INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1980, p. 448-459 Vol. 30, No. 2 W20-7713/80/02-0448/12$02.00/0 Characterization of Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, and Bacillus amyloliquefaciens by Pyrolysis Gas-Liquid Chromatography, Deoxyribonucleic Acid-Deoxyribonucleic Acid Hybridization, Biochemical Tests, and API Systems A. G. O’DONNELL,’j-J. R. NORRIS,’ R. C. W. BERKELEY,”D. CLAUS,2T. KANEK0,3N. A. LOGAN,4 AND R. NOZAK13 Agricultural Research Council, Meat Research Institute, Langford, Bristol, BS18 7DY, United Kingdom’; Deutsche Sammlung uon Mikroorganismen, Gesellschaft fur Biotechnologische Forschung mbH, 0-3400 Gdttingen, West Germany2;Department of Microbiology, Institute of Physical and Chemical Research, Wako-shi,Saitama-ken 351, Japan3;and Department of Bacteriology, University of Bristol, The Medical School, Bristol BS8 1 TD,United Kingdom‘ Eight strains each of Bacillus subtilis, Bacillus pumilus, Bacillus lichenifor- mis, and Bacillus amyloliquefaciens were analyzed by using pyrolysis gas-liquid chromatography. Statistical analysis with canonical variates gave four well-sep- arated groups, which represented the four species. Further analysis of the same strains by deoxyribonucleic acid-deoxyribonucleic acid hybridization and API identification systems confirmed the discrimination obtained with pyrolysis gas- liquid chromatography. However, analysis by biochemical tests performed in the classical way gave only three groups since it was not possible to achieve separation of the strains representing B. subtilis from those of B. amyloliquefaciens when these tests were used. Pyrolysis, a process whereby molecules are way have been described (10, 16), but as yet thermally degraded in an inert gas atmosphere, there is no agreement on the best statistical has enhanced the use of conventional gas-liquid approach, and much work remains to be done in chromatography by enabling nonvolatile com- this field. pounds to be analyzed. Pyrolysis gas-liquid chro- This paper reports on the usefulness of low- matography (PGLC) was fmt proposed as an resolution PGLC when coupled to multivariate approach to microbial differentiation by Oyama data analysis for differentiating closely related (15) during the development of a system aimed groups of bacteria and provides evidence for the at detecting life on Mars. However, its potential separation of Bacillus antyloliquefaciens from in microbiology was not appreciated until Reiner Bacillus subtilis. (17) was able to distinguish different species of Mycobacterium and different serotypes of Esch- MATERIALS AND METHODS erichia coti in a reproducible manner. Since Bacterial strains and growth conditions. The then, PGLC has been used in the differentiation majority of the strains used (Table 1) were from the of numerous types of bacteria (10, 18, 22) and collection of the late T. Gibson; the cultures were held fungi (5,231.The recent application of PGLC to on soil extract agar slants at the Meat Research Insti- tute. Also included were strains from the American aerobic sporeformers by Oxborrow et al. (12-14) Type Culture Collection,the Deutsche Sammlung von indicates that, providing the cultural and chro- Mikroorganismen, and T. Kaneko. Organisms were matographic conditions remain constant, PGLC grown on membrane filters (type HAWP 047; 0.45 pm; can be applied usefully to the characterization Millipore Corp.), as described by Oxborrow et al. (12). of bacilli. Each culture was incubated for 14 h at 30°C on nutri- The variation between pyrograms of the same ent agar (Oxoid) containing 2 g of glucose per liter. strain and the high level of redundancy found in Cultures showing sporulation were replated. Only non- PGLC data require the application of data pro- sporulated cultures were used for PGLC analysis. cessing techniques capable of highlighting sig- Examination of strains by PGLC. (i) Sample nificant variations in the heights of specific preparation.Samples were harvested from the mem- peaks. Several methods for handling data in this brane filters by using a sterile platinum loop and were stored in sterile distilled water at -4°C before analysis. t Present address: School of Chemistry, The University, Bacterial suspensions containing 80 to 100 pg of cells Newcastle-upon-Tyne, NE1 7RU, United Kingdom. were applied directly to the platinum coil of a Chem- 448 VOL. 30,1980 CHARACTERIZATION OF BACILLUS 449 TABLE1. Bacillus strains used in this study Strain no. MRI no.o Identity Comments' in study 1 32 B. subtilis DSM 10 (neotype) 2 38 B. subtilis Gibson 636 3 39 B. subtilis Gibson 1111 4 40 B. subtilis Gibson 1156 5 41 B. subtilis Gibson 1137 6 42 B. subtilis Gibson 1115 7 43 B. subtilis Gibson 1136 8 44 B. subtilis Gibson 1152 9 37 B. pumilus DSM 27 (type) 10 58 B. pumilus Gibson 1130 11 59 B. pumilus Gibson 1036 12 60 B. pumilus Gibson 10 13 61 B. pumilus Gibson 47 14 62 3.pumilus Gibson 67 15 63 B. pumilus Gibson 604 16 64 B. pumilus Gibson 768 17 35 B. licheniformis DSM 13 (neotype) 18 49 B. licheniformis Gibson 307 19 50 B. licheniformis Gibson 1142 20 51 B. licheniformis Gibson 1174 21 52 B. licheniformis Gibson 46 22 53 B. licheniformis Gibson 1160 23 54 3. licheniformis Gibson 5 24 55 B. licheniformis Gibson 1158 25 72 B. amyloliquefaciens From Kaneko as B. rnegaterium 203 26 73 B. amy loliquefaciens From Kaneko as Fukumoto strain F 27 74 B. amyloliquefaciens From Kaneko as B. subtilis H 28 75 B. amyloliquefaciens From Kaneko as B. subtilis K 29 76 B. amy loliquefaciens From Kaneko as B. subtilis N 30 95 B. amyloliquefaciens From Gordon as ATCC 23843 31 96 B. amyloliquefaciens From Gordon as ATCC 23845 32 97 B. amyloliquefaciens From Gordon as ATCC 23842 a MRI, Meat Research Institute. DSM, Deutsche Sammlung von Mikroorganismen; ATCC, American Type Culture Collection. ical Data Systems 190 pyroprobe by using a microsy- each cluster of peaks in each pyrogram. Although ringe (Hamilton). Repeated firing of the probe in air setting the base line was 811 arbitrary procedure, once at 50°C ensured evaporation of excess water. established for this study, it was set for all of the (ii) Chromatography. Chromatographic analysis pyrograms in the same way (Fig. 1). A set of 23 was carried out with a Perkin-Elmer F17 gas chroma- reproducibly resolved peaks was chosen, and their tograph fitted with dual glass columns (3 m by 5-mm heights were measured to the nearest millimeter. The inside diameter) packed with 10% Carbowax 20M- criteria for choosing these peaks were that they TPA on Chromosorb W 85-100 mesh AW-DMCS showed the same relative retention time on each pyr- (Phase Separations Ltd., Queensferry, England). Py- ogram and that their heights could be measured in rolysis was carried out in a stream of nitrogen (20 ml/ every case. To remove variation due to sample size, min) at 850°C for 10 s. An injection temperature of these 23 peaks were standardized to a common total 250°C was used. Refiring of a clean probe resulted in peak height. This was done by dividing each of the 23 no shadow chromatograms. After an initial hold at peaks on each py-rogram by the sum of the 23 peaks 75°C for 2 min, the oven temperature was increased and multiplying the quotient by 1,OOO. In this way 10"C/min to 200°C and held at that temperature. pyrograms of different sample sizes could be com- Raising the temperature to 230°C after an analysis pared. The standardized data were analyzed by using removed the compounds with higher boiling points, the ICL system 4/70 computer at Rothamsted Exper- thereby cleaning the column. The total analysis time imental Station. Figures 2 and 3 show the mean peak was approximately 50 min. Output from the column heights of aJl of the pyrograms for each species (i.e., was detected by a flame ionization detector with an the species means) and illustrate the qualitative simi- attenuation of 32 and was recorded at 1 cm/min on larity and high redundancy of standardized peak two parallel chart recorders set at full-scale deflections height data from PGLC. Since it was not possible to of 2 and 5 mV. select a single peak that differentiated all of the spe- (iii) Data collection. Each culture was plated in cies, it was necessary to use statistical methods which duplicate, and the suspensions from each plate were used several or all of the peaks simultaneously. analyzed twice. A base line was set manually across Data analysis: canonical variates. Each pyro- 450 O’DONNELL ET AL. INT. J. SYST.BACTERIOL. FIG.1. Pyrogram of €3. amytoliquefaciens strain 26 showing base line and chosen set ofpeaks. ~ B 10 12 +4 ~ 46 RETENTION TIME (mln) f 120 43 100 I i i d io ti ti Ib r’o io I)CTENTION TIME (mln) FIG. 2. Line diagrams representing the mean peak heights for B. subtilis and B. amyloliquefaciens. These means were derived from all of the analyses used to define each group. gram with its particular set of peak heights (in this description is given by Marriott (11) and Blackith and case 23) can be represented as a single point in multi- Reyment (2). dimensional space, with each peak height representing DNA-DNA hybridization. For deoxyribonucleic one dimension of that space. In this study the 32 sets acid (DNA)-DNA hybridizations, organisms were of coordinates representing the strain means define a grown in a medium consisting of 5 g of polypeptone multidimensional scatter; only two or three dimen- (Wako Pure Chemical Industries Co.) per liter, 5 g of sions of this scatter can be represented visually. Can- beef extract (Wako Pure Chemical Industries Co.) per onical variates analysis redefines the distances be- liter, and 2 g of yeast extract (Difco Laboratories) per tween groups of points in terms of Mahalanobis DZ(a liter.