Identification of bacteria using linear programmed thermal degradation

Linear Programmed Thermal Degradation Mass Spectrometry

2839

Identification of Bacteria Using Linear Programmed Thermal Degradation Mass Spectrometry. The Preliminary Investigation Terence H. Risby Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania 16802

and Alfred L. Yergey" Scientific Research Instruments Corporation, 6707 Whitestone Road, Baltimore, Maryland 2 1207 (Received May 20, 1976) Publication costs assisted by Scientific Research Instruments Corporation

A novel process for the identification of bacteria has been developed. The process makes use of evolution patterns of molecular fragments generated during the well-controlled thermal degradation of bacterial cells. The process has been shown to be effective in distinguishing ten well-characterized bacterial organisms from each other and in grouping known members of the same genus together. The reproducibility of the method has been demonstrated.

Introduction The identification of bacterial organisms is important for a set of human problems which range from maximizing food production to controlling and preventing. disease. The methods commonly used for bacteria identification depend upon the nature of the organisms, the time available for the task, and the degree of identification required. Classical physical methods of identification include morphological differences detected by microscopy and enhanced by staining techniques.' This type of identification, while fairly rapid, is unsatisfactory for the speciation of most organisms. Biochemical methods used for identification are based on the ability of organisms to ferment specific sugars, or on other characteristic chemical reacti0ns.l These methods, although generally specific, require a t least 1.5 days for the reactions to be carried to a point satisfactory for identification. Some of the newer chemical techniques used for the identification of bacteria to the strain level are immunofluorescence, radio-antibody analysis, and serology.2 All of these methods are so specific that they have no general applicability and can be used only when the identification is nearly complete. Modern physical approaches to bacterial identification fall into two broad classes: thermal methods such as microcalorimetry' and pyrolysis methods including use of a gas chrom a t ~ g r a p h and/or ~ - ~ a mass spectrometer to analyze the pyr ~ l y s a t e . ~ -Other ll thermal analytical methods such as thermogravimetry, differential thermal analysis, and thermogravimetry-mass spectrometry, while used for polymer analysis, have found little or no application to bacteria analysis.12-14 Pyrolysis gas chromatography is the most widely used physical method at this time, and bacterial identifications based on its use are frequently dependent upon characteristic ratios of the heights of peaks in a gas chromatogram rather than upon the existence of characteristic peaks.7 The use of mass spectrometer with or without a gas chromatograph to analyze directly the products of a pyrolysis carried out in or near the ion source results in some improvement in identification of organisms. Nevertheless, all these methodologies employ isothermal pyrolysis, which means the bacteria identification is based upon the analysis of the complex mixture of cellular degradation products produced at a single temperature.

The purpose of this investigation is to test the feasibility of identifying bacterial organisms through the use of a new methodology. The methodology is based on the hypothesis that a sample of complex biological materials will decompose in an orderly and reproducible manner when heated gradually. The hypothesis of a sequence of characteristic reactions occurring reproducibly in complex materials has been validated in a study of the hydrodesulfurization of bituminous c0a1s.l~ This hypothesis has not been previously tested for the more complex materials represented by bacterial cells. The results of such decomposition processes in bacteria are expected to be a sequence of characteristic molecular fragments that could be monitored mass spectrometrically to produce temperature-dependent profiles of the masses of the decomposition products. Because of the temperature dependence of the mass profiles, it was felt the bacterial cell decomposition products having the same molecular weight might evolve at different temperatures. This anticipated separation of decomposition product masses resulting from sequential degradations, absent in isothermal pyrolysis, represents a parameter for identifying bacterial organisms that has not been available previously. I t was thought that these temperature-dependent profiles of specific ion intensities could be characteristic of the organisms being pyrolyzed and could therefore be used to identify them. In addition, it was thought that some of the profiles from known different but taxonomically related species could be used for the classification of organisms. Experimental Section

Apparatus. A standard Scientific Research Instruments Biospect chemical ionization quadrupole mass spectrometer was used to obtain the mass spectra. The instrumental conditions used were: 1 Torr of methane reagent gas, 0.1-mA emission current, and 160 "C source temperature. Consistent sensitivity and resolution for the mass range in this work was established using a mixture of methylstearate (protonated parent ion, m l e 299) and chromium tris(l,l,l-trifluoro)2,4-pentanedionate (protonated parent ion, mle 512). The solids probe used to introduce samples into the chemical ionization source was heated by a linear temperature programmer. A temperature ramp of 20 "Clmin operating between ambient and 400 "C was used for all of the work re-

The Journal of Physical Chemistry, Vo/. 80, No. 26, 1976

Terence H. Risby and Alfred L. Yergey

2840

ported here. Control of the probe tip temperature was effected by a feedback loop between the power source and a thermocouple junction at the probe tip. Data were recorded using a Systems Industries (Sunnyvale, Calif.) System 150 mass spectrometry data system. Scan conditions used in the mass range 250-750 amu were as follows: data acquisition times: 3 ms/amu from 250 to 350 amu, 10 ms/amu from 351 to 450 amu, 20 ms/amu from 451 to 750 amu; time between scans 1 s; time per scan 8.3 s. Because the source temperature is maintained a t 160 "C, and the rate of temperature increase is 20 OC/min, it is apparent that a linear temperature increase in the sample does not hold over the entire temperature range. The nonlinear portion of temperature increase is very reproducible and, because the time per scan is known and constant, it can be shown that after approximately the sixtieth scan, a linear relationship between temperature and scan number is obtained. Organisms. Table I lists ten organisms obtained from American Type Culture Collection (ATCC), Rockville, Md., along with the accession number of each organism. These ten organisms were prepared a t the ATCC by using a 10%innoculation of 250-ml nutrient broth, culturing for 24 h, harvesting the organisms, spinning down the cells, washing in 0.1 M phosphate buffer, and lyopholyzing for 18 h in two vials. Upon receipt, the vials were stored at 4 "C. Samples for analysis were prepared by opening a sample vial, extracting a portion of dried cells with a flamed spatula, placing the cells onto a flamed watch glass, replugging the sample vial, placing the vial into a flamed test tube, and sealing the tube with a sterile gauze swatch. Only one sample vial was open in the room a t a time, and all items in contact with the cells were flamed between openings. The sample of Citrobacter freundii used in the reproducibility studies was obtained from Dr. Frank Kocka, Billings Hospital, Chicago, Ill. Cells for analysis were obtained by streaking plates of trypticase-soy-5% sheep blood agar plates (Bioquest lot no. K4ZMMA). After approximately 18-h growth at 37 OC, each plate was divided into three sections by scoring with a sterile loop. The growth in each sector was harvested by scraping the cells from the agar and placing the cells into approximately 1ml of methanol in conical tubes. The tubes containing the cells were stored at 4 OC. The concentration of cells in the tubes was determined by using McFarland nephelometer standards and the concentrations were shown to be greater than 3 X loTcells/cm3,the most optically dense standard available. Analysis. The samples consisted of either slightly moist lyophilized cellular material (3 X lo7 cells, 10-100 pg of dry material) placed into a capillary or a suspension of cells in methanol drawn into an approximately 6-p1 sample tube by touching the surface of the suspension with the sample tube. A lower limit of the amount of cellular material in a 6-p1aliquot of the suspension was estimated to be 2 X 10: cells. Solvent was removed by pumping a t approximately 0.1 Torr at ambient temperature. The capillary sample tubes were introduced into the chemical ionization source in the tip of the solids probe. A run was initiated immediately after the probe was seated by starting the temperature programmer and the computer data acquisition. Data taking was terminated when the temperature reached 400 "C (150 scans), by which time the material had evolved from the probe tip. After a run was completed, the data stored by the computer could be displayed in a variety of formats shown schematically The Journal of Physical Chemistry, Vol. 80, No. 26, 1976

TABLE I: Organisms Used in the Characterization Process

Organism

ATTC no.

Sequence no.

Pseudomonas putida Pseudomonas putida Pseudomonas fluorescens Escherichia coli Escherichia coli Bacillus megaterium Bacillus subtilis Arthrobacter oxydans Arthrobacter globiformis Erivinia amylovora

12633 15073 13525 4147 11775 14581 6051 14358 8010 15580

1 2 3 4 5 6 7 8 9 10

in Figure 1.The objective of the data analysis was to find those temperaturehime dependent specific ion plots, Figure l D , that would be characteristic of particular bacteria. Selecting those ions to be used in making specific ion plots was done by selecting mass spectra (Figure 1C) from several prominent points in the total ion plot (Figure 1B). Candidate ions for specific ion temperaturehime profiles were those ions significantly higher than the background intensity. A table was made by combining the list of candidate ions from each of the organisms studied. Specific ion profiles were then prepared for each of the organisms at all of the mass numbers contained in the summary table of candidate ions. Study of these specific ion profiles was undertaken to find those that were judged to have the greatest ability to distinguish and classify bacteria.

Results and Discussion A total of 77 ions from all of the organisms studied were found to have intensities significantly greater than background levels. These 77 ions were used as candidate ions and specific ion temperaturehime profiles were produced for each of these masses for the ten bacterial organisms in Table I. Those profiles that could be used as characterizers of the organisms were then selected. Because the purpose of this study was to demonstrate the possibility of identifying bacteria by the use of particular specific ion temperaturehime profiles, it was decided to limit the search of the 770 profiles to the finding of a single profile for each of the bacteria in Table I that would distinguish that bacterial strain from all others in the table. This limitation is a reasonable one because the organisms listed in Table I are taxonomically quite diverse in that five genera are represented, but at the same time several genera are represented by several species e.g. Pseudomonas and several species are represented by two strains, i.e., Pseudomonas p u t i d a and Escherichia coli. If the degree of differentiation is great enough that the use of a single profile can be used to distinguish intraspecific strains, then, at the level of a feasibility study, use of a single profile to distinguish between species and genera is acceptable. The considerably more demanding task of finding multiple characterizing profiles for each organism is left to future work when computer-assisted sorting methods can be applied to the data. Figure 2 is a flow chart that shows how a set of characteristic specific ion profiles are used to distinguish the bacteria in Table I from each other. At m l e 259, six organisms are seen to be inseparable from each other, two separate from the remaining eight but not from each other, and two are completely distinguishable. Figure 3 shows the temperaturehime profile for the m / e 259 ion of all ten organisms numbered from top


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Figure 3. Profiles of ions at m/e 259.

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Figure 1. Data formats: (A) data axes; (5)total ion plot; (C) sequential mass spectra; (D) specific ion plots (temperature/time profile).

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Figure 2. Flow chart for differentiating 10 bacteria using temperatureltime profiles.

to bottom as they are numbered in Table I. The basis for Figure 2 is readily seen. Referring back to Figure 2, it is seen that m/e 261 (Figure 4)allows organism one to be separated

from the other five in its subgroup. For the sake of clarity, only those organisms being differentiated at a particular mass number have ion profiles illustrated at that mass number. The remaining five temperature/time dependent profiles required for differentiation are shown in Figures 5-9. Note that the differentiating features of the profiles chosen are those that have sharp maxima, or maxima that are displaced to temperatures different from the rather broad maximum that occurs in the region of the 120th scan (approximately 300 "C). All of the differentiating characteristics of the profiles shown in Figures 3-9 occur above the 160 O C temperature that is the beginning of linear dependence of temperature on time. In addition to distinguishing the ten organisms from each other, the specific ion temperature/time profiles can be used to obtain a posteriori, the normal taxonomic relationships between the organisms. Figure 10 is a flow chart that shows the grouping. The profiles at m/e 259 produce separations into The Journal of Physical Chemistry, Vol. 80, No. 26, 1976


2842

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Figure 5. Profiles of ions at m/e 299

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ions at m/e 523.

Figure 6. Profiles of ions at m/e 31 1.

Figure 8. Profiles

three groups: organisms 8 and 9 known to be of the same genus, organisms 1 , 2 , 3 , 4 , 5 ,and 10 known to have three genera among its members, and organisms 6 and 7 known t o belong to the same genus. The profiles a t mle 276 (Figure 11) separates the species Erwinnia organisms from the other five in the large group. The profiles a t m/e 308 (Figure 12) are used to group the known members of genus Bacillus together. In light of the great differences between the profiles of the two Bacillus organisms a t mle 259, and in fact a t many other masses not shown here, this seems to be an excellent choice. The profiles at m/e 313 show a large resemblance between the known members of genus Escherichia and differences between the three known members of genus Pseudomonas. The combination of separation a t m/e 313 and resemblance a t mle 259 is used as the means of grouping the three Pseudomonas organisms of this study.

The most likely source of error in using a temperatureltime profile a t a particular mass number is the potential presence of plasticizers in the samples. Control of experimental conditions was such that other contaminents or systematic errors were not likely to contribute to erroneous results, however, the almost ubiquitous presence of plasticizers in a laboratory could have resulted in their being in the sample. These plasticizers could produce profiles that are apparently characteristic of a bacterial organism, but in fact are characteristic of the history of the sample. Care was taken that no plastic materials contacted the organisms used in this work after they were received. Since all of the organisms were grown under identical conditions, it would be expected that any plasticizer contamination would be uniformly present in all the bacteria, thus a particular specific ion plot would not show the amount of characteristic behavior seen in Figures 3-9 and 11-13.

The Journal of Physical Chemistry, Vol. 60, No. 26, 1976


2843

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Figure IO. Flow chart for grouping 10 bacteria according to genus using temperatureltime profiles. Nevertheless, several plasticizers exist that would be expected to have the same chemical ionization masses as several of the ions used as characterizers.l: The most likely interfering plasticizer is butylbenzylphthlate, molecular weight 312. The interference would occur a t mle 313, a mass that was used to separate the Escherichia organisms from the Pseudomonas organisms. The estimated boiling point of butylbenzylphthlate is 227 "C a t 1Torr; this temperature is reached a t about the 90th scan under the experimental conditions used. There are no features common to all of the profiles at m/e 313 to indicate a particular single compound evolving a t about the 90th scan. Since the single maximum for organism 2 and the lower temperature maximum for organism 1 that occur a t

about the 90th scan do not coincide with each other when superimposed, it appears unlikely that they have a common origin in such a single simple contaminant. The long-term repeatability of the results from linear programmed thermal degradation mass spectrometric (LPTDMS) analysis of bacteria was established by repeating the analyses many times during the course of a year. During this time, the detailed procedures for the method were developed, and so the location of specific maxima shifted, but the overall shapes of specific ion profiles were the same. Having developed the methodology, the reproducibility of a particular analysis was of a very high order. This was tested using the Citrobacter freundii organisms grown on blood agar plates. Sample cells prepared in this way will constitute a more severe test of reproducibility since relatively small numbers of cells were harvested from portions of a single plate. This allowed a greater opportunity for variations to appear than with a large mass of cells spun down together. The Journal of Physical Chemistry, Vol. 80, No. 26, 1976


2044

TABLE 11: Correlation Coefficients for Replicate Runs of Citrobacter freundii

Replicate no.

1

3

4

5

Av

1.000 0.978 0.985 0.948 0.987

1.000 0.987 0.972 0.994

1.000 0.975 0.998

1.000 0.978

1.000

1.000 0.961 0.990

1.000 0.981

1.000

1.000

1 2

0.969 0.971

3 4

0.984

0.936 0.983

5

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0.930 0.965 0,946 0.979

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0.960 0.961 0.906 0.968

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120 1 ' 1

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Figure 13. Profiles for ions at m/e 313.

Reproducibility of temperature/time profiles a t various masses was shown by calculating linear correlation coefficients between each of several replicate profiles and an averaged profile. A vector representative of a particular profile in a given replicate was produced by taking the ion intensity a t every tenth scan number above the 60th, and normalizing each point in the vector to the base ion for that entire run. The vectors formed in this way for each of the replicates were then used to produce an average vector. Linear correlation coefficients between each replicate and all other replicates and the average vector were then calculated. This was done for several mass numbers for the Citrobacter jreundii samples. Table TI shows the results for two different mass numbers, m / e 259 and 261. These results are in a format that is symmetrical about the diagonal with the number located a t the intersection of any row and column representing the value of the linear correlation coefficient for those two runs, e.g,, the value of the linear correlation coefficient for the second and third replicates a t m / e 259 is 0.978. The linear correlation coefficient of each replicate with the average vector is given in the row labeled average. The linear correlation coefficients of each of these replicates both with the average value and with each of the other replicates is seen to be very high. The existence of these high values of correlation coefficients demonstrates that the The Journal of Physical Chemistry, Vol. 80,No. 26, 1976

LPTD-MS method is capable of giving a high order of reproducibility to determinations, and therefore the quality of bacterial identification based upon a number of characteristic profiles can be expected to be high. The fact that this high degree of reproducibility was obtained from organisms harvested from a small section of an agar plate increases t,he confidence one has in the ability to easily grow bacterial organisms using a generally accepted method for preparation. The purpose of this work was to demonstrate the feasibility of identifying various bacterial organisms by use of ion intensity profiles produced in the course of an LPTD-MS analysis. This goal has clearly been achieved. The degree of separation between sevral closely related organisms has been demonstrated to be high, and the separation has been shown to be relatively free from the most likely source of interfering masses. The degree of separation, while being high, has been shown to have a relationship with the taxonomy of the bacteria studied. Organisms known to be related to each other have been shown to have points of commonality in the ion profiles generated. Finally, the type of data generated in an LPTD-MS analysis have been shown to be very reproducible insofar as the linear correlation coefficients between replicate runs and the average of several runs agree with each other.

NOTE ADDED IN PROOF:I t has been brought to our attention that some work related to the present research was reported at the 2d Western Regional Meeting of the American Chemical Society (Oct, 1966) and reviewed briefly.18 This earlier work is not cited in Chemical Abstracts nor in any o f the other literature searched in conjunction with the present work. The results obtained by Friedman et al. were quite different than those obtained in this work since they were working with the low mass products of air pyrolysis. The earlier work resulted in observing few qualitative differences between six different bacterial samples, and the results from any bacterial pyrolysis were difficult to reproduce. Acknowledgments. We are grateful to Scientific Research Instruments Corporation for financial support of this work. We wish also to acknowledge helpful discussions about the calculations of correlation coefficients with Professor F. W. Lampe of Pennsylvania State University and Dr. Gordon J. Fergusson of Scientific Research Instruments, as well as to thank Mr. Brian A. Jerome for help in data acquisition. References and Notes (1)

S.T. Cowan, "Manual for the Identificationof Medical Bacteria",Cambridge

University Press, New York, N.Y., 1965. (2) R . E. Strange, Adv. Mlcrobiol. Phys., 88, lQ5 (1972).

2845

High Pressure Mass Spectrometry (3) W. J. Russell, J. F. Zettler, G. C. Blanchard, and E. A. Boling, "New Approaches to the Rapid Identification of Microorganisms", C. Tiborilleni, Wiley, New York, N.Y., 1975, pp 393-406. (4) P. D. Zemany, Anal. Chem., 24, 1709 (1952). (5) E. Reiner. Nature (London),206, 1272 (1965). (6) E. Reiner and W. H. Ewing, Nature(London), 217, 191 (1966). (7) E. Reiner, J. J. Hicks, R. E. Beam, and H. L. David, Am. Rev. Respir. Dis., 101, 656 (1971). ( 8 ) P. G. Vincent and M. L. Kulik, Appl. Microbiol., 20, 957 (1970). (9) H. L. C. Meuzelaar and P. G. Kistermaker, Anal. Chem., 45, 587 (1973). (10) H. R. Schulten, H. D. Becky. H. L. C. Meuzelaar, and A. J. H. Boerboom, Anal. Chem., 45, 191 (1973). (11) J. P. Anhalt and C. Fenselau, Ana!. Chem., 47, 219 (1975). (12) B. D. Mitchell and A. C. Birnie, Differential Thermal Analysis", R. C.

McKenzie, Ed., Academic Press, London, 1970, Chapter 24. (13) C B. Murphy, ref 12, Chapter 23. (14) E. K. Gibson and S. M. Johnson, Thermochim.Acta, 4, 49 (1972). (15) A. L. Yergey, F. W. Lampe, M. L. Vestal, E. J. Gilbert, and G. J. Fergusson, "Gassification of Fossil Fuels under Oxidative, Reductive and Pyrolytic Conditions", Final Report to EPA, Project No. 68-02-0206 (US. Government Access: EPA-650/2-73-042). (16) S. R. Prescott,J. E. Campana, P. C. Jurs, T. H. Risby and A. L. Yergey, Anal. Chem.,'40, 827 (1976). (17) "Modern Plastics Encyclopedia", McGraw-Hili, New York, N.Y., 1946, pp 460-477. (18) H. L.Friedman,G. A. Griffith, and H. W. Goldstein in "Thermal Analysis", Vol. 1, R. F. Schwanker, Jr., and P. D. Gam, Ed., Academic Press, New York, N.Y., 1969, p 405.

Arrival Time Distributions in High Pressure Mass Spectrometry. 5. Effect of €/P on Measured Apparent Heats and Entropies of Reaction G. G. MeiseIs,* R. K. Mltchum,+ and J. P. Freeman* Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588 (Received May 25, 1976) Publication costs assisted by the University of Nebraska

The temperature dependence of the equilibrium involving the proton transfer between methane and carbon dioxide and their protonated analogues has been investigated at a series of values of the field strength EIP. The apparent values of A S and AH were found to depend on EIP; experimental uncertainty in the former was too large to establish systematic trends. The latter decreased approximately linearly with EIP; limiting values of AH = -1.58 f 0.06 kcal/mol and A S = 1.7 f 0.3 eu are in good agreement with those obtained by flowing afterglow and ICR techniques.

Introduction High pressure mass spectrometry has been employed extensively in recent years for the measurement of ionic equilibria in the gas phase.?-4 Values for A S and AH are derived from studies of the temperature dependence of the equilibrium constant K . Such measurements may be subject to systematic these fall into two categories. One results from uncertainties in the conditions prevailing in the ion source. These are of interest here. Another is associated with the problems of ion sampling, discrimination, collision induced dissociations, and similar processes which are primarily external tc; the reaction region. Two complications exist within the reaction region itself. The first is caused by the time dependence of the approach to eq~ilibrium.~,g Time resolved measurements are now accepted as essential. The second difficulty arises from the occasional use of extraction fields in high pressure mass spectrometers of the chemical ionization type, or in drift tube mass spectrometers. It is well known that average ion energies are shifted upward by applied fields and that the ion velocity distributions lose Maxwell-Boltzmann character at higher values of ElP.9JoIn this investigation we report our findings on the effect of EIP on measured values of the equilibrium Present address: NCTR-FDA, Jefferson, Ark. 72079. Present address: Department of Chemistry, University of Hawaii, Honolulu, Ha. +

constant under conditions where equilibrium is totally achieved. We have chosen to study the equilibrium

CO*H+ + CH4

COz

+ CHb+

(1)

for several reasons. It is one of the earliest studies by high pressure mass spectrometry in the laboratory of Professor Franklin." A careful study of this system has also been made by the flowing afterglow technique,12and has received support from another investigation employing ion cyclotron resonance.13The previous findings should provide a reliable reference point against which to evaluate our observations. Lastly, the enthalpy change associated with the equilibrium of reaction 1is relatively small, so that the equilibrium reaction should proceed rapidly and ions should be thermalized quickly. The small exothermicity of the reaction also leads to the hope that processes external to the ion source such as collision induced dissociation, etc., will affect both ions approximately equally in spite of the substantial difference in mass.

Experimental Section The experimental procedures have been described previ0usly.1~Briefly, an Atlas CH-4 mass spectrometer was modified by the installation of a high pressure ion source and differential pumping system. Gases were maintained in sepThe Journal of Physical Chemistry, Vol. 80, No. 26, 1976

Identification of bacteria using linear programmed thermal degradation

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