A
NALYSIS OF
V
OLATILE
M
ETABOLITES DURING
P
HAGE
-B
ACTERIA
I
NFECTION VIA
G
AS
C
HROMATOGRAPHY
/M
ASS
S
PECTROMETRY
Jacqueline Black, Lambert Kabwar, James Erdmann, and Dr. Franco Basile
Department of Chemistry, University of Wyoming
I
NTRODUCTION
Current detection methods of potential biological weapons
require hours to days for specific identification, as well as
the use of toxic chemicals or complex and bulky
instrumentation. The specificity of the phage-bacteria
infection process and the known fact that small metabolite
molecules are produced during this infection stage
1could
provide the opportunity for rapid analysis with use of no
toxins and relatively small and simple instrumentation. This
platform can be coupled with solid-phase microextraction
(SPME) and mass spectrometry (MS) in a field setting for
rapid detection of both potential biological weapons and
microorganisms of clinical and/or food safety relevance.
M
ETHODS
• E. coli: E. coli was grown on Trypticase Soy Agar (TSA) plates in an
incubator at 37°C for 8-10 hours. After incubation, 108 cells were obtained in 5mL of a lambda buffer consisting of 8mM MgSO4 • 7H2O, 100mM NaCl, and 50mM TrisH+ at pH of 7.5 using an absorbance reading of approximately 0.20 at 600nm.
• MS2 Infection: E. coli was infected with a 100:1 phage:bacteria ratio from a
1010 pfu/mL stock solution of MS2.
• Standards: A standard volatile organic compound (VOC) mixture was
created with 20, 20, 20, 20, 5, and 5 μg/mL of acetone, pyridine, isopropanol, 1-butanol, ethanol, acetic acid, and propanoic acid, respectively.
• Sampling: The headspace of a headspace vial or modified microcentrifuge
tube was sampled with a SPME divinylbenzene (DVB)/Carbowax fiber. The mixture was held at 35-40°C with the fiber exposed to the headspace for 5 or 30 minutes.
• Analysis: The headspace was analyzed using GC/MS (Model DSQ, Thermo)
with 10 seconds of fiber exposure to the injector at 150°C. The oven was ran at 40°C for two minutes, ramped at 20°C/minute to 120°C, and then held for a total run time of 10 minutes.
D
ISCUSSION
/M
ETHOD
D
EVELOPMENT
The method is based on the detection of VOCs formed during the infection of bacteria with phage. SPME is used to sample the headspace during the phage-bacteria infection for VOCs (Figure 6A),
which are then analyzed using GC/MS (Figure 6B).
Lytic bacteriophages infect their specific host, where they then multiply and cause the host cell to rupture (Figure 1). This process occurs very rapidly, with the time from a single infection to cell
rupture lasting less than 30 minutes. Sonkar et al.
1recently demonstrated a measurable metabonomic response of a bacterium, Pseudomonas aeruginosa strain K, to a bacteriophage, pf1, using nuclear
magnetic resonance (NMR), showing that phage infection causes additional production and/or release of metabolites. SPME coupled with field-portable mass spectrometry is proposed as a reliable and
specific approach to detect such volatile metabolites (i.e., VOCs) in a variety of scenarios (e.g., national defense, clinical, food safety).
L
ITERATURE
C
ITED
1. Sonkar, K., R.N. Purusottam, and N. Sinha. 2012. Metabonomic study of host–phage interaction by nuclear magnetic resonance- and statistical total correlation spectroscopy-based analysis. Analytical Chemistry 84:4063–4070.
A
CKNOWLEDGMENTS
The authors thank Raj Mahat for his indispensable help with setting up and using the GC/MS. We also thank Dr. Franco Basile’s other graduate students, Rudy Mingon, Mitch Helling, Rong Zhou, Liang Lu, and Chenglin Liu for help with method optimization, planning and laboratory navigation. This project was supported in part by grants from the National Center for Research Resources (P20RR016474) and the National Institute of General Medical Sciences (P20GM103432) from the National Institutes of Health. The GCMS used in this project was purchased with funds from a NSF-CAREER award (CHE-0844694 to FB). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
R
ESULTS
20140418-05ecoli RT: 0.04 - 10.03 Mass: 35.00 - 180.00 NL: 3.13E6
1 2 3 4 5 6 7 8 9 10 Time (min) 40 60 80 100 120 140 160 180 m /z
20140418-07blank2 RT: 0.04 - 10.01 Mass: 35.00 - 180.00 NL: 4.02E6
1 2 3 4 5 6 7 8 9 10 Time (min) 40 60 80 100 120 140 160 180 m /z
Acetone(0.30)
iPrOH(0.70)
Acetic Acid(0.73)
1-BuOH(1.20)
Pyridine(1.76)
Propanoic Acid (N/D)
A.
B.
Figure 4: A.) Ion map of the lambda buffer (blank). B.) Ion map of 108 cells/mL
of E. coli in lambda buffer.
Figure 2: Chromatogram of VOC standard mixture.
C H3 O Figure 3: Mass spectrum of Decanal from E. coli in lambda buffer (eluted at 7.64 min.)
A.
B.
Figure 5: A.) Chromatogram of the lambda buffer (blank). B.) Chromatogram
of 1:100 bacteria-to-phage infection of E. coli with MS2 in lambda buffer.
RT:0.00 - 10.03 0 1 2 3 4 5 6 7 8 9 10 Time (min) 0 10 20 30 40 50 60 70 80 90 100 Re lativ e A bu nd anc e 1.76 1.20 0.73 0.70 4.64 NL: 1.08E8 TIC MS sample5 RT:0.00 - 10.02 0 1 2 3 4 5 6 7 8 9 10 Time (min) 0 10 20 30 40 50 60 70 80 90 100 Rela tiv e A b u n d ance 1.03 6.43 7.64 5.52 3.40 4.52 9.49 NL: 1.69E6 TIC MS 20140327-1 20140327-1#1810-1818 RT:7.62-7.65 AV:9 SB:47 7.51-7.60 , 7.68-7.77 NL:1.09E4 T:+ c Full ms [35.00-150.00] 40 50 60 70 80 90 100 110 120 130 140 150 m/z 0 10 20 30 40 50 60 70 80 90 100 R el ati v e A bu nd an ce 41.06 43.06 57.05 55.05 70.05 71.09 82.06 68.03 44.03 83.00 67.02 81.05 54.06 112.14 95.08 110.11 45.05 72.07 84.02 128.12 97.06 138.28 Decanal Time (min) m/z Time (min) m/z RT: 0.00 - 10.01 0 1 2 3 4 5 6 7 8 9 10 Time (min) 0 10 20 30 40 50 60 70 80 90 100 Re lativ e A bu nd anc e 1.04 1.20 7.40 1.43 6.41 7.61 7.09 4.35 5.49 NL: 4.58E6 TIC MS 20140418-07blank2 RT: 0.00 - 10.03 0 1 2 3 4 5 6 7 8 9 10 Time (min) 0 10 20 30 40 50 60 70 80 90 100 Re lativ e A bu nd anc e 1.03 7.31 1.18 1.25 6.42 7.09 7.61 5.09 NL: 3.79E6 TIC MS 20140418-06phage
A
BSTRACT
Preliminary results are presented in the development of a rapid
and
field-portable
method
for
bacteria
detection
and
identification.
Figure 1: Schematic of bacteriophage infection. Detection of Volatile Organic
Compounds (VOC’) during the phage-bacteria infection will be indicative of the presence of bacteria.
SPME SPME
GC oven
Mass Analyzer
Figure 6: A.) SPME sampling of VOC in headspace. B.) SPME analysis in GC/MS.
A.
B.
Source: http://torion.com
Figure 7: Proposed field portable MS to perform detection of VOC. TRIDION-9
