1. 1 23
Applied Microbiology and
Biotechnology
ISSN 0175-7598
Appl Microbiol Biotechnol
DOI 10.1007/s00253-013-5393-9
2-methylbutanal, a volatile biomarker, for
non-invasive surveillance of Proteus
Raju Aarthi, Raju Saranya & Krishnan
Sankaran

2. 1 23
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3. METHODS AND PROTOCOLS
2-methylbutanal, a volatile biomarker, for non-invasive
surveillance of Proteus
Raju Aarthi & Raju Saranya & Krishnan Sankaran
Received: 20 June 2013 /Revised: 1 November 2013 /Accepted: 9 November 2013
# Springer-Verlag Berlin Heidelberg 2013
Abstract Pathogen detection needs a paradigm shift from
time-consuming conventional microbiological and biochemi-
cal tests to much simpler identification methods with higher
sensitivity and specificity. In this regard, a simple detection
method for frequently isolated nosocomial uropathogen,
Proteus spp., was developed using the characteristic volatile
2-methylbutanal released in Luria Bertani broth. The instant
reaction of the compound with 5-dimethylaminonaphthalene-
1-sulfonylhydrazine (DNSH) has been adapted to develop a
sensitive fluorescence assay named “ProteAl” (Prote,
“Proteus” & Al, “Aldehyde”). The assay was performed by
direct addition of the fluorescence reagent to the culture after
7 h of growth. The distinct green fluorescence by Proteus
(other organisms show orange fluorescence) served as the
simplest and quicker identification test available for Proteus.
In the laboratory, it exhibited 100 % specificity and 100 %
sensitivity during testing of 95 strains including standard and
known clinical isolates representing frequently encountered
uropathogens.
Keywords 2-methylbutanal . DNSH . Fluorescence
detection . Proteus spp. . Surveillance . Urinary tract
infections
Introduction
Urinary tract infections (UTI) have been persistent due to
populated communities living under impoverished conditions
without access to clean water and proper sanitation (Sheela
and Johanna 2013). Despite advancement in clinical research,
these diseases emerge either as severe epidemics or pan-
demics. Of late, causative bacteria have been reported to be
multidrug resistant to most of the commonly used broad-
spectrum antibiotics (Gerard and Arlene 2007). Hence, sur-
veillance becomes essential as a measure to control pathogens
and prevent emergence of their resistance to potent drugs. The
existing protocols and instruments for field detection and
identification of such pathogens are laborious, time-
consuming, and demand technical skill to serve as effective
surveillance tools (Guernion et al. 2001).
Among the infectious diseases, prevalence of urological
infections are high, especially in women, but grossly ignored
as it is not fatal in the early stages. Escherichia coli, Proteus,
Klebsiella, Enterobacter, Staphylococcus, and Pseudomonas
are the common causative organisms of UTI (Samia 2012).
Though uropathogenic E. coli causes 70–80 % of UTI
(Zalewska 2011), Proteus is the prime cause of nosocomial
infection in patients especially with long-term catheterization
or due to structural and/or functional abnormalities of the
urinary tract (Nicolle 2005). The infection may be either
limited to the bladder (cystitis) or may involve the renal
parenchyma (pyelonephritis) (Christopher et al. 2000). The
global incidence of UTI is estimated to be 150 million annu-
ally (Sheela and Johanna 2013), and the necessity of contain-
ing spread of Proteus is of immediate significance.
Conventional microbiological, immunological, and bio-
chemical techniques used for identification of UTI-causing
bacteria provide qualitative as well as quantitative informa-
tion. However, they are low-throughput, they consume 2–
3 days of critical treatment time, are prone to manual errors,
are subjective and less affordable (Vijayalakshmi et al. 2010).
Biosensors like glucometer, sensing array (James et al. 2011),
and electronic nose (Lee et al. 2010) provide rapid detection
methods, but these sensors have not been developed for de-
tecting pathogens (Andre et al. 2002). These limitations and
R. Aarthi :R. Saranya :K. Sankaran (*)
Centre for Biotechnology, Anna University, Sardar Patel Road,
Guindy, Chennai 600 025, India
e-mail: ksankran@yahoo.com
Appl Microbiol Biotechnol
DOI 10.1007/s00253-013-5393-9
Author's personal copy

4. the necessity for constant surveillance of UTI pathogens
prompted us to develop an appropriate non-invasive instru-
mentation methodology.
Volatile organic compounds (VOCs) released by bacteria
are either unique or characteristic under certain experimental
growth conditions (Thorn and John 2012). Since non-
destructive (Sethi et al. 2013) and remote identifications are
preferred for early diagnostics and surveillance, identification
of such volatile compounds offers a promising approach
(Laurent et al. 2004). The reported VOCs of Proteus mirabilis
and Proteus vulgaris, which are the prevalent UTI-causing
species, include aldehydes (benzaldehyde, acetaldehyde,
formaldehyde, 2-methylbutanal), ketones (2-
aminoacetophenone, acetone), alcohols (ethanol, 1-butanol,
1-pentanol), acids (phenyl acetic acid), compounds like hy-
drogen sulphide, methyl mercaptan, dimethyl sulphide, di-
methyl disulphide, ethyl butanoate, n-propyl acetate, iso-
prene, trimethyl amine, and ammonia (Thorn et al. 2011).
These act either as chemical messengers or secondary metab-
olites, and one or more of these are produced under specific
growth conditions (Oland et al. 2004). Hence, the hypothesis
for developing pathogen identification methods using charac-
teristic VOCs released in the medium under specific growth
conditions were explored, choosing Proteus spp. as a model.
Existing analytical instrumentation methods for identifying
and characterizing traces of VOCs are high-performance liq-
uid chromatography (HPLC), gas chromatography (GC) and
mass spectrometry (MS) (Sichu 2009), selected ion flow tube
mass spectrometry (SIFT-MS) (Thorn et al. 2011), followed
by Fourier transform infrared (FT-IR) (Bungert et al. 2001).
Obviously, sophistication and affordability limit their use in
surveillance. Simpler colorimetric methods using reagents like
2, 4-dinitrophenylhydrazine (DNPH) or fluorimetric methods
using reagents like 5-dimethylaminonaphthalene-1-
sulfonylhydrazine (DNSH), commonly known as dansyl hy-
drazine, provide cost-effective and high-throughput detection
methods for carbonyl compounds (Norbert et al. 1998). The
latter are much more sensitive and suit simpler instrumenta-
tion using LEDs and photodiodes. Among the hydrazine dyes
available for fluorescence methods, DNSH was reported to be
superior (Martin et al. 2000). In this study, a VOC-based
biomarker for Proteus species was identified, and a specific
fluorimetric assay that can be used in day-to-day clinical
diagnosis and field-level screening was developed.
Materials and methods
Collection and identification of strains
a. Standard strains: Standard strains of P. mirabilis (three), P.
vulgaris (two), Shigella flexneri (four), Salmonella paratyphi
(one), Salmonella enterica subspecies (two), E. coli (nine),
Klebsiella pneumoniae (four), Klebsiella oxytoca (one),
Staphylococcus aureus (four), Streptococcus pyogenes
(one), Staphylococcus epidermidis (one), Staphylococcus
chromogenes (one), Staphylococcus haemolyticus (one),
Pseudomonas aeruginosa (three), Streptococcus pneumoniae
(one), and Listeria monocytogenes (one) were obtained from
Microbial Type Culture Collection (MTCC), Chandigarh, and
Sri Ramachandra University, Chennai, Tamilnadu, India.
(Details of the strains are provided in Table 1)
b. Clinical isolates: P. mirabilis (twenty), P. vulgaris (two), E.
coli (eighteen), S. aureus (one), P. aeruginosa (four),
Salmonella typhi (four), Enterobacter (two), Citrobacter
(two), and Klebsiella spp. (three) were obtained from M/s
Lister Metropolis Laboratory, Chennai, Tamilnadu,
India (Table 1). The strains were confirmed by standard mi-
crobiological (growth on selective medium and motility) and
biochemical tests (Methyl Red/Voges-Proskauer (MR/VP)
Test, urease, catalase, Triple sugar iron, and Indole test).
Furthermore, all the Proteus strains were confirmed using
Sensititre GNID identification plate from TREK diagnostics
systems, UK, and also by 16S rRNA sequencing.
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
hydroxide (Himedia, India) and autoclaved at 121 °C and
15 lbs pressure for 20 min.
Extraction of volatile organic compounds (VOCs)
from culture
In 1 mL of sterile LB medium contained in 2 mL centrifuge
tube, 20 μL (105
cells) of each organism was inoculated
separately and incubated in a orbital shaker set at 37 °C and
170 rpm. Following 7 h of incubation, an equal volume of
chloroform or dichloromethane (DCM) or ethyl acetate was
added and vortexed for 1 min to extract the VOCs. The
solvent phase was collected and analyzed.
Gas chromatography–mass spectrometry (GC-MS) analysis
GC-MS was performed using Agilent 7890A GC system with
7000C Triple Quad MS. GC: The start and stop temperatures
were set at 60 °C and 150 °C, respectively, and separated on
Agilent HP5 MS column. Helium (99.9 %) was used as the
carrier gas at the flow rate of 1.2 ml/min. MS: The samples
were scanned at the range of 35–150m/z between 1.5 to
10 min with electron ionization EI source with ionization
energy, −70 ev. The ion source temperature was 230 °C with
Appl Microbiol Biotechnol
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5. the interface temperature of 180 °C. The event time and
solvent cut time were 14.66 s and 1.5 min, respectively.
Fourier transform-infrared (FT-IR) analysis
The FT-IR vibrational spectra of the solvent extracts were read
using an IR Prestige model FT-IR spectrometer (Make:
Shimadzu, Japan). The co-elution of 2-methylbutanal in the
medium was taken as a control. IR spectrum was recorded by
introducing the sample into the IR path. The spectrum was
taken from 400 to 4,000 cm−1
with a resolution of 4 cm−1
.
Dye reagent specific for carbonyl compounds
Fluorogenic reagents for the derivatization of carbonyl com-
pounds are in routine practice for sensitive and selective
detection. The fluorimetric dye, DNSH (M/s Sigma Aldrich,
USA), was chosen for the study. The dye solution was pre-
pared by dissolving 0.02 g of the dye in 1 mL of HPLC-grade
acetonitrile (M/s Merck, India) to give a final concentration of
75.3 mM. When the dye was reacted directly with carbonyl
compounds, acids, and alcohols, the sensitivity was not ade-
quate for detecting lower concentrations of VOC from
Table 1 Validation of ProteAl using standard and clinical strains
Well no. Organism ProteAl (RFU) Well no. Organism ProteAl (RFU) Well no. Organism ProteAl (RFU)
Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trail 2
A1 Medium blank 7,234 8,241 C9 K. pneumoniae
(MTCC 2653)
8,281 7,089 F5 P. aeruginosaa
(326543) 8,204 7,096
A2 E. coli (ATCC 25922) 7,826 8,315 C10 P. mirabilisa
(328271) 18,401 11,032 F6 P. aeruginosaa
(326604) 8,121 7,336
A3 P. aeruginosa
(ATCC 27853)
7,730 7,662 C11 K. pneumoniae
(MTCC 661)
8,345 9,021 F7 P. aeruginosaa
(121602592)
7,873 7,327
A4 S. flexneri (ATCC 29508) 8,375 7,729 C12 P. aeruginosa
(MTCC 424)
8,685 7,085 F8 P. mirabilisa
(5164) 13,838 16,615
A5 S. flexneri (MTCC 9543) 8,401 7,734 D1 P. vulgarisa
(121103217) 11,232 15,289 F9 S. typhimuriuma
(327753) 8,219 8,007
A6 P. mirabilis (ATCC 7002) 13,774 15,241 D2 P. aeruginosa
(MTCC 1934)
8,338 7,844 F10 S. typhimuriuma
(328897) 8,471 8,208
A7 S. paratyphi (MTCC 3220) 7,931 7,869 D3 S. flexneri (MTCC 1457) 8,447 7,256 F11 P. mirabilisa
(5166) 16,296 11,272
A8 S. enterica (MTCC 3231) 8,259 7,549 D4 S. flexneri (MTCC 9543) 8,129 6,951 F12 S. typhimuriuma
(121703058)
8,386 7,909
A9 P. mirabilis (ATCC 29906) 19,267 14,619 D5 S. pneumoniae
(MTCC 655)
8,617 9,430 G1 S. typhimuriuma
(18946) 8,173 7,854
A10 E. coli (MTCC 723) 7,394 7,017 D6 S. pyogenes
(MTCC 1927)
9,819 9,099 G2 Enterobactera
(14736) 8,590 7,178
A11 E. coli (MTCC 443) 7,623 8,229 D7 S. enterica (MTCC 3224) 9,818 9,980 G3 P. mirabilisa
(5169) 10,112 16,873
A12 E. coli (ATCC 13534) 8,218 8,382 D8 P. mirabilisa
(15322) 13,310 15,802 G4 Enterobactera
(339969) 8,068 8,246
B1 P. vulgaris(ATCC 6380) 13,416 12,288 D9 L. monocytogenes
(MTCC 839)
8,002 9,433 G5 P. mirabilisa
(281) 12,381 18,743
B2 S. aureus (MTCC 3160) 7,605 7,901 D10 S. aureus (ATCC 25923) 8,309 8,242 G6 Citrobactera
(24361) 8,534 7,408
B3 K. pneumoniae
(ATCC 13883)
8,440 8,005 D11 E. coli (MTCC 901) 8,390 7,989 G7 Citrobactera
(328327) 8,716 7,517
B4 P. vulgaris (MTCC 1771) 12,981 12,476 D12 P. mirabilisa
(806970) 12,492 13,234 G8 E. colia
(311475) 7,946 7,112
B5 S. aureus (MTCC 3160) 8,577 8,474 E1 E. colia
(21728) 7,956 9,475 G9 E. colia
(21595) 8,864 8,502
B6 S. aureus (MTCC 6908) 8,654 7,718 E2 P. mirabilisa
(122101203) 13,055 15,298 G10 P. mirabilisa
(282) 11,938 20,535
B7 S. chromogenes
(MTCC 6153)
8,669 8,876 E3 E. colia
(21748) 8,043 8,335 G11 E. colia
(121201233) 8,783 7,321
B8 S. haemolyticus
(MTCC 8924)
7,432 8,412 E4 E. colia
(25922) 8,001 8,254 G12 P. mirabilisa
( 803) 13,259 13,946
B9 P. mirabilis
(ATCC 336874)
18,682 16,120 E5 P. mirabilisa
(5155) 10,596 16,132 H1 E. colia
(318253) 8,066 7,642
B10 S. epidermidis
(MTCC 435)
7,624 8,792 E6 S. aureus (25923) 8,450 9,152 H2 P. mirabilisa
(487) 17,231 16,465
B11 P. mirabilisa
(6878) 18,187 15,111 E7 P. mirabilisa
(3401488) 10,587 15,056 H3 E. colia
(318304) 8,336 7,977
B12 E. coli (MTCC 568) 7,395 8,635 E8 P. aeruginosaa
(27853) 8,060 9,715 H4 E. colia
(318429) 8,806 7,372
C1 E. coli (MTCC 1687) 9,822 8,178 E9 P. mirabilisa
(5156) 24,917 12,583 H5 P. mirabilisa
(981447) 16,962 17,126
C2 P. vulgarisa
(307316) 12,610 24,749 E10 E. colia
(340266) 8,166 8,217 H6 E. colia
(318510) 8,257 7,626
C3 E. coli (MTCC 433) 8,941 8,254 E11 E. colia
(111406070) 8,081 8,570 H7 E. colia
(320149) 8,276 8,818
C4 P. mirabilisa
(121101096) 20,973 24,003 E12 E. colia
(111706439) 8,045 7,422 H8 P. mirabilisa
(494750) 14,519 14,311
C5 E. coli (MTCC 9537) 9,591 8,299 F1 Klebsiellaa
(340053) 8,083 7,470 H9 E. colia
(320487) 9,455 7,401
C6 K. pneumoniae
(MTCC 3384)
9,132 8,347 F2 Klebsiellaa
(4483) 8,618 7,389 H10 E. colia
(320652) 8,568 7,327
C7 P. mirabilisa
(332049) 11,746 22,184 F3 Klebsiellaa
(121103186) 8,276 7,809 H11 E. colia
(320904) 8,257 7,788
C8 K. oxytoca (MTCC 2275) 8,151 7,629 F4 P. mirabilisa
(5163) 16,255 15,389 H12 E. colia
(320923) 8,449 7,231
RFU relative fluorescence unit
a
M/s Lister Metropolis Laboratory
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6. bacteria. However, when the dye solution (75.3 mM) was
added to the sample followed by glacial acetic acid (for acid
catalysis) (Norbert et al. 1998), the fluorescence yield in-
creased two times, and the conversion of orange to green
fluorescence could be visualized in UV transilluminator.
Standardization of DNSH assay for carbonyl compounds
To standardize the DNSH assay and determine its sensitivity,
16 pure compounds, carbonyl as well as non-carbonyl (benz-
aldehyde (28 μM), hexanal (42 μM), decanal (25 μM),
acetophenone (21 μM), 2-pentanone (27 μM), 2-heptanone
(26 μM), 2-nonanone (29 μM), 2-undecanone (24 μM), 2-
tridecanone (20 μM), methanol (31 μM), ethanol (22 μM),
propanol (32 μM), butanol (24 μM), propionic acid (24 μM),
butyric acid (24 μM), and phosphoric acid (20 μM)) were
reacted with the dye, and the resultant fluorescence was
scanned for excitation at 300–400 nm. Subsequently, the
samples were excited at the fixed maximum excitation wave-
length for carbonyl compounds and scanned at 500–600 nm
for respective emission maximum. Thus, excitation was fixed
at 336 nm, and emission was fixed at 531 nm. For detecting
carbonyl compounds from culture, DNSH assay was per-
formed with bacterial strains after 7 h of growth, and the
fluorescence was read in a fluorimeter set at the above excita-
tion and emission wavelengths (Ex/Em). The fluorescence
was measured using a fluorimeter, (Model: Enspire, Perkin
Elmer, USA), and the same was imaged using a UV transil-
luminator for visualization.
Fluorescence-based DNSH assay (ProteAl) for detection
of Proteus species
The DNSH assay for detecting the aldehyde released by
Proteus species was performed in a 96-well plate. Each well
was filled with 180 μL of LB medium, and 20 μL of 105
cells
of the test strains were inoculated. The plate was incubated at
37 °C and 100 rpm for 7 h in an orbital shaker. The optical
densities of 7 h bacterial culture were measured at 600 nm
using Multiscan reader (Thermo, Finland), and then the
DNSH assay was performed by adding 5.0 μL of the dye
solution and 2.5 μL of glacial acetic acid. The fluorescence
was measured after 5 min using the fluorimeter, and the plates
were also imaged. The assay is referred to as ProteAl (Prote,
“Proteus” & Al, “Aldehyde”). To profile VOC release with
respect to time, the assay was performed every 1 h of bacterial
growth. ProteAl assay was performed with various concen-
trations of 2-methylbutanal, and a standard graph was gener-
ated using the fluorescence data obtained for each concentra-
tion. A quantitative estimation of the VOC in the culture at
different time point (from fourth hour) was obtained using the
standard graph.
Testing the volatility of 2-methylbutanal from culture
To check whether the target of the assay is a volatile carbonyl
compound released by the bacteria, the assay plate was incu-
bated open at different temperatures: at room temperature
(≈27 °C), in the refrigerator (≈4 °C), and on ice, and the assay
was performed after 1 and 2 h. The same was tested with 2-
methylbutanal and compared.
Laboratory validation of ProteAl
After optimizing and testing the assay conditions, a set of 95
strains including 39 standard and 56 known clinical strains
representing frequently encountered uropathogens (Table 1)
were validated. The experiment was repeated twice, and the
RFU values are given in Table 1.
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: true positive, b: false positive, c: false
negative, d: true negative (Abdul and Anthony 2008). When
the growth (OD) of the strains where similar, the 99 % confi-
dence for the positive (Proteus) and negatives were calculated
using the formula.
X Æ 2:58 δ=√n
À Á
Where X is sample mean; δ, population standard deviation,
and n, sample size (Jose 2009).
Results
VOC-based detection, which has the distinct advantage of
being non-invasive and suitable for surveillance over existing
techniques, is yet to emerge as a diagnostic approach in
bacterial identification (Lieuwe et al. 2013). Highly sensitive
fluorescence-based chemical methods are now becoming pop-
ular in a variety of analytical applications. Hence, such a
fluorescence method has been developed in this study to
detect the carbonyl compound, 2-methylbutanal from the
cultures of Proteus. The results of identification of the char-
acteristic carbonyl compound under defined growth condi-
tions, the standardization of the assay, ProteAl, and its labo-
ratory validation are given below.
Release of carbonyl compound(s) by Proteus cultures
confirmed using GC-MS and FT-IR analyses
The volatile compounds extracted in DCM were directly
subjected to GC-MS analysis. The gas chromatograms
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7. revealed the presence of various compounds at different re-
tention time (Rt), among which 2-methylbutanal was found
specific to Proteus when the mass spectra were analyzed.
Figure 1a shows the gas chromatogram of Proteus having
three peaks at 1.57, 1.78, and 2.92 min. Furthermore, when
the mass spectrum at each Rt was analyzed, the fraction at
1.78 min showed a compound with a molecular mass ion of 86
(shown in Fig. 1b). Matching retention indices and fragmen-
tation pattern with the spectral library indicated that the com-
pound could be 2-methylbutanal. Its low abundance, however,
was well above the detection limit of the fluorescence assay
developed. Similarly, it has been reported that 2-
methylbutanal is one of the VOCs released by Proteus when
grown in similar complex medium (Thorn et al. 2011).
The FT-IR spectra of 2-methylbutanal, extracts of LB, P.
vulgaris, and P. mirabilis after eliminating DCM peaks are
shown in Fig. 2. In the spectra of 2-methylbutanal, the peak at
1,723 cm−1
is characteristic to strong C=O stretching,
representing the presence of carbonyl group. The two peaks
at 2,684 and 2,829 cm−1
are attributed to the medium intensity
=C–H stretching indicating an aldehyde. The absorption
peaks at 1,421 and 2,976 cm−1
are representing a variable
C–H bending and a strong C–H stretching, respectively,
which corresponds to alkane. The other peaks in the spectrum
are similar to that of the blank indicating the organic com-
pounds released from the medium. The spectra of P. vulgaris
and P. mirabilis also shows peaks at 1,721 and 1,725 cm−1
for
C=O stretching. The absorption peaks corresponding to =C–H
stretching (2,686 and 2,827 cm−1
for P. vulgaris; 2,686 and 2,
830 cm−1
for P. mirabilis) indicates aldehyde. Similarly, the
absorption peaks representing a variable C–H bending (1,
423 cm−1
for P. vulgaris and 1,427 cm−1
for P. mirabilis)
and a strong C–H stretching (2,985 cm−1
for P. vulgaris and 2,
986 cm−1
for P. mirabilis) corresponding to alkanes were
observed. This comparative analysis of 2-methylbutanal with
Proteus confirms the presence of an aldehyde in the solvent
extract for Proteus spp. under the described conditions.
Detection of Proteus by the fluorescence shift of ProteAl
reagent
Confirming the release of 2-methylbutanal by Proteus spp.
and sensitive fluorescence reagents for carbonyls, a simple
fluorescence method for their detection in Proteus cultures
using DNSH was devised. The Em λmax of DNSH under
acidic condition (pH 3.4) was 564 nm, and when it reacted
with carbonyl compounds (pure or in culture), the Em λmax
shifted to between 510 and 535 nm (bright green
Fig. 1 GC analysis of DCM
extract from Proteus culture and
the mass spectrum of the material
at retention time 1.78 min. a
Shows the gas chromatograms of
volatile organic compounds in the
DCM extracts of Proteus. The
characteristic peak at 1.78 min in
Proteus was further analyzed for
identification of mass. b is the
mass spectrum of the unique
compound for Proteus at Rt
1.78 min in GC. The fragment
peak at 57m/z is the base peak
showing 100 % abundance and
corresponding to
2-methylbutanal. No other
carbonyl compound was detected
from the other peaks
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8. fluorescence), while, for a variety of acids and alcohols, it was
between 545 and 570 nm (orange fluorescence). Figure 3a
below shows the representative spectra of the carbonyl and
non-carbonyl compounds, and Fig. 3b shows those of bacte-
rial cultures. The shift to green fluorescence from orange,
specific to carbonyl compounds among commonly reported
Fig. 2 FT-IR spectra of P.
vulgaris and P. mirabilis solvent
extract in comparison with
2-methylbutanal shows the IR
spectrum of the DCM-extract of
medium blank, 2-methylbutanal,
P. vulgaris, and P. mirabilis
samples. The Proteus samples
showed the presence of carbonyl
group along with the =C–H
stretch corresponding to an
aldehyde which is similar to the
standard 2-methylbutanal.
Together, the analysis was
suggestive of the presence of
2-methylbutanal as the volatile
organic compound in low
abundance in the cultures of
Proteus grown in LB
Fig. 3 Determination of Ex/Em λmax for pure compounds and bacterial
cultures. The emission spectra on the left (excitation 336 nm) in a are of
pure carbonyl (hexanal and 2-heptanone), acid (propionic acid), and
alcohol (butanol) compounds after reaction with DNSH under the assay
conditions. The emission spectra on the right b are of the cultures of
Proteus, UPEC, and Salmonella after reaction with DNSH under the
assay conditions. The spectra of Proteus samples are similar to that of
carbonyl compounds with the λmax around 520 nm, indicating the pos-
sible release of such compounds. The inset figures show the fluorescence
after the assay for the carbonyl and non-carbonyl compounds and for the
positive (Proteus) and non-Proteus cultures. The distinct greenish fluo-
rescence after DNSH reaction is diagnostic of Proteus
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9. VOC types, was also convenient for visual observation. For
the routine assay, ProteAl excitation was set at 336 nm, and
the fluorescence shift was measured between 520 and 530 nm.
ProteAl was highly sensitive and non-invasive
Since surveillance demands high-throughput methods, the
assay was adapted to the standard 96-well microtiter plate
format so that it could be read using a fluorescence plate
reader or imaged with a UV transilluminator. The comparative
fluorescence spectrum of ProteAl for 2-methylbutanal and
Proteus VOC showed a similar trend as shown in Fig. 4a.
The sensitivity of the method was ≈1 nmol, and the measure-
ments of 2-methylbutanal was linear up to ≈200 μmol with
0.99 regression Fig. 4b. The amount of VOC released by
Proteus was calculated using the standard graph. The assay
at various time points of growth, from 0–24 h, as shown in
Fig. 4c, revealed that detectable amounts of the compound
was present in the culture from fourth hour (≈1 nmol) in the
mid-log phase and increased linearly up to 10 h (≈15 nmol).
From the point of view of diagnostic test, detection requires
5 h of growth for a sensitive fluorimeter and 7 h for observa-
tion using UV illuminator, even when the inocula/samples
contain as low as 102
cells.
2-methylbutanal was found to be the volatile component
responsible for green fluorescence in ProteAl
The fact that ProteAl was reacting to only volatile carbonyls
in the culture was established by the green fluorescence seen
Fig. 4 Quantification of 2-methylbutanal using ProteAl assay. a Shows
the fluorescence emission spectra of DNSH reacted with 2-methylbutanl
matched with that of the spectrum obtained from the reaction of DNSH
with the culture. b is the standard graph for 2-methylbutanal using
ProteAl assay showing sensitivity up to 1 nmol and good linearity up
to 20 nmol. c Shows the graph of the fluorescence response for bacterial
cultures using ProteAl performed every hour up to 24 h; only Proteus
showed the release of 2-methylbutanal in nanomoles to reach a maximum
of 13 nmol in broth culture and assay conditions. Being a volatile
compound, the actual amount of 2-methylbutanal released by the organ-
ism could be hundreds of nanomoles. The set of data in this composite
figure compares the properties of pure 2-methylbutanal with those of
DCM-extract from the Proteus culture
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10. in samples maintained at 4 °C and on ice but not in those at
room temperature when assayed after 1 and 2 h, as shown in
the Fig. 5a below, due to prevention of evaporation. The same
was the case when experimented with 2-methylbutanal as
shown in Fig. 5b.
ProteAl was found to be 100 % sensitive and specific to
Proteus in laboratory validation with known clinical bacterial
isolates.
As can be seen from the Fig. 6, laboratory-level validation
using 39 standard strains and 56 samples of clinical bacterial
isolates consisting of commonly occurring uropathogens such
as E. coli, Proteus spp., P. aeruginosa, Klebsiella spp.,
Enterobacter, Citrobacter, Staphylococcus spp., and
Salmonella spp. showed absolute specificity and sensitivity
(using the formula) for the genus Proteus. The concentrations
of 2-methylbutanal for the cut-off with 100 % sensitivity and
specificity is approximately 55 μM, whereas the RFU is
greater than10,000 for positives and less than 10,000 for
negatives.
The confidence interval for the sensitivity and specific-
ity of the assay for positive, Proteus and the negatives
were calculated using 28 samples (taken in triplicate).
Thus, the 99 % confidence interval for sensitivity and
specificity of all positives and negatives are between
0.941 and 1.039.
Discussion
The need for surveillance of Proteus using non-invasive
methods
Among the uropathogens that are prevalent and significant in
causing UTI, Proteus is particularly dangerous because it is a
hospital-acquired pathogen, and our laboratory analyses of
clinical strains generally show MDR phenotype. In a study
conducted in the lab, out of 56 uropathogen isolates collected
from Lister Metropolis laboratory, 20 were Proteus spp., and
all of them were resistant to most of the commonly and
currently used antibiotics like amikacin, cephotaxime,
amoxyclav, and ciprofloxacin. The possibility of using char-
acteristic VOCs released under described conditions by
Proteus was examined and a high-throughput methodology
in the popular 96-well format which is user-friendly and less
hazardous for the technician was developed. Since headspace-
trapping methods were found to be less practical, cumber-
some, subject to more variations than liquid assays, and re-
quires instrumentation development, it was decided to identify
VOC marker in the culture itself. Manipulating cultures for
obtaining the spent medium to identify the VOCs, lead to
severe losses (50–60 %), and therefore even the essential
Fig. 5 Volatility of 2-methylbutanal released by Proteus in comparison
with pure compound. a Shows that the fluorescence intensity of DNSH-
derivatized carbonyl compound(s) in the Proteus cultures kept at room
temperature (27 °C), fridge (4 °C), and on ice (0 °C) reduces drastically as
a function of temperature as well as duration of storage indicating volatile
nature. b Shows the fluorescence intensity of standard 2-methylbutanal
experimented similar to Proteus culture at different temperatures
Fig. 6 Validation of ProteAl using standard and clinical strains shows
the performance of ProteAl using 39 standard strains and 56 clinical
isolates as given in Table 1. The Proteus spp. were distinguished among
the uropathogens by the characteristic green fluorescence due to reaction
of DNSH with 2-methylbutanal from the medium blank and negatives, all
showing orange fluorescence
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11. centrifugation step was avoided. Direct addition of reagent
components quickly into the culture was found to be the best
approach for mass screening requiring minimum exposure
and to develop a cost-effective method.
Potential of 2-methylbutanal as surveillance marker
for Proteus
Characteristic metabolites (biomarkers) like VOCs secreted as
defense against antagonists or as signalling molecules by the
organisms under specific conditions through specific bio-
chemical pathways (Laurent et al. 2004) are promising targets
for such techniques. In the case of Proteus, 2-methylbutanal
was found to be one such characteristic volatile compound,
under the experimental growth conditions, released as a sec-
ondary metabolite from isoleucine degradation (David 2005).
The identification of 2-methylbutanal has also been veri-
fied using GC-MS and FT-IR analysis in comparison with the
pure compound confirms the release of 2-methylbutanal by
Proteus spp. The volatility in the medium and our extensive
literature survey also supported the release of 2-methylbutanal
by Proteus encountered in UTI infections (Thorn et al. 2011).
It is reported that different organisms produce characteristic
VOCs only under defined growth conditions, and this is true
also for Proteus. 2-methylbutanal is produced by Proteus
grown in LB but not when grown in minimal medium. Such
selectivity also adds to the specificity of the technique. Our
quantitative estimation of 2-methylbutanal from the standard
graph for the pure compound in LB showed that the culture
concentration of the compound is in the range of 1–20 nmol,
indicating that it is a secondary metabolite synthesized in
moderate levels (detected from fourth hour of growth).
Being moderately volatile at 37 °C, the test is required to be
conducted immediately after growth or after immediate chill-
ing. Even freezing and thawing resulted in the loss of the
compound.
ProteAl as surveillance method for Proteus
As 2-methylbutanal is characteristic to Proteus under the
described conditions, a simple, sensitive, and non-invasive
fluorimetric assay has been designed. Though reports suggest
a variety of dyes like DNPH, DNSH, nitroaromatic hydra-
zines, 2-diphenylacetyl-1, 3-indandione-1-hydrazone
(DAIH), and halogenated phenyl hydrazine reagents specific
for carbonyl compound, DNSH, has been found to be best
suited owing to its lower level detection in atmospheric sam-
ples (Laurent et al. 2004). We have adapted the dye reaction
performed in aqueous phase by reacting the VOC with the dye
in the pH range 3.6 to 3.9. It is noteworthy that the maximum
fluorescence yield was observed only when the dye in aceto-
nitrile is added first followed by acetic acid (final pH 3.6 to
3.9). However, in order to simplify the assay, when DNSH and
glacial acetic acid was prepared as a reagent at the same pH
range and was added, the fluorescence yield was observed to
be halved. This is due to the carboxylic group in acetic acid
competing with the carbonyl group in aldehyde or ketone to
react with the hydrazine group in DNSH when premixed
(William and Stone 1958).
As the aqueous phase concentration of the VOC was found
to be maximal at 7 h for moderate inoculum of 105
bacteria,
the same was set as the minimum time required before the test
for visual observation using UV transilluminator. Using sen-
sitive fluorimeters, it was possible to detect fluorescence
changes from fifth hour even for a lower inoculum of 102
cells. The simplest manipulation of just the addition of dye to
culture enables this to be conveniently adapted for high-
throughput assay formats and automation required for surveil-
lance, screening, or clinical testing. The 96-well format has
been routinely performed with ease, and this was employed
for laboratory validation with 95 known clinical isolates.
Laboratory validation of ProteAl
As our cheminformatics investigation of volatile compounds
produced by different bacteria indicated that 2-methylbutanal
could be characteristic of Proteus, our validation experiment
with other common bacteria seen in urine samples confirmed
it. The 100 % specificity as well as 100 % sensitivity among
the 95 strains tested supported the usefulness of this assay for
the identification of Proteus under the growth conditions
reported here. Though the initial results are promising, the
actual clinical and environmental utility of the method re-
quires large size of the samples with many diverse organisms.
ProteAl is meant for detection of uropathogenic Proteus. It
can also be used for other clinical and environmental samples.
When it is employed for testing the urine samples or even
identifying the organisms grown on the plates from urine
samples, a small amount of the urine sample or a colony
isolated from it should be grown for 6–7 h before the fluores-
cent reagent is added. The fluorescence can be either read in a
plate reader or imaged from a UV-transilluminator. As fluo-
rescence measurements are becoming quite popular, such
instrumentation is becoming cheaper and more affordable.
However, more work is needed to standardize the assay with
urine samples and validate with samples from normal as well
as in a variety of disease conditions.
Acknowledgments We are grateful to Mr. Suresh Lingham, M/s
Trivitron Pvt Ltd. for clinical samples, Dr. Sridhar, Dept. of Microbiology,
Sri Ramachandra University, for providing standard bacterial cultures; Dr.
Mathiyarasu and Sankararao, CECRI, Karaikudi, Dr. T. Sivakumar, Prof.
B. Sivasankar, and B. Palanisamy, Anna University, and Prof.
Mohanakrishnan, University of Madras, for analysis and analytical data.
We acknowledge the financial support from Centre with Potential for
Excellence in Environmental Science (CPEES) of University Grants
Commission. Our deepest gratitude to our family and friends.
Appl Microbiol Biotechnol
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12. Author disclosure statement 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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