inner diameter, and 0.25 μm film thickness) with helium carrier gas at a flow rate of 1.1 ml/min was used. The starting temperature was 50°C, increasing at a rate of 10°C/minute up to 250°C. The GC-MS was set in splitless mode and a quadrupole ion trap with ionization energy of 70 eV was used in the filament.

VOCs were identified using the National Institute of Standards and Technology (NIST) reference library. To analyze the GC-MS data, Xcalibur 3.0 with Foundation 3.0 SP2 software (Thermo Fisher Scientific) was used, and the kovats retention index (RI) was calculated for each chromatographic peak.

When calculating the RI, a series of standards were used: n-alkanes were injected into the GC-MS the day before starting experiments, using the same temperature profile that would be used for the analysis of VOCs. The NIST17 Mass Spectral Library (NIST7/2017/EPA/NIH) was used to identify each compound according to its RI. Since there may be several types of volatile compounds have similar RI, to validate the final results extensive studies were also performed by a phytochemist to determine if the compounds were organic. The common VOCs released from the sterile environment (Blank samples) and tests were not considered.

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.v1 [32].

One chromatogram of the six chromatograms obtained is displayed in 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.

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.

Table 1.

The identified VOCs for E. coli, S. aureus and C. albicans, and the percentage of the total area that their average peak covered (peak area %), after 2hours in MB and TSB media.

In total, 25 types of VOCs by E. coli, 33 types by S. aureus and 28 types by C. albicans were generated in this period.

Compound	E. coli in MB	E. coli in TSB	S. aureus in MB	S. aureus in TSB	C. albicans in MB	C. albicans in TSB
(e)-2-hexyl ester-butanoic acid	1.84	0.79	-	-	6.75	3.78
1-(1,5-dimethyl-4-hexyl-4-methyl-benzene	3.19	0.41	-	-	-	-
1,2-benzenedicarboxylic acid	-	-	0.39	-	-	0.2
1,2-butadiene	-	-	-	1.73	-	-
1,3-butadiene	-	-	-	-	-	26.68
1,3-heptadiene	-	-	-	4.88	-	4.55
1,5-decadiene	-	-	-	-	-	0.86
1,9-decadiene	0.05	0.05	-	-	-	0.39
1-decyne	-	0.07	0.85	-	1.55	1.55
1-penten-3-ol	-	0.02	-	5.14	-	-
2,3-pentandione	-	1.33	-	-	-	-
2,5-(1,1-dimethylethyl)-phenol	0.13	0.1	-	0.11	-	0.43
2,5-dimethyl pyrazine	-	-	-	20.19	-	3.07
2,6-bis(1,1-dimethylethyl)-4-methyl-phenol	-	0.04	0.5	-	-	0.64
2,6-dibutyl-2,5-cyclohexadiene-1,4-dione	0.03	-	-	-	-	-
2-ethenyl-6-methyl-pyrazine	1.1	0.63	6.58	6.63	-	3.63
2-ethyl hexanol	-	-	-	2.32	-	-
2-heptanone	0.05	-	-	2.31	-	-
2-hexan-1-ol	-	-	-	-	-	0.22
2-methyl-2-undecanethiol	0.24	0.13	1.98	1.09	-	-
3-methyl-1,5-heptadiene	-	-	-	-	3.77	1.03
3-propionyloxypentadecane	0.57	0.18	7.36	1.3	2	0.61
4-t-butyl-2-(1-methyl-2- nitroethyl)cyclohexane	0.93	0.73	12.17	5.4	10.98	2.45
5.5-dodecadinyl-1, 12-diol	-	-	0.48	0.5	1.19	6.38
allyl butylhydroquinone	-	-	-	0.31	-	-
anisol	-	0.05	-	1.19	-	-
benzaldehyde	2.13	1.34	3.22	8.98	-	0.64
benzene acetaldehyde	-	-	8.74	7.04	-	-
benzophenone	0.03	-	-	-	-	-
bisabolene	1.21	0.03	-	-	-	-
butyl cyclohexyl acetate	-	-	-	0.4	-	-
butyraldehyde	-	-	-	-	-	0.67
cadinene	-	-	-	-	-	1.79
carbamic acid	-	-	-	-	48.49	0.5
caryophyllene	-	-	-	0.09	-	-
cedran-1,8-diol	0.14	0.09	2.63	0.39	-	0.48
cedrol	-	-	0.71	0.23	-	-
copaene	0.01	-	-	-	-	-
cyclohexene, 4-ethenyl-	-	-	29.64	2.18	-	0.47
decanol	-	0.93	-	-	-	-
decene	-	-	-	2.85	-	-
dimethyl octenal	-	-	-	1.39	-	-
dimethyl ethyl cyclohexanol	-	-	-	1.04	-	0.59
dodecane	0.06	-	-	0.96	-	-
dodecanol	-	0.27	-	-	-	-
dodecenol	-	-	-	-	-	0.5
eicosane	-	-	-	-	-	0.12
ethyl butanoate	-	-	-	-	5.31	4.64
heptadecane	-	-	12.35	5.33	-	-
humulen	-	-	0.71	-	-	-
indole	82.61	90.97	-	0.48	-	-
limonene	0.68	-	-	-	-	-
longifolene	-	-	-	-	4.96	0.43
longifolene	-	-	-	0.52	-	-
methone	-	-	-	7.49	-	1.4
muurola-4,5-diene	0.47	-	-	-	-	-
naphthalenol	-	0.84	-	0.21	-	0.44
neryl acetate	0.06	0.03	-	-	-	-
nonadecanone	-	-	0.62	-	-	-
ocimene	-	-	-	-	-	2.24
octacosane	0.41	0.06	1.37	1.22	1.2	1.02
octyl acetate	-	-	-	-	-	0.4
pentadecane	0.03	-	0.86	0.68	-	4.1
phthalic acid, butyl ester	0.19	0.13	0.97	-	-	0.22
phenyl ethyl pyrrole	-	0.06	-	-	-	-
sesquiphellandrene	1.1	-	-	-	-	4.79
tetra butyl cyclohexyl acetate	-	-	1.7	-	-	-
tetradecane	0.01	-	-	-	-	-
tetradecanol	-	-	-	-	-	0.34
zingiberene	1.63	-	1.03	-	2.87	7.91
α-acetoxydihydrocoumarin	-	0.52	1.88	0.25	-	-
β-santalol	-	-	-	-	4.87	1.34

Table 2.

The identified VOCs for E. coli, S. aureus and C. albicans, and the percentage of the total area that their average peak covered (peak area %), after 4 hours in MB and TSB media.

In total, 9 types of VOCs by E. coli, 19 types by S. aureus and 42 types by C. albicans were generated in this period.

Compound	E. coli in MB	E. coli in TSB	S. aureus in MB	S. aureus in TSB	C. albicans in MB	C. albicans in TSB
(e)-2-hexyl ester-butanoic acid	-	-	-	-	6.64	4.24
(z)-2-octene-1-ol	-	-	-	-	0.69	0.54
(z)-4-decan-1-ol	-	-	-	-	0.70	0.46
1,2-benzenedicarboxylic acid	-	-	-	-	0.29	0.31
1,2-butadiene	-	-	0.37	4.02	-	-
1,3-butadiene	-	-	-	-	1.40	-
1,3-heptadiene	-	-	81.33	-	0.23	0.47
1,5-decadiene	-	-	-	-	1.01	3.00
1,9-decadiene	-	0.01	-	-	0.20	3.59
1-decyne	2.36	-	0.59	16.47	2.81	1.40
1-methoxy-2-propanol	-	-	0.20	-	-	-
2-(phenylmethylene)-octanal	-	-	-	-	1.55	1.63
2,3-pentandione	-	-	7.07	21.67	-	-
2,5-(1,1-dimethylethyl)-phenol	-	-	-	-	0.69	0.43
2,5-dimethyl pyrazine	-	-	-	-	-	3.48
2-acetyl-1-pyrroline	-	0.07	-	-	-	-
2-ethenyl-6-methyl-pyrazine	0.01	-	0.80	-	7.33	-
2-ethyl hexanol	-	-	-	1.05	-	-
2-heptanone	0.02	0.33	-	-	-	-
2-hexan-1-ol	-	-	-	-	0.33	0.23
2h-tetrazole-5-carboxylicacid, 2-phenyl	-	-	0.48	-	1.44	1.59
2-methyl-2-undecanethiol	-	-	0.24	-	-	-
3-methyl-1,5-heptadiene	-	-	0.60	-	0.76	0.53
4-t-butyl-2-(1-methyl-2- nitroethyl)cyclohexane	-	-	-	-	6.05	5.81
5.5-dodecadinyl-1, 12-diol	-	-	-	-	5.16	0.51
6-methyl-5-hepten-2-one	-	-	-	-	0.31	0.20
benzaldehyde	-	-	-	-	0.83	0.68
butyraldehyde	-	-	0.38	-	-	-
cadinene	-	-	-	-	0.79	0.34
carbamic acid	-	-	-	-	18.10	8.19
caryophyllene	-	0.02	-	4.88	-	-
cedrol	-	-	-	-	0.36	0.36
cis-dihydro-α-terpinyl acetate	-	-	-	17.90	-	-
cyclohexene, 4-ethenyl-	-	-	0.08	6.77	-	-
dimethyl octenal	-	-	-	-	0.58	-
dimethylethyl cyclohexanol	-	-	-	-	0.79	0.22
dodecenal	-	-	-	-	0.52	0.39
dodecenol	-	-	-	-	0.61	2.54
eicosane	-	-	-	-	-	0.29
ethyl butanoate	0.01	-	7.57	12.21	6.63	0.73
indole	97.05	99.46	-	0.82	0.34	-
levomenthol	-	-	-	3.74	-	-
longifolene	-	-	-	-	0.31	0.26
longifolol	-	-	-	-	5.66	22.07
methyl isopropyl hexenal	-	-	-	-	1.11	0.59
naphthalenol	-	-	-	-	-	0.37
octacosane	-	-	-	-	1.74	1.60
octyl acetate	-	-	-	-	0.34	-
pentadecane	-	-	-	0.87	1.80	1.38
phthalic acid, butyl ester	-	-	-	-	0.60	0.36
tetradecanol	-	-	-	-	0.61	0.26
tridecanol	-	-	-	-	0.34	0.34
zingiberene	-	-	-	-	1.27	1.12
β-santalol	-	0.03	-	5.16	6.52	14.15
sesquiphellandrene	-	-	-	-	2.00	0.70

Table 3.

The identified VOCs for E. coli, S. aureus and C. albicans, and the percentage of the total area that their average peak covered (peak area %), after 24 hours in MB and TSB media.

In total, 16 types of VOCs by E. coli, 26 types by S. aureus and 27 types by C. albicans were generated in this period.

Compound	E. coli in MB	E. coli in TSB	S. aureus in MB	S. aureus in TSB	C. albicans in MB	C. albicans in TSB
(e)-2-hexyl ester-butanoic acid	0.04	0.03	0.21	0.04	0.50	-
(z)-2-octene-1-ol	-	-	-	-	-	0.18
(z)-4-decan-1-ol	-	-	-	-	-	0.60
1,2-benzenedicarboxulic acid	-	-	-	-	0.13	0.28
1,2-butadiene	-	-	3.71	0.10	-	-
1,3-heptadiene	-	-	21.75	-	-	-
1,5-decadiene	-	-	-	-	0.10	0.26
1,9-decadiene	0.02	0.03	-	-	-	-
1-decyne	-	0.02	0.81	59.78	-	0.75
1-methoxy-2-propanol	-	-	6.74	0.02	-	-
2-(phenylmethylene)-octanal	-	-	-	-	0.71	0.86
2,3-pentandione	0.03	0.48	15.53	0.66	-	-
2,5-dimethyl pyrazine	-	-	0.62	-	-	0.55
2-acetyl-1-pyrroline	-	6.37	-	1.11	-	-
2-decenal	-	-	0.06	0.01	-	-
2-ethenyl-6-methyl-pyrazine	0.06	-	1.35	-	0.39	-
2-ethyl hexanol	-	-	0.13	0.02	-	0.51
2-heptanone	-	0.21	-	-	-	-
2h-tetrazole-5-carboxylicacid, 2-phenyl	-	-	3.21	-	-	-
2-methyl tetradecane	0.05	0.03	-	-	-	-
2-methyl-1-propanol	-	-	0.15	0.04	5.89	16.03
2-methyl-2-undecanethiol	0.10	-	2.54	-	-	61.65
2-octyl-1-ol	-	-	-	-	0.15	-
2-octyne	-	-	-	-	-	0.27
3-methy-4-pentene-3-ol	-	-	-	-	-	0.13
3-methyl-1,5-heptadiene	-	-	0.19	-	-	-
3-methyl-1-pentene	-	-	-	-	-	0.41
-4-t-butyl-2-(1-methyl-2- nitroethyl)cyclohexane	-	-	-	-	80.87
5.5-dodecadinyl-1, 12-diol	0.01	0.03	0.77	0.05	-	-
butyraldehyde	-	-	1.41	0.09	0.14	0.24
carbamic acid	-	-	-	-	0.48	-
caryophyllene	-	0.21	-	0.14	-	1.25
cedrol	-	-	-	-	1.93	2.68
cis-dihydro-α-terpinyl acetate	-	0.64	-	34.77	-	-
cyclohexene, 4-ethenyl-	-	-	3.54	0.15	-	-
ethyl acetoacetate	-	-	-	-	-	0.46
ethyl butanoate	0.06	0.31	28.72	0.46	-	1.64
indole	99.61	88.86	0.07	0.02	-	-
levomenthol	-	-	-	1.66	-	-
longifolol	-	-	-	-	0.33	0.24
octacosane	-	-	-	-	0.33	0.44
pentadecane	-	0.35	0.17	0.08	-	-
thiophene	-	-	0.18	-	-	-
zingiberene	-	-	-	-	0.20	-
β-santalol	-	0.49	-	0.21	-	1.21

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).

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.

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 compounds [36]. 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 environment [11].

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 metabolism [41].

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.v1 [32].

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

I would like to thank the authors for their response. Although most questions are answered, one question remains open to me:

- Considering they have perfomed their tests in triplicate, which statistical test did the authors use to compare outcomes between and within blank and actual samples?

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

The paper shows some interesting preliminary results. The authors should be complimented for their good English. The works cries out for more repeats and the authors appreciate this. Within the abstract, and in the text throughout, chemical names should not have capital letters, more importantly some of the compound names need checking, particularly 1,3-heptadiene-3-yne which is in the abstract, it cannot be right.

Also check in tables, spelling and also are you sure:

1,2-Benzene dicarboxulic acid, this should be 1,2-benzenedicarboxylic acid

1,3-Butadiyene, do you mean 1,3-butadiene?

Likewise 1,2-Butadiyene ?

1,3-Heptadiene-3-yne? Can’t be

Dibutyl phatalate should be phthalate,  also its other name is 1,2-Benzenedicarboxylic acid, dibutyl ester. Best to be consistent with names, I only draw attention to this  case as an example, because 1,2-benzene dicarboxylic acid has been stated to be found, and when they have the same naming, its brings out the thought that the acid may have come from the ester (I say this, rather than the other way around as the butyl ester is a common plasticizer, and can be a contaminant).

Longifolrne spelling! And it appears just below, they are the same? Do they have the same rts?

Longifolene

Phatalic acid, butyl ester, spelling

Sesquiphellandrene  and beta- Sesquiphellandrene, are both listed are they the same?

Table 2,3

Check as above.

There are some compounds, which may be speculatively  assigned, eg 4-t-butyl-2-(1-methyl-2- nitroethyl)cyclohexane? Are the authors really sure

Supplementary : E. coli:5.5-Dodecadinyl-1, 12-diol, can’t be right, check, plus check names of chemicals as above

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

The researchers tackle an area of significant interest, rapid determination of bacterial species, especially associated with life threatening illness, using a relatively new approach, that of VOC analyses. In general the paper reads well.

The analytical method for VOC analyses is ok. Could the authors confirm whether the cfu/ml count is approx. for the different species at the same time points to enable a good comparison of VOCs to be made.  One major weakness to the work is that only one analysis for each species at one time point and media was undertaken, the literature gives examples of several analyses being undertaken for similar studies. The title is too ambitious, rather than diagnoses being purported, maybe an” initial study of…” would be more accurate.

The authors could comment on how their research would ultimately fit into a clinical test, if the VOCs were to be analysed in breath, would the same volatile profile be expected?

In the text it was stated that “Extensive studies were also performed by a phytochemist to determine if the compounds were organic” , some explanation would be good as to what this means.

Some other matters, VOC abbreviation used twice in the abstract, also in quite a few places the chemical names have capital letters and are misspelt and merged with another word.

In Results, what is uncommon determined by, some more discussion on stats…

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

This paper looks at the detection of VOC's produced by Escherischia coli, Staphylococcus aureus and Candida albicans using solid phase micro-extraction coupled with gas chromatography / mass spectrometry.

Its seems as though the group only ran this experiment once so while they have pointed out the detection of specific volatiles at differing time points this needs to be repeated to ensure that it is indeed a true and repeatable release from the micro-organisms.

The work is outlined clearly and the results listed comprehensively however there has been no indepth discussion surrounding why these volatiles have been produced in vitro, would they be produced in vivo nor an extensive list of references to back this up.

While they state Indole as the most important volatile produced by E.coli they do not go into any detail regarding how this would relate to an actual breath test since indole can be released by mouth flora. Also as indole is produced from tryptophan discussion around the levels of tryptophan in the lung and therefore its actual availability to E.coli growing in the lung is pertinent. Growth in a minimal media with varying tryptophan levels would give more insight.

It would have also been beneficial to look at the effect of co-culturing these micro-organisms as this may alter the release of the detected VOC's and initiated the production of others.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

General Comments:

This study aimed to identify typical volatile organic compounds (VOCs) produced by Staphylococcus aureus, Escherichia coli and Candida albicans by extraction of cultured bacterial strain headspace using solid phase microextraction (SPME) fibers analyzed by Gas Chromatography-Mass Spectrometry (GC-MS). Although the aim of study is obvious and very relevant data is shown, some extension on utilized statistics is needed. Additionally, a replication and/or validation of findings is crucial. The authors do show that some of the VOCs, e.g. Indole, are revealed at multiple time points, but confirmation of observations by repetition of experiments is recommended.

Major Comments:

To avoid possible false discoveries a repetition of experiments is recommended.

Which (statistical) methods were used in order to compare blank vs. actual samples?

Which criteria were applied in order to confirm a ‘match’ between detected volatiles and the NIST library?

The authors nicely show the overlap and/or discrepancy between their findings and current available literature, but what about the interpretation of findings? Can the authors extend and/or speculate about possible biological mechanisms behind the revealed VOCs

Minor comment:

Please consider as reference: Neerincx et al; Identification of Pseudomonas aeruginosa and Aspergillus fumigatus mono- and co-cultures based on volatile biomarker combinations; J Breath Res. 2016 Jan 29;10(1):016002.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.