saranya ppr

1 23
Applied Microbiology and
Biotechnology
ISSN 0175-7598
Volume 99
Number 10
Appl Microbiol Biotechnol (2015)
99:4423-4433
DOI 10.1007/s00253-015-6573-6
Simple and specific colorimetric detection
of Staphylococcus using its volatile 2-[3-
acetoxy-4,4,14-trimethylandrost-8-en-17-
yl] propanoic acid in the liquid phase and
head space of cultures
Raju Saranya, Raju Aarthi & Krishnan
Sankaran

1 23
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METHODS AND PROTOCOLS
Simple and specific colorimetric detection of Staphylococcus
using its volatile 2-[3-acetoxy-4,4,14-trimethylandrost-8-en-17-yl]
propanoic acid in the liquid phase and head space of cultures
Raju Saranya1
& Raju Aarthi1
& Krishnan Sankaran1
Received: 15 November 2014 /Revised: 13 March 2015 /Accepted: 24 March 2015 /Published online: 22 April 2015
# Springer-Verlag Berlin Heidelberg 2015
Abstract Spread of drug-resistant Staphylococcus spp. into
communities pose danger demanding effective non-invasive
and non-destructive tools for its early detection and surveil-
lance. Characteristic volatile organic compounds (VOCs) pro-
duced by bacteria offer new diagnostic targets and novel ap-
proaches not exploited so far in infectious disease diagnostics.
Our search for such characteristic VOC for Staphylococcus
spp. led to the depiction of 2-[3-acetoxy-4,4,14-
trimethylandrost-8-en-17-yl] propanoic acid (ATMAP), a
moderately volatile compound detected both in the culture
and headspace when the organism was grown in tryptone soya
broth (TSB) medium. A simple and inexpensive colorimetric
method (colour change from yellow to orange) using methyl
red as the pH indicator provided an absolutely specific way for
identifying Staphylococcus spp., The assay performed in liq-
uid cultures (7-h growth in TSB) as well as in the headspace of
plate cultures (grown for 10 h on TSA) was optimised in a 96-
well plate and 12-well plate formats, respectively, employing
a set of positive and negative strains. Only Staphylococcus
spp. showed the distinct colour change from yellow to orange
due to the production of the above VOC while in the case of
other organisms, the reagent remained yellow. The method
validated using known clinical and environmental strains (56
including Staphylococcus, Proteus, Pseudomonas, Klebsiella,
Bacillus, Shigella and Escherichia coli) was found to be
highly efficient showing 100 % specificity and sensitivity.
Such simple methods of bacterial pathogen identification are
expected to form the next generation tools for the control of
infectious diseases through early detection and surveillance of
causative agents.
Keywords Staphylococcus . Colorimetric . Methyl red .
Surveillance . Nosocomial infection
Introduction
Infectious diseases and their severity due to drug-resistant
pathogens are emerging threats to health care and economy
(Anne 2006). Emerging infectious diseases cause severe epi-
demics or pandemics with their wide spectrum of infections.
Hence, surveillance becomes essential as a measure to control
pathogens and prevent emergence of their resistance to potent
drugs. This is especially applicable for highly populated coun-
tries like India (Vincent Ki et al. 2008). The existing protocols
and instruments for field detection and identification of such
pathogens are low throughput, time-consuming and demand
technical skill to serve as effective surveillance tools
(Guernion et al. 2001).
Despite major advances in the medical arena,
Staphylococcus spp. (Kloos and Schleifer 1986) are a chal-
lenge for modern day medicine due to the complexity of dis-
ease process, presence and expression patterns of their respec-
tive virulence factors. Staphylococcal infections can range
from a minor boil or skin abscess to life-threatening infections
such as septicaemia or endocarditis. This bacterium is respon-
sible for invasive infections including bacteremia related to
intravenous catheters, pneumonia and ventriculitis or menin-
gitis in the neonate. Problematically, methicillin-resistant
Staphylococcus aureus (MRSA) has become a major cause
Electronic supplementary material The online version of this article
(doi:10.1007/s00253-015-6573-6) contains supplementary material,
which is available to authorized users.
* Krishnan Sankaran
ksankran@yahoo.com
1
Centre for Biotechnology, Anna University, Sardar Patel road,
Guindy, Chennai 600 025, India
Appl Microbiol Biotechnol (2015) 99:4423–4433
DOI 10.1007/s00253-015-6573-6
Author's personal copy

of hospital-acquired infections (Gomez et al. 2007). It is being
recognised with increasing frequency in community-acquired
infections, which is a dangerous trend. Therefore, develop-
ment of rapid and sensitive technique for early detection
(Marlowe and Bankowski 2011), screening, and preventive
surveillance of Staphylococcus spp. is very important for ef-
fective treatment.
Conventional methods of bacteriological testing
employing time-consuming microbiological culturing
and bacterial isolation are the reasons why alternative
detection and identification technologies are needed
(Velusamy et al. 2010). Modern and sophisticated
methods are not found favourable for clinical diagnosis
due to either high cost, or being laborious or requiring
special skills (Kohne et al. 1984). Recent developments
in biosensors (sensitivity panels, arrays, electronic nose)
provide rapid detection but these sensors are not devel-
oped for pathogen detection (Prakash and Saxena 2013).
These limitations and the necessity for constant surveil-
lance prompted us to develop an appropriate methodol-
ogy to detect Staphylococcus spp. (Kelechi et al. 2011).
Volatile organic compounds (VOCs) released by bac-
teria are either unique or characteristic under certain
experimental growth conditions (Senecal et al. 2002).
In recent years, VOCs raise a growing interest since
they are measurable through non-invasive and non-
destructive methods that can be used for early diagnos-
tics and continuous surveillance. Though bacterial
VOCs include carbonyl compounds, carboxylic acids,
amines, amides and sulphur compounds, only specific
subset is released by particular bacteria under specified
growth conditions. In other words, VOC signature or
biomarkers VOC are distinct possibilities remaining un-
exploited (Carey et al. 2011). Specific release of VOC
in appropriate growth conditions is the novel approach
taken in this study. However, we have used this novel
approach to develop rapid detection of Staphylococcus.
Identification of the VOCs released from in vitro bac-
terial cultures is performed in the laboratories using gas
chromatography (GC) (Boots et al. 2014), GC coupled
with mass spectrometry (Tait et al. 2013), proton-transfer
reaction mass spectrometry (Bunge et al. 2008), and se-
lected ion flow tube coupled with mass spectrometry
(Allardyce et al. 2006). These methods fail and are not
suitable for clinical practice or environmental testing for
obvious reasons, thus failing in effective surveillance.
Adapting simpler colorimetric methods employing re-
agents like acid-base indicators provides a cost-effective and
high-throughput detection method and inexpensive diagnostic
tool for effective surveillance. Therefore, after designing con-
ditions for specific release of a carboxylic compound, we have
developed a rapid and inexpensive colorimetric assay for de-
tecting Staphylococcus species.
Our laboratory investigations of waste dump sites (includ-
ing hospital wastes) clearly showed the predominance of
Staphylococcus. Therefore, we chose to study this organism
for VOC-based detection, in line with what was developed for
Proteus (Aarthi et al. 2014), but focusing on colorimetric for
better field deployment.
Materials and methods
Collection and identification of strains
Standard strains Strains of Salmonella paratyphi (MTCC
3220); Salmonella enterica sub spp. (MTCC 3231);
Salmonella typhimurium (MTCC 3224); Escherichia coli
(MTCC 568, MTCC 901, MTCC 723, MTCC 443);
Staphylococcus aureus (MTCC 3160, MTCC 6908);
Staphylococcus epidermidis (MTCC 435); Staphylococcus
chromogenes (MTCC 6153); Klebsiella pneumoniae
(MTCC 2653, MTCC 661); Proteus mirabilis (MTCC 1429,
MTCC 425); Proteus vulgaris (MTCC 1771); Staphylococcus
haemolyticus (MTCC 8924); Klebsiella oxytoca (MTCC
2275); Pseudomonas aerunginosa (MTCC 424, MTCC
1934); Shigella flexneri (MTCC 1457, MTCC 9543);
Streptococcus pneumoniae (MTCC 655); Streptococcus
thermorphilus (MTCC 1938); Listeria monocytogenes
(MTCC 839, MTCC 1143); Enterobacter aerogenes
(MTCC 111); Lactobacillus fermentum (MTCC 903);
Lactobacillus acidophilus (MTCC 447); Bacillus subtilis
(MTCC 1790) and Streptococcus pyogenes (MTCC 1927)
were obtained from Microbial Type Culture Collection
(MTCC), Chandigarh, India. Escherichia coli (ATCC
25922), Bacillus cereus (ATCC 21769), Staphylococcus
aureus (ATCC 25923), Shigella flexneri (ATCC 29508),
Proteus mirabilis (ATCC 29906),Proteus vulgaris (ATCC
6380), Klebsiella pneumoniae (ATCC 13883) and
Pseudomonas aerunginosa (MTCC-27853) were obtained
from Sri Ramachandra University, Chennai, Tamil Nadu,
India.
Clinical isolates Clinical diarrheagenic Escherichia coli
(EPEC) strains were isolated from stool samples of
children, who were hospitalised with acute or persistent
diarrhoea at the Institute of Child Health (IHC) and
Hospital for Children (HC), Chennai, Tamil Nadu,
India. Salmonella typhimurium strain was obtained
from Sri Ramachandra University, Chennai, and
uropathogens such as uropathogenic Escherichia coli
(UPEC), Klebsiella, Proteus, Pseudomonas aeruginosa,
Citrobacter and Staphylococcus aureus were obtained
from M/s Trivitron Healthcare Ltd., Chennai, Tamil
Nadu, India.
4424 Appl Microbiol Biotechnol (2015) 99:4423–4433

Environmental samples Escherichia coli, Pseudomonas
aerunginosa, Staphylococcus aureus, Proteus and Bacillus
were isolated from soil in different places including hospital
wastes. Environmental strains were collected from three dif-
ferent areas including Madipakkam (hospital waste),
Pallikaranai (domestic waste) and Taramani (laboratory
waste) located within 15 km from our laboratory. Around
10 g of soil samples were collected from each location by
digging the ground approximately 6 in.. From this, 1 g of
the soil was suspended in 10 ml of sterile distilled water and
was serially diluted and plated onto Luria-Bertani (LB) agar
plates. Colonies with different morphology (size, colour, tex-
ture etc.) visually were picked and identified by standard mi-
crobiological (colony morphology, gram staining and growth
on differential medium), and biochemical (methyl red/Voges-
Proskauer (MR/VP) test, urease, catalase, triple sugar iron,
phenylalanine deaminase test) tests.
Preparation of nutrient broth
Nutrient broth (NB) was prepared by dissolving 5 g of peptic
digest of animal tissue (HiMedia, India), 1.5 g of beef extract
(HiMedia, India), 1.5 g of yeast extract (HiMedia, India) and
5 g of NaCl (Merck, India) in 1 L of distilled water, after the
pH was adjusted to 7.3 with 1 M sodium hydroxide, the broth
was autoclaved at 15 lbs pressure for 20 min.
Preparation of Luria-Bertani broth
Luria-Bertani (LB) broth was prepared by dissolving 10 g of
tryptone (HiMedia, India), 5 g of yeast extract (HiMedia,
India), and 10 g of NaCl (Merck, India) in 1 L of distilled
water, and the pH was adjusted to 7.2 with 1 M sodium hy-
droxide (HiMedia, India) and autoclaved at 121 °C and 15 lbs
pressure for 20 min.
Preparation of soya bean casein digest medium Soya bean
casein digest medium or tryptone soya broth (TSB) (HiMedia,
India) was prepared by dissolving 30 g of the powder in 1 L of
distilled water and the pH was adjusted to 7.25 with 1 M
sodium hydroxide (HiMedia, India) and autoclaved at
121 °C and 15 lbs pressure for 20 min.
Preparation of modified soya bean casein digest
agar Tryptone soya broth agar (TSA) was prepared by dis-
solving 30 g of soya bean casein digest medium or tryptone
soya broth (TSB) (HiMedia, India) with 2 % NaCl and 2 %
agar (HiMedia, India) in 1 L of distilled water and the pH was
adjusted to 7.25 with 1 M sodium hydroxide (HiMedia, India)
and autoclaved at 121 °C and 15 lbs pressure for 20 min.
Dye reagent specific for Staphylococcus The colorimetric
dye, methyl red (M/s Merck), was chosen for the study. The
dye was prepared by dissolving 0.01 g of 2-(N,N-Dimethyl-4-
aminophenyl)azobenzenecarboxylic acid (also called C.I.
Acid Red 2, commonly known as methyl red) in 100 ml of
distilled water. When the dye was directly added to the culture,
the distinct colour change from yellow to orange could be
visualised instantly. For testing, 50 μL of the dye was mixed
with 200 μL of sample in 96-well microtitre plate.
Standardisation of colorimetric assay To standardise, the
assay was performed after 7 h of growth of bacterial strains.
Absorbance was read at 462 nm using multiscan reader (mod-
el: Enspire, PerkinElmer, USA) and the same plate was im-
aged using a camera (Canon SX160 IS) for visualisation.
Extraction of volatile organic compounds (VOCs) from
culture To 1 ml of sterile TSB medium contained in 2-mL
centrifuge tube, 20 μL (105
cells) of each organism were in-
oculated separately. After incubation in a rotary shaker set at
37 °C and 120 rpm for 7 h, 1 mL of chloroform or dichloro-
methane (DCM) or ethyl acetate, was added and vortexed for
1 min to extract the VOCs. The solvent phase was collected
and analysed using GC-MS and FT-IR.
Comparison of Staphylococcus cultures in NB, LB
and TSB
The methyl red assay for detecting Staphylococcus spe-
cies was performed in NB, LB and TSB. Each well was
filled with 180 μL of medium and 20 μL of 105
cells
of the test strains were inoculated. The plate was incu-
bated at 37 °C and 100 rpm for 7 h in an orbital
shaker. The optical densities (600 nm) of bacterial cul-
tures were measured after 7 h using Multiscan reader
(Thermo, Finland) and then the methyl red assay was
performed by adding 50 μL of the 0.01 % dye solution.
The absorbance was measured at 462 nm immediately
using the plate reader and the plates were also imaged.
Similarly as mentioned above, DCM extract of
Staphylococcus cultures in NB, LB and TSB were pre-
pared. The solvent phase was collected and analysed
using gas chromatography (GC).
Purification of VOC from Staphylococcus
Dichloromethane fraction was purified by column chromatog-
raphy using chloroform/methanol (60:40) as eluent. It was
checked on thin layer chromatography (TLC) and showed a
single spot and also confirmed the compound of interest by
methyl red staining. Chromatographic developments were
carried out in a glass beaker. The percentage composition of
the solvent system was acetone/dichloromethane/ethanol
(6:60:10); 0.5 μL of TSB medium (negative control) and pu-
rified and crude samples of Staphylococcus extract culture
Appl Microbiol Biotechnol (2015) 99:4423–4433 4425

components reacted with methyl red (0.01 %) were spotted on
chromatogram and dried. The development of the chromato-
gram was allowed to proceed until the solvent had travelled 6–
7 cm beyond the starting line. The chromatogram of purified
and crude produced single spot.. The plates were removed
from the chamber and allowed to dry in air. Then, it was
subjected to gas chromatography-mass spectrometry (GC-
MS) analysis.
Gas chromatography-mass spectroscopy (GC-MS)
analysis An Agilent 6890 N GC system with Jeol
(Turbovac) mass selective detector was used for analysis of
all the extracts. GC separation was achieved using HP-5 ms
capillary columns (30 m×0.25 mm i.d., film thickness
0.25 μm) using Helium as a carrier gas. The GC system was
programmed in the following temperature mode: initial tem-
perature 60 °C, hold 1 min, linear ramp 10 °C per minute to
180 °C and then ramped at 4 °C per minute to 300 °C and held
for 15 min. The MS detection was carried out in full scan
mode, and used the mass-to-charge ratio (m/z) of 50–650 with
a cycle time of 2.28 scans s−1
and EI ionisation of 70 eV. The
ion source temperature was 230 °C with the interface temper-
ature of 180 °C. The event time and solvent cut time were
14.66 s and 1.5 min, respectively. Identification was based
on comparing the mass spectra of the chromatographic peaks
with those reported in the mass spectral library.
Fourier transform-infrared (FT-IR) analysis
The FT-IR vibrational spectra of the solvent extracts were read
using a Thermo Nicolet IR-100 spectrometer (make:
ThermoNicole Corporation, Madison). IR spectrum was re-
corded by introducing the sample into the IR path. The spec-
trum was taken from 450 to 4000 cm−1
with a resolution of
1 cm−1
.
Colorimetric assay for detection of Staphylococcus
species The methyl red assay for detecting the acidic com-
pound released by Staphylococcus species was performed in
a 96-well plate. Each well was filled with 180 μL of TSB
medium and 20 μL of 105
cells of the test strains were inoc-
ulated. The plate was incubated at 37 °C and 100 rpm for 7 h
in an orbital shaker. The optical densities (600 nm) of bacterial
cultures were measured after 7 h using Multiscan reader
(Thermo, Finland). The liquid culture had approximately 109
cells after 7 h growth and then the Methyl red assay was
performed by adding 50 μL of the 0.01 % dye solution. The
absorbance was measured at 462 nm immediately using the
plate reader and the plates were also imaged. To profile VOC
release with respect to time, the assay was performed every 1 h
of bacterial growth. A quantitative estimation of the VOC in
the culture at different time point (from the fourth hour) was
obtained using the standard graph.
Testing the volatility of the volatile metabolite
from culture
To check whether the target of the assay is a volatile acid released
by the bacteria, the assay plate was incubated open at room
temperature (≈27 °C), on ice, and 60 °C, assay was performed
every 15 min up to 2 h.
Laboratory validation
After optimising and testing the assay conditions, a set of 95
strains including 39 standard and 56 known clinical and envi-
ronmental strains representing frequently encountered patho-
gens (Table 1) were validated. The experiment was repeated
twice, and the absorbance is given in Table 1.
Headspace analysis of volatile organic compound
Bacteria were plated during log phase growth on tryptic soy agar.
Bacterial suspensions were prepared by inoculating 5 mL of
TSB with a single colony and allowing it to grow overnight.
From this, 10 μL of 105
cells of the culture was spread onto a
60-mm TSA Petri dish. The plate culture was a lawn after 10-h
incubation of 105
cells spread on the plate. A control (10 μL of
TSB without the bacterial inoculum) was included in parallel for
each experiment. Silica-coated discs (TLC, Merck) was inserted
into the lid of the Petri dish. The Petri dish was closed and
housed in an incubator at 37 °C for 10 h and after incubation,
5 μL of methyl red dye was added in silica-coated discs and the
change in colour was observed. The same was adapted in 12-
well plate with 1 ml of TSA for better field deployment.
Sensitivity and specificity calculation and the confidence
level
Sensitivity and specificity of the assay was calculated using
the formula, sensitivity=[a/(a+c)]×100 and specificity=[d/
(b+d)]×100, where a is true positive, b is false positive, c is
false negative and d is true negative (Abdul and Anthony
2008). When the growth (OD) of the strains was similar, the
99 % confidence for the positive (Staphylococcus) and nega-
tives were calculated using the formula.
X Æ 2:58 δ=
ffiffiffi
n
pÀ Á
where X is sample mean, δ is population standard deviation
and n is sample size (Jose 2009).
Results
Developing a colorimetric method based on VOC was our
aim. Staphylococcus spp. was our interest as it is a common

Table1Validationofassayusingstandard,clinicalandenvironmentalstrains
Wellno.OrganismAbsorbance(a.u.)Wellno.OrganismAbsorbance(a.u.)Wellno.OrganismAbsorbance(a.u.)
Trial1Trial2Trial1Trial2Trial1Trail2
A1Mediumblank00C9P.aeruginosa(273)#
−0.012−0.021F5S.aureus(3160)*0.7120.756
A2E.coli(ATCC25922)0.0010.002C10P.mirabilis(328271)*−0.0080.006F6P.aeruginosa(326604)*0.0110.006
A3S.aureus(256)#
0.6880.712C11K.pneumoniae(MTCC661)0.0150.010F7P.aeruginosa(121602592)*−0.009−0.003
A4P.mirabilis(122101203)*0.0010.008C12Shigellaflexneri(MTCC1457)0.0010.015F8Bacilluscereus(ATCC21769)0.0100.008
A5E.coli(21748)*0.0030.001D1P.vulgaris(121103217)*0.012−0.005F9S.typhimurium(327753)*0.0050.006
A6K.pneumoniae(MTCC2653)−0.0050.014D2P.aeruginosa(MTCC1934)0.0030.009F10S.typhimurium(328897)*0.0060.007
A7E.coli(25922)*0.0080.011D3S.flexneri(MTCC9543)0.0010.002F11S.aureus(251)#
0.6950.686
A8P.mirabilis(5155)−0.0020-001D4S.aureus(MTCC6908)0.7560.732F12S.typhimurium(121703058)*0.0210.009
A9P.aeruginosa(MTCC424)−0.005−0.021D5S.chromogenes(MTCC6153)0.6780.659G1S.typhimurium(MTCC3224)0.0110.016
A10P.mirabilis(3401488)*0.0020.008D6S.pyogenes(MTCC1927)0.0140.017G2Enterobacter(14736)*0.0050.012
A11P.aeruginosa(MTCC27853)−0.012−0.004D7S.enterica(MTCC3231)0.0110.001G3S.aureus(ATCC25923)0.7310.756
A12S.aureus(253)#
0.6540.692D8P.mirabilis(15322)*−0.010−0.021G4S.enterica(3231)*0.0010.008
B1E.coli(340)#
0.0060.004D9Streptococcusthermorphilus(MTCC1938)0.0080.002G5P.mirabilis(881)#
0.0080.004
B2E.coli(111406070)*0.0030.009D10Listeriamonocytogenes(MTCC1143)0.0010.005G6Citrobacter(24361)*0.0120.006
.B3P.mirabilis(12210120)*0.0010.015D11E.coli(MTCC901)0.0100.012G7Citrobacter(328327)*0.0060.008
B4L.monocytogenes(MTCC839)0.0210.014D12Bacillussubtilis(MTCC1790)0.0210.011G8S.aureus(23160)*0.6850.726
B5S.aureus(MTCC3160)0.7030.712E1S.haemolyticus(MTCC8924)0.7240.689G9E.coli(21595)*0.0260.052
B6E.coli(318233)*0.0010.004E2P.mirabilis(813)#
0.0050002G10P.mirabilis(MTCC425)0.0090.007
B7E.coli(318560)*0.0100.014E3Lactobacillusacidophilus(MTCC447)−0.014−0.006G11E.coli(121201233)*0.0040.011
B8Streptococcuspneumoniae(MTCC655)0.0010.004E4E.coli(304)#
0.0030.009G12P.mirabilis(853)#
−0.011−0.013
B9P.mirabilis(MTCC1429)−0.014−0.006E5E.coli(308)#
0/0010.003H1S.flexneri(ATCC29508)0.0030.009
B10S.epidermidis(MTCC435)0.6880.672E6P.mirabilis(981447)*0.0030.009H2S.paratyphi(MTCC3220)0.0040.008
B11P.mirabilis(803)#
0.0020.010E7Lactobacillusfermentum(MTCC903)0.0080.015H3S.enterica(4231)0.0140.008
B12E.coli(318253)#
0.0020.001E8E.coli(350148)*0.0060.008H4P.mirabilis(ATCC29906)−0.006−0.004
C1P.mirabilis(487)*0.0070.011E9P.mirabilis(494750)*0.0090.007H5E.coli(MTCC723)0.0110.012
C2E.coli(318304)#
0.0200.011E10E.coli(320488)*0.0060.009H6E.coli(MTCC443)0.0210.018
C3E.coli(MTCC568)0.0050.001E11E.coli(320923)*0.0050.004H7E.coli(ATCC13534)0.0080.007
C4Enterobacteraerogenes(MTCC111)0.0120.005E12S.aureus(25923)#
0.7210.758H8P.vulgaris(ATCC6380)0.0080.005
C5E.coli(38510)*0.0010.005F1Klebsiella(340053)*0.0110.018H9E.coli(13534)0.0150.011
C6E.coli(320149)*0.0040.003F2K.oxytoca(MTCC2275)0.0130.016H10K.pneumoniae(ATCC13883)0.0110.012
C7P.mirabilis(494750)*0.0070.008F3Klebsiella(121103186)*0.0020.001H11P.vulgaris(MTCC1771)−0.007−0.011
C8E.coli(320487)*0.0070.004F4P.mirabilis(5163)*−0.015−0.005H12S.aureus(52601)*0.6580.702
*M/sListerMetropolisLaboratory
#
Environmentalsamples

nosocomial as well as community pathogen. The results of the
study are given below.
Characterisation of the VOCs extracted
from Staphylococcus using GC-MS and FT-IR analysis
When the volatile compounds extracted from cell-free culture
supernatant in DCM were subjected to GC-MS analysis, among
the presence of various compounds, 2-[3-acetoxy-4,4,14-
trimethylandrost-8-en-17-yl] propanoic acid (ATMAP) was
found to be specific for Staphylococcus in comparison with the
other common pathogens like Pseudomonas, Proteus, Shigella,
Salmonella, and Escherichia coli employed in this study. The
mass spectra showing the gas chromatogram of Staphylococcus
having peaks at 14.3, 16.12, 17.72, 19.17 and 21.83 min is given
in Fig. 1a. Furthermore, when the mass spectrum at each Rt was
analysed, the fraction at 19.17 min showed a compound with an
ion mass of 355 (shown in Fig. 1b). Matching retention indices
and fragmentation pattern with the spectral library and from lit-
erature (Venkatachalam et al. 2013) indicated that the compound
could be ATMAP. Reconfirmation of the compound was done
after purification by GC-MS analysis shown in Fig. 2a. Its low
abundance, however, was well above the detection limit of the
colorimetric assay developed.
FT-IR analysis of the DCM-extracted sample
for functional group identification
The FT-IR spectra of extracts of Staphylococcus spp. grown in
TSB after eliminating DCM peaks are shown in Fig. 2b. In the
spectra the peak at 1646 cm−1
was characteristic of aryl–
COOH representing the presence of carboxylic acid group.
Another characteristic peak was also shown at 1373 cm−1
that
represents C–O stretching of acids. The two peaks at 1285 and
1048 cm−1
are attributed to the medium intensity –C–0
stretching indicating the presence of carbonyl functional
group. The absorption peak at 975 cm−1
represents a O–H
bending corresponding to an acid. The intense peak at
3407 cm−1
(O–H stretching) showed that there was an inter-
molecular hydrogen bonding of carboxylic acids at low
frequencies.
Comparison of Staphylococcus cultures in NB, LB
and TSB
The gas chromatogram of Staphylococcus extracts in NB, LB
and TSB is shown in Fig. 3. The comparative analysis of
chromatograms revealed the absence of 19 min peak in LB
and NB compared to TSB medium which confirms the release
of the characteristic compound under the desired conditions.
Detection of Staphylococcus by colorimetric method
Once the release of ATMAP by Staphylococcus spp. was
characterised, a simple colorimetric method for the detection of
the bacteria in cultures using methyl red was devised. The meth-
yl red changed its yellow colour at neutral pH to orange in
Staphylococcus spp. cultures presumably due to acidity caused
by the release of acidic compounds. The concomitant absorption
with peak at 462 nm was seen only for Staphylococcus and not
Fig. 1 GC analysis of DCM
extract from Staphylococcus
culture and the mass spectrum of
the material at retention time
19.17 min. a Gas chromatograms
of VOCs released by
Staphylococcus spp. in the DCM
extracts. b Mass spectrum of the
unique compound for
Staphylococcus spp. at 19.17 min.
The fragment peak at 73m/z is the
base peak showing 100 %
abundance and corresponding to
ATMAP

for 25 other pathogens tested. Figure 4a shows the spectral re-
sults of Staphylococcus and a few other common pathogens. For
the routine assay, optical measurement was set at 462 nm.
The volatile component released by Staphylococcus being
responsible for the acidity and detection in colorimetric
assay
The fact that the acidity of the medium as revealed by pH
measurement using pH metre, was predominantly due to the
presence of volatile molecule was evident from the distinct
colour change with methyl red. This could be seen after 1 or
2 h in samples maintained on ice but not in those at room
temperature and left at 60 °C, presumably due to evaporation,
as shown in Fig. 4c.
Screening of bacterial strains using colorimetric assay
The assay performed at various time points during 24 h of
growth after the inoculation of 105
cells, as shown in
Fig. 4b, revealed that colour change of methyl red was visu-
ally observed by 4 h and measured by 3 h. Under the assay
conditions, the colour intensity increased linearly up to 10 h
and plateaued after that. In case of lower inocula size, the time
of detection increased progressively. For inoculum containing
102
cells, corresponding to low abundance or early stage of
infection, visible and instrumental detection was possible after
7 and 6 h, respectively.
Since surveillance requires high-throughput method, the
assay was adapted to the standard 96-well microtitre plate
format and read using a colorimetric plate reader or im-
aged with a camera. The assay was found to be 100 %
sensitive and specific to Staphylococcus in laboratory val-
idation with known clinical and environmental bacterial
isolates. Figure 5 shows the laboratory validation results
of 39 standard strains and 56 samples of clinical and
environmental bacterial isolates consisting of common
pathogens like Escherichia coli, Proteus spp.,
Pseudomonas aeruginosa, Klebsiella spp., Enterobacter,
Citrobacter, Staphylococcus spp. and Salmonella spp. As
can be seen, the assay showed absolute specificity and
sensitivity for the genus Staphylococcus; only the 13
Staphylococcus strains were identified unequivocally
among 96 bacteria; 99 % confidence interval for sensitiv-
ity and specificity of all positives and negative samples
were between 0.941 and 1.039.
Headspace analysis for the volatile compound
for the detection of Staphylococcus
Since our main interest is to develop a remote assay
without getting into physical contact with the culture
Fig. 2 a Gas chromatogram of
Staphylococcus culture extract
after purification The inset figure
shows the chromatogram with a
single spot produced by purified
and crude samples. b FT-IR
spectra of Staphylococcus spp.
solvent extract

or the bacterium, we tested the applicability of the
method in the head space. The silica-coated discs when
placed in the inside of the lid and exposed to the head-
space of bacterial plate cultures, the silica adsorbed the
VOC including the propionic acid derivative. After 10-h
exposure, spotting of methyl red produced red coloured
spot only in the case of Staphylococcus and remained
yellow for other strains, thus indicating that headspace
detection for Staphylococcus is possible using this meth-
od. Since this is of great value in diagnostics, we have
adapted it to a 12-well format and the results for 12
different strains, including Staphylococcus are shown
in Fig. 6.
Discussion
Preventive control of infectious diseases is becoming a
necessity because of the increasing virulence and persis-
tence of the causative organisms like bacteria. The
daunting challenge of multi drug resistance, especially
when the prospect of developing new antibiotics is
bleak, makes the development of tools to prevent the
diseases imperative. In this regard, Staphylococcus in-
fections due to both community and hospital-acquired
strains are prime targets because of their high preva-
lence, quick spread, morbidity (Hospital statistics
2012), and mortality (Elixhauser and Steiner 2007).
Apart from their early identification, surveillance for
their presence and spread require techniques that are
quite simple, easy and inexpensive, non-destructive and
automated using instrumentation. The study described
here has addressed this lacuna by developing a simple
colorimetric method on the basis of a detailed molecular
study for the presence of extracellular VOC in the cul-
ture liquid and the headspace. Though the test involves
simply the addition of the pH indicator, methyl red, to
the culture or as a spot on the silica plate exposed to
the headspace, the absolute specificity and sensitivity
among 96 different strains belonging to 25 commonly
encountered pathogenic bacteria is remarkable.
Moreover, the results are unambiguous. The specificity
of VOC release with respect to the medium used for
culturing is an important factor in this test.
VOC secreted by bacteria under specific conditions
appear to be its finger print, which could be exploited
as promising targets for preventive diagnosis. In case of
Staphylococcus, we found ATMAP to be one such char-
acteristic volatile compound released as secondary me-
tabolite in response to growth in TSB medium but not
in LB or NB, as revealed by the GC analysis of DCM
extract. This is evident from the TLC and GC results
shown in Figs. 2 and 3, respectively. Among the com-
pounds in the DCM extract from Staphylococcus
Fig. 3 Comparison of gas chromatogram of Staphylococcus cultures
extract in NB (a), LB (b) and TSB (c) showing the absence of 19-min
peak on the GC traces. d Colorimetric assay on Staphylococcus cultures
in TSB, minimal NB and LB. The figure shows the distinct colour change
of Staphylococcus from yellow to orange in TSB unlike others

cultures, only one acid-positive spot corresponding to
the purified preparation of ATMAP could be seen in
TLC. Its release in millimolar concentration appears to
be the cause for reduction in pH (from 7.4 to 5.2), as
indicated by the pH measurement of the culture medium
(supplemental Table S1) and the neutral pH of the
DCM-extracted culture medium. Taking these evidences
collectively, the characterised volatile compound appears
to be responsible for the pH reduction.
The reason for this specificity is not clear now. It is mod-
erately volatile at 37 °C and hence, the test has to be carried
out immediately after growth or after immediate chilling for
the best and accurate results.
Though reports suggest a variety of dyes like 4-
pyrene methanol (Cubero-Herrera et al. 2006), dansyl
cadaverine (Lee et al. 1989), 1-pyrenyl diazomethane
(Nimura and Kinoshita 1988), and 4-bromomethyl-7-
methoxycoumarin (Dunges 1977) reagents specific for
carboxylic acids, bronsted acid-base dye and methyl
red, was found to be best suited for the method. This
is because the above reagents generally require aprotic
solvents, higher temperatures and prolonged heating for
derivatization, conditions not suitable for field-level
tests. Moreover, they are either expensive, require strin-
gent storage conditions, hazardous or require costlier
instrumentation and working skill.
The characterisation of ATMAP as the volatile organ-
ic molecule responsible for the acidity (we found that
the pH of the culture changed from 7.3±0.2 to 5.2±0.2
in grown culture at the time of detection) was interest-
ing from the point of view of the metabolism of the
organism. Though this secondary metabolite is produced
in sufficient quantities to change the pH of the medium
(weakly buffered) by about 2 units, there is not much
information on the anabolic biochemical pathway or the
importance of the metabolite.
Fig. 4 a Determination of absorption maximum for bacterial cultures of
Staphylococcus, Proteus and Salmonella after reaction with methyl red
dye. b Graph of the absorbance response for bacterial cultures using
methyl red assay performed every hour up to 24 h. c Absorbance
intensity of methyl red reaction with volatile acids in the
Staphylococcus cultures kept at room temperature (27 °C), 60 °C and
on ice (0 °C) reduces drastically as a function of temperature as well as
duration of storage indicating volatile nature

The test has been developed in a high-throughput mode
by performing in 96-well microtitre plate format for
liquid-based detection and moderately throughput for using
headspace. Though the results can be obtained visually,
for instrumentation including automation, the results of
the liquid assay can be readily obtained using microplate
reader, which are common in laboratories, or through im-
aging using a camera and image analysis software. In the
case of headspace assay, apart from visual judgement, it is
possible to use available optoelectronic devices like strip
Fig. 5 Validation of assay using
standard and clinical strains. The
performance of methyl red assay
using standard strains, clinical
isolates and environmental
samples as given in Table 1. The
Staphylococcus spp. were
distinguished among other
species by the characteristic
change in colour (orange) from
the medium blank and negatives,
all showing yellow colour
Fig. 6 Colour difference for
different bacterial strains resulting
from colorimetric assay exposure
to 12-well plate growing cultures
after 10 h

readers or even electronic noses that are being developed
to detect characteristic VOCs.
Acknowledgments We are grateful to Mr. Suresh Lingham, M/s
Trivitron Pvt Ltd. for clinical samples and Dr. Sridhar, Dept. of Microbi-
ology, Sri Ramachandra University, for providing standard bacterial cul-
tures. Our sincere thanks to P. Dineshkumar and S. Akshay for their
contribution to our study. We acknowledge the financial support from
Centre with Potential for Excellence in Environmental Science
(CPEES) of University Grants Commission. One of the authors, R Aarthi,
acknowledges CSIR for providing CSIR-SRF. We also express our
deepest gratitude to our family and friends.
Conflict of interest There is no conflict of interests among the authors
for submitting this article. This work was supported by the Centre with
Potential for Excellence in Environmental Science (CPEES) of
University Grants Commission, India. They have no involvements in
the study design; in the collection, analysis and interpretation of data; in
the writing of the article and in the decision to submit the article for
publication.
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