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A Highly Discriminatory Method for the Direct Comparison of Two Closely Related Bacterial Populations by Pyrolysis Mass Spectrometry
Zbl. Bakt. 285, 285-290 (1997) © Gustav Fischer Verlag, Jena
A Highly Discriminatory Method for the Direct Comparison of Two Closely Related Bacterial Populations by Pyrolysis Mass Spectrometry ROGER FREEMAN!, PENELOPE R. SISSOW, MICHAEL R. BARERI, ALAN C. WARD 1 , and NIGEL F. LIGHTFOOT l Public Health Laboratory, Institute of Pathology, Newcastle General Hospital, Newcastle-upon-Tyne, UK 2 Department of Microbiology, Medical School, Newcastle upon Tyne, UK 1
Summary The ability of pyrolysis mass spectrometry (Py-MS) to discriminate between paired groups of closely related bacteria has been examined. The technique was challenged with increasing levels of biological difference; from that between different subcultures of the same isolate of Staphylococcus aureus, through that between separate isolates of the same strain and eventually to that between different staphylococcal species and Streptococcus pyogenes. By using an analytical method in which the spectral data from any two groups can be directly compared at a time, it was shown that Py-MS was capable of measuring statistically significant differences between staphylococci and Streptococcus pyogenes, between different Staphylococcus species and between two isolates of the same strain of S. au reus from different sources. Two subcultures of the same isolate were found to be indistinguishable. The differing magnitude of the Py-MS-derived differences corresponded to the "biological differences", being much larger between staphylococci and streptococci than between staphylococcal species and only just statistically significant between isolates of the same strain from different sources. The technique described allows the assessment of the magnitude and significance of PyMS-derived differences between any two bacterial populations.
Introduction
Fine distinctions between different bacterial populations have been achieved using pyrolysis mass spectrometry (Py-MS). Good discrimination has been documented at species, sub-species and strain levels of difference. The method has even been shown to differentiate between a parent strain of Escherichia coli and genetically constructed derivatives, each differing only in the possession of single plasmids (4). However, most studies to date have used the method to make relative comparisons between several groups of organisms, either for taxonomic purposes (7) or, increasingly, for the detection of clusters or closely similar isolates, indicating putative strains, in epidemiological analyses (1-3). Attempts have been made to use Py-MS to directly compare only two organisms, whether species, subspecies or strains (6), but the principal
286
R. Freeman et al.
component (PC) and canonical variate (CV) analyses usually employed rely on the presence of a minimum of three groups in order to construct ordination diagrams and thus express the similarities and differences measured. The differences measured between any two groups may therefore be influenced by the obligatory presence of the third group. An analytical method has been applied which directly compares the PC and CV data from only two organisms at a time. This allows mathematically-based statements of similarity and difference between the two groups to be made which are not subject to any distortion. This method has been used to analyse a constructed series of bacterial comparisons in which different levels of biological difference were addressed.
Methods A single laboratory-maintained strain of Staphylococcus aureus (S. aureus 'SG') was used for the first experiment. This organism was originally isolated from a nasal swab and was known to be resistant to penicillin but otherwise sensitive to all common appropriate antibiotics, including fusidic acid.
Experiment 1 Staphylococcus aureus SG was grown overnight at 37°C in nutrient broth. The resultant broth culture was sampled with an automatic pipette and 0.1 mL aliquots were spread across nutrient agar plates supplemented with fusidic acid at a concentration of 0.5 mg/L. Identical aliquots were also inoculated onto control nutrient agar plates lacking the antibiotic. After overnight incubation at 37°C the fusidic-acid containing medium was inspected for colonies of presumptive fusidic acid-resistant spontaneous mutants and four such distinct colonies, designated M1, M2, M3 and M4, were then randomly selected and re-subcultured onto nutrient agar plates lacking fusidic acid to produce confluent growth after further overnight incubation. A subculture of the parental strain (P) was obtained by touching several colonies from the control nutrient agar plates and inoculating onto a further nutrient agar plate to produce confluent growth and incubated in parallel. Subcultures of each of the presumptive fusidic acid-resistant mutants and the parent organism were tested for susceptibility to fusidic acid by a disc-diffusion sensitivity method. The following day, each of the five subcultures (P, M1, M2, M3 and M4) was sampled ten times with a sterile loop and triplicate pyrolysis foils (Horizon Instruments Ltd., Heathfield, Sussex, England, UK) were prepared from each sample. This meant that each subculture (four distinct fusidic acid-resistant mutants and one control parent subculture) was represented by 10 samples, each of which had been smeared in triplicate giving a total of 150 foils to be pyrolysed. After insertion into pyrolysis tubes (Horizon Instruments), all foils were heated at 80 DC for 5 minutes prior to pyrolysis mass spectrometry. Experiment 2 Two subcultures, designated FH 1a and FH 1b, of a single clinical isolate of S. aureus previously shown to be of a strain responsible for a point-source outbreak (5) and a third culture, designated FH 2, of the same outbreak strain, but isolated from a separate patient, were each subcultured in quadruplicate onto nutrient agar. After overnight incubation at 37°C, each subculture was sampled in triplicate onto pyrolysis foils, resulting in twelve foils per original organism (FH la, FH 1b and FH 2). The foils were then processed as before. The outbreak strain used is known to be free of plasmids and of phage type 29152/52A.
Py-MS discrimination between two groups
287
Experiment 3
The strain of S. aureus used in Experiment 1 (S. aureus SG) and single strains of S. epidermidis, S. hominis and Streptococcus pyogenes were each subcultured, at 37 DC, in quadruplicate overnight onto fresh blood agar. Triplicate pyrolysis foils were prepared from each subculture (that is, twelve foils from each organism) and processed as before prior to pyrolysis. Pyrolysis mass spectrometry
Foils from each Experiment were examined in distinct batches. Pyrolysis mass spectrometry was carried out on a Horizon Instruments Py-MS 200X pyrolysis mass spectrometer at a Curie point of 530 DC, as previously described (1). For each foil the pyrolysis sequence number, integrated ion counts at unit mass intervals of 50 to 200 and the total ion count were recorded on floppy disk. The 30 foils from each subculture in Experiment 1 were labelled as separate groups in subsequent analyses. In Experiments 2 and 3 the 12 foils corresponding to each subculture were labelled as separate groups during subsequent analysis. Data analysis
A series of analyses was pursued whereby the spectral data for each individual group were compared severally with the spectral data from all other groups within an experimental batch. The same analytical sequence was followed on each occasion. As a first step, differences in spectra solely attributable to variations in the amount of material pyrolysed were identified and corrected. The normalised spectral data for each group were then analysed for within-group and between-group variation and all 150 mass ions were ranked in an order of discrimination, taking those mass ions with the highest between-group to within-group ratio as being the most discriminatory. These data were then subjected to principal component (PC) and canonical variate (CV) analysis using the Genstat 5 program. The principal component (PC) and canonical variate (CV) scores from the spectral data of the two groups selected for each comparison were then plotted as stacked histograms along an axis, the midpoint of which (designated as zero) was equidistant between the means to the two groups. The difference between the arithmetic means of the two groups was calculated. These differences are equivalent to values of X2 with one degree of freedom.
Results The results of the comparisons made between the various groups within the different experiments and the statistical significance of the differences measured are set out in the Tables 1 and 2. It can be seen from Table 1 that the differences measured between the parent strain of S. au reus (P) and the four separate daughter colonies (Ml-4), each exhibiting spontaneous chromosomal mutational resistance to fusidic acid, all fell within the 95% confidence limit. Differences measured between the individual mutant colonies were also below significance by this criterion. An ordination diagram PCCV 1 versus PCCV 2 (not shown) also failed to show any significant differentiation between the five groups. In Table 2 the results show that comparison between two subcultures of the same recent clinical isolate (FH 1a and FH 1b) were found to differ by less than a significant amount, whereas the difference measured between two separate isolates (from two different patients) of the same epidemic strain of S. aureus was shown to be sig-
288
R. Freeman et al.
Table 1. The differences between the means of principal component - and canonical variate scores of Py-MS spectral data of a colony of a parent strain of S. aureus (P) and four separate colonies of spontaneously fusidic-acid-resistant mutants, M1-M4 (Experiment 1) Groups compared
Difference between the means
PvM1 PvM2 PvM3 PvM4 M1vM2 M1 vM3 M1vM4 M2vM3 M2vM4 M3vM4
1.44 2.92 2.86 3.14 2.16 2.43 3.01 1.19 2.47 2.01
Table 2. The differences between the means of combined principal component- and canonical variate scores of Py-MS spectral data, comparing samples of two subcultures of the same culture of Staphylococcus aureus (FH 1a and FH 1b), two cultures of the same strain of S. aureus (FH 1a and FH 2) and S. aureus, S. epidermidis, S. hominis and Streptococcus pyogenes each with one another (Experiments 2 and 3) Groups compared FH 1a v FH 1b FH 1a v FH2
Difference between the means 3.23 6.17*
S. epidermidis v S. hominis
13.64**
S. aureus v S. epidermidis
11.87**
S. aureus v S. hominis
20.22**
S. epidermidis v Streptococcus pyogenes
23.08**
S. hominis v Streptococcus pyogenes
25.4**
S. aureus v Streptococcus pyogenes
47.2*'"
'" indicates difference is> 95% confidence limit; < 99% confidence limit. ** indicates difference is> 99% confidence limit.
nificant at the 5% level, but not at the 1 % level. Similarly, no significant differentiation was seen on the ordination diagram (not shown). The order of differences found when comparisons were made between members of different staphylococcal species and between Staphylococcus species and Streptococcus pyogenes are shown in Table 2. All these differences were well in excess of the value required for 1 % confidence limits (10.83). It can be seen that there was a greater difference between S. aureus and S. hominis than between S. aureus and S. epiderm idis, this latter difference being of the same order as that between S. epidermidis and S. hominis. The difference between S. aureus and Streptococcus pyogenes was found to
Py-MS discrimination between two groups
289
be much greater than the differences between S. epidermidis or S. hominis and Streptococcus pyogenes.
Discussion The analytical method employed in this study circumvents the inability to compare only two groups inherent in conventional, ordination diagram-based Py-MS analyses. A direct comparison, with mathematically-based confidence limited on the significance of the results, can now be made between any two bacterial populations. It may be that the degree of difference measured relate to the magnitude of the biological difference between the groups being compared, given the absence of any distorting effect of the presence of any third group. In the series presented here, Py-MS was shown to be unable to reliably discriminate between a parent strain of S. au reus and four single colonies selected for spontaneous mutation to fusidic-acid resistance. The mutant colonies were also indistinguishable one from another. A similar result was obtained when two subcultures from a single recent clinical isolates of S. aureus were compared (FH la and Fh Ib).It is important to note that in the fusidic acid-resistance series (Experiment 1) both parent organism and resistant mutants had been re-subcultured onto antibiotic-free medium prior to testing. The suggestion (7) that subculture onto medium containing a sub-inhibitory amount of the antibiotic may improve the possibility of making the distinction because of the encouragement of phenotypic differences between parent and mutants (in this case, production of an altered translocase enzyme (8) by the mutants) requires examination. The possibility that Py-MS is capable of detecting small, presumably phenotypic, differences between two cultures of the same strain with different culture histories is suggested in Experiment 2, in which the cultures of the same strain of S. aureus from two different patients were found to be just distinguishable. Much further work needs to be done to establish the level of correlation between the mathematically-based differences measured by this analysis and the biological meaning of these differences. For instance, this method of analysing the data may allow a mathematically precise definition of a strain. Each apparently related isolate within a cluster on an ordination diagram can, if necessary, be individually compared with each other isolate. The demonstration that subcultures of the same isolate were significantly less different than separate cultures of the same strain suggest that the previously adopted convention of strain definition (3), requiring as much difference between two subcultures of the same isolate as between two different isolates, may have been too strict. References
1. Goodacre, R. and R. C. W. Berkeley: Detection of small genotypic changes in Escherichia coli by pyrolysis mass spectrometry. FEMS Microbiol. Lett. 71 (1990) 133-138 2. Magee, J. T., J. M. Hindmarch, B. I. Duerden, and L. Goodwin: Classification of oral pigmented anaerobic bacilli by pyrolysis mass spectrometry and biochemical tests. J. Med. Microbiol. 37 (1992) 56-61 3. Freeman, R., P. R. Sisson, N. E Lightfood, and J. McLaughlin: Analysis of epidemic and sporadic strains of Listeria monocytogenes by pyrolysis mass spectrometry. Lett. Appl. Microbiol. 12 (1991) 133-136
290
R. Freeman et a!.
4. Freeman, R., M. Goodfellow, F. K. Gould, S.]. Hudson, and N. F. Lightfoot: Pyrolysis mass spectrometry for the rapid epidemiological typing of clinically significant bacterial pathogens. J. Med. Microbio!. 32 (1990) 283-286 5. Freeman, R., F. K. Gould, P. R. Sisson, and N. F. Lightfoot: Strain differentiation of capsule type 23 Streptococcus pneumoniae by pyrolysis mass spectrometry. Lett. App!. Microbio!. 13 (1991) 28-31 6. Magee, J. T., J. M. Hindmarch, B. I. Duerden, and D. W. R. Mackenzie: Pyrolysis mass spectrometry as a method for inter-strain discrimination of Candida albicans. ]. Gen. Microbio!' 134 (1988) 2841-2847 7. Gould, F. K., R. Freeman, P. R. Sisson, B. D. Cookson, and N. F. Lighfoot: Inter-strain comparison by pyrolysis mass spectrometry in the investigation of Staphylococcus aureus nosocomial infection.]. Hosp. Inf. 19 (1991) 41-48 8. Verbist, L.: The antimicrobial activity of fusidic acid. J. Antimicrob. Chemother. 25, Supp!. B. (1990) 1-5
Prof. R. Freeman, Public Health Laboratory, Institute of Pathology, Newcastle General Hospital, Westgate Road, Newcastle-upon-Tyne NE4 6BE
A Highly Discriminatory Method for the Direct Comparison of Two Closely Related Bacterial Populations by Pyrolysis Mass Spectrometry ROGER FREEMAN!, PENELOPE R. SISSOW, MICHAEL R. BARERI, ALAN C. WARD 1 , and NIGEL F. LIGHTFOOT l Public Health Laboratory, Institute of Pathology, Newcastle General Hospital, Newcastle-upon-Tyne, UK 2 Department of Microbiology, Medical School, Newcastle upon Tyne, UK 1
Summary The ability of pyrolysis mass spectrometry (Py-MS) to discriminate between paired groups of closely related bacteria has been examined. The technique was challenged with increasing levels of biological difference; from that between different subcultures of the same isolate of Staphylococcus aureus, through that between separate isolates of the same strain and eventually to that between different staphylococcal species and Streptococcus pyogenes. By using an analytical method in which the spectral data from any two groups can be directly compared at a time, it was shown that Py-MS was capable of measuring statistically significant differences between staphylococci and Streptococcus pyogenes, between different Staphylococcus species and between two isolates of the same strain of S. au reus from different sources. Two subcultures of the same isolate were found to be indistinguishable. The differing magnitude of the Py-MS-derived differences corresponded to the "biological differences", being much larger between staphylococci and streptococci than between staphylococcal species and only just statistically significant between isolates of the same strain from different sources. The technique described allows the assessment of the magnitude and significance of PyMS-derived differences between any two bacterial populations.
Introduction
Fine distinctions between different bacterial populations have been achieved using pyrolysis mass spectrometry (Py-MS). Good discrimination has been documented at species, sub-species and strain levels of difference. The method has even been shown to differentiate between a parent strain of Escherichia coli and genetically constructed derivatives, each differing only in the possession of single plasmids (4). However, most studies to date have used the method to make relative comparisons between several groups of organisms, either for taxonomic purposes (7) or, increasingly, for the detection of clusters or closely similar isolates, indicating putative strains, in epidemiological analyses (1-3). Attempts have been made to use Py-MS to directly compare only two organisms, whether species, subspecies or strains (6), but the principal
286
R. Freeman et al.
component (PC) and canonical variate (CV) analyses usually employed rely on the presence of a minimum of three groups in order to construct ordination diagrams and thus express the similarities and differences measured. The differences measured between any two groups may therefore be influenced by the obligatory presence of the third group. An analytical method has been applied which directly compares the PC and CV data from only two organisms at a time. This allows mathematically-based statements of similarity and difference between the two groups to be made which are not subject to any distortion. This method has been used to analyse a constructed series of bacterial comparisons in which different levels of biological difference were addressed.
Methods A single laboratory-maintained strain of Staphylococcus aureus (S. aureus 'SG') was used for the first experiment. This organism was originally isolated from a nasal swab and was known to be resistant to penicillin but otherwise sensitive to all common appropriate antibiotics, including fusidic acid.
Experiment 1 Staphylococcus aureus SG was grown overnight at 37°C in nutrient broth. The resultant broth culture was sampled with an automatic pipette and 0.1 mL aliquots were spread across nutrient agar plates supplemented with fusidic acid at a concentration of 0.5 mg/L. Identical aliquots were also inoculated onto control nutrient agar plates lacking the antibiotic. After overnight incubation at 37°C the fusidic-acid containing medium was inspected for colonies of presumptive fusidic acid-resistant spontaneous mutants and four such distinct colonies, designated M1, M2, M3 and M4, were then randomly selected and re-subcultured onto nutrient agar plates lacking fusidic acid to produce confluent growth after further overnight incubation. A subculture of the parental strain (P) was obtained by touching several colonies from the control nutrient agar plates and inoculating onto a further nutrient agar plate to produce confluent growth and incubated in parallel. Subcultures of each of the presumptive fusidic acid-resistant mutants and the parent organism were tested for susceptibility to fusidic acid by a disc-diffusion sensitivity method. The following day, each of the five subcultures (P, M1, M2, M3 and M4) was sampled ten times with a sterile loop and triplicate pyrolysis foils (Horizon Instruments Ltd., Heathfield, Sussex, England, UK) were prepared from each sample. This meant that each subculture (four distinct fusidic acid-resistant mutants and one control parent subculture) was represented by 10 samples, each of which had been smeared in triplicate giving a total of 150 foils to be pyrolysed. After insertion into pyrolysis tubes (Horizon Instruments), all foils were heated at 80 DC for 5 minutes prior to pyrolysis mass spectrometry. Experiment 2 Two subcultures, designated FH 1a and FH 1b, of a single clinical isolate of S. aureus previously shown to be of a strain responsible for a point-source outbreak (5) and a third culture, designated FH 2, of the same outbreak strain, but isolated from a separate patient, were each subcultured in quadruplicate onto nutrient agar. After overnight incubation at 37°C, each subculture was sampled in triplicate onto pyrolysis foils, resulting in twelve foils per original organism (FH la, FH 1b and FH 2). The foils were then processed as before. The outbreak strain used is known to be free of plasmids and of phage type 29152/52A.
Py-MS discrimination between two groups
287
Experiment 3
The strain of S. aureus used in Experiment 1 (S. aureus SG) and single strains of S. epidermidis, S. hominis and Streptococcus pyogenes were each subcultured, at 37 DC, in quadruplicate overnight onto fresh blood agar. Triplicate pyrolysis foils were prepared from each subculture (that is, twelve foils from each organism) and processed as before prior to pyrolysis. Pyrolysis mass spectrometry
Foils from each Experiment were examined in distinct batches. Pyrolysis mass spectrometry was carried out on a Horizon Instruments Py-MS 200X pyrolysis mass spectrometer at a Curie point of 530 DC, as previously described (1). For each foil the pyrolysis sequence number, integrated ion counts at unit mass intervals of 50 to 200 and the total ion count were recorded on floppy disk. The 30 foils from each subculture in Experiment 1 were labelled as separate groups in subsequent analyses. In Experiments 2 and 3 the 12 foils corresponding to each subculture were labelled as separate groups during subsequent analysis. Data analysis
A series of analyses was pursued whereby the spectral data for each individual group were compared severally with the spectral data from all other groups within an experimental batch. The same analytical sequence was followed on each occasion. As a first step, differences in spectra solely attributable to variations in the amount of material pyrolysed were identified and corrected. The normalised spectral data for each group were then analysed for within-group and between-group variation and all 150 mass ions were ranked in an order of discrimination, taking those mass ions with the highest between-group to within-group ratio as being the most discriminatory. These data were then subjected to principal component (PC) and canonical variate (CV) analysis using the Genstat 5 program. The principal component (PC) and canonical variate (CV) scores from the spectral data of the two groups selected for each comparison were then plotted as stacked histograms along an axis, the midpoint of which (designated as zero) was equidistant between the means to the two groups. The difference between the arithmetic means of the two groups was calculated. These differences are equivalent to values of X2 with one degree of freedom.
Results The results of the comparisons made between the various groups within the different experiments and the statistical significance of the differences measured are set out in the Tables 1 and 2. It can be seen from Table 1 that the differences measured between the parent strain of S. au reus (P) and the four separate daughter colonies (Ml-4), each exhibiting spontaneous chromosomal mutational resistance to fusidic acid, all fell within the 95% confidence limit. Differences measured between the individual mutant colonies were also below significance by this criterion. An ordination diagram PCCV 1 versus PCCV 2 (not shown) also failed to show any significant differentiation between the five groups. In Table 2 the results show that comparison between two subcultures of the same recent clinical isolate (FH 1a and FH 1b) were found to differ by less than a significant amount, whereas the difference measured between two separate isolates (from two different patients) of the same epidemic strain of S. aureus was shown to be sig-
288
R. Freeman et al.
Table 1. The differences between the means of principal component - and canonical variate scores of Py-MS spectral data of a colony of a parent strain of S. aureus (P) and four separate colonies of spontaneously fusidic-acid-resistant mutants, M1-M4 (Experiment 1) Groups compared
Difference between the means
PvM1 PvM2 PvM3 PvM4 M1vM2 M1 vM3 M1vM4 M2vM3 M2vM4 M3vM4
1.44 2.92 2.86 3.14 2.16 2.43 3.01 1.19 2.47 2.01
Table 2. The differences between the means of combined principal component- and canonical variate scores of Py-MS spectral data, comparing samples of two subcultures of the same culture of Staphylococcus aureus (FH 1a and FH 1b), two cultures of the same strain of S. aureus (FH 1a and FH 2) and S. aureus, S. epidermidis, S. hominis and Streptococcus pyogenes each with one another (Experiments 2 and 3) Groups compared FH 1a v FH 1b FH 1a v FH2
Difference between the means 3.23 6.17*
S. epidermidis v S. hominis
13.64**
S. aureus v S. epidermidis
11.87**
S. aureus v S. hominis
20.22**
S. epidermidis v Streptococcus pyogenes
23.08**
S. hominis v Streptococcus pyogenes
25.4**
S. aureus v Streptococcus pyogenes
47.2*'"
'" indicates difference is> 95% confidence limit; < 99% confidence limit. ** indicates difference is> 99% confidence limit.
nificant at the 5% level, but not at the 1 % level. Similarly, no significant differentiation was seen on the ordination diagram (not shown). The order of differences found when comparisons were made between members of different staphylococcal species and between Staphylococcus species and Streptococcus pyogenes are shown in Table 2. All these differences were well in excess of the value required for 1 % confidence limits (10.83). It can be seen that there was a greater difference between S. aureus and S. hominis than between S. aureus and S. epiderm idis, this latter difference being of the same order as that between S. epidermidis and S. hominis. The difference between S. aureus and Streptococcus pyogenes was found to
Py-MS discrimination between two groups
289
be much greater than the differences between S. epidermidis or S. hominis and Streptococcus pyogenes.
Discussion The analytical method employed in this study circumvents the inability to compare only two groups inherent in conventional, ordination diagram-based Py-MS analyses. A direct comparison, with mathematically-based confidence limited on the significance of the results, can now be made between any two bacterial populations. It may be that the degree of difference measured relate to the magnitude of the biological difference between the groups being compared, given the absence of any distorting effect of the presence of any third group. In the series presented here, Py-MS was shown to be unable to reliably discriminate between a parent strain of S. au reus and four single colonies selected for spontaneous mutation to fusidic-acid resistance. The mutant colonies were also indistinguishable one from another. A similar result was obtained when two subcultures from a single recent clinical isolates of S. aureus were compared (FH la and Fh Ib).It is important to note that in the fusidic acid-resistance series (Experiment 1) both parent organism and resistant mutants had been re-subcultured onto antibiotic-free medium prior to testing. The suggestion (7) that subculture onto medium containing a sub-inhibitory amount of the antibiotic may improve the possibility of making the distinction because of the encouragement of phenotypic differences between parent and mutants (in this case, production of an altered translocase enzyme (8) by the mutants) requires examination. The possibility that Py-MS is capable of detecting small, presumably phenotypic, differences between two cultures of the same strain with different culture histories is suggested in Experiment 2, in which the cultures of the same strain of S. aureus from two different patients were found to be just distinguishable. Much further work needs to be done to establish the level of correlation between the mathematically-based differences measured by this analysis and the biological meaning of these differences. For instance, this method of analysing the data may allow a mathematically precise definition of a strain. Each apparently related isolate within a cluster on an ordination diagram can, if necessary, be individually compared with each other isolate. The demonstration that subcultures of the same isolate were significantly less different than separate cultures of the same strain suggest that the previously adopted convention of strain definition (3), requiring as much difference between two subcultures of the same isolate as between two different isolates, may have been too strict. References
1. Goodacre, R. and R. C. W. Berkeley: Detection of small genotypic changes in Escherichia coli by pyrolysis mass spectrometry. FEMS Microbiol. Lett. 71 (1990) 133-138 2. Magee, J. T., J. M. Hindmarch, B. I. Duerden, and L. Goodwin: Classification of oral pigmented anaerobic bacilli by pyrolysis mass spectrometry and biochemical tests. J. Med. Microbiol. 37 (1992) 56-61 3. Freeman, R., P. R. Sisson, N. E Lightfood, and J. McLaughlin: Analysis of epidemic and sporadic strains of Listeria monocytogenes by pyrolysis mass spectrometry. Lett. Appl. Microbiol. 12 (1991) 133-136
290
R. Freeman et a!.
4. Freeman, R., M. Goodfellow, F. K. Gould, S.]. Hudson, and N. F. Lightfoot: Pyrolysis mass spectrometry for the rapid epidemiological typing of clinically significant bacterial pathogens. J. Med. Microbio!. 32 (1990) 283-286 5. Freeman, R., F. K. Gould, P. R. Sisson, and N. F. Lightfoot: Strain differentiation of capsule type 23 Streptococcus pneumoniae by pyrolysis mass spectrometry. Lett. App!. Microbio!. 13 (1991) 28-31 6. Magee, J. T., J. M. Hindmarch, B. I. Duerden, and D. W. R. Mackenzie: Pyrolysis mass spectrometry as a method for inter-strain discrimination of Candida albicans. ]. Gen. Microbio!' 134 (1988) 2841-2847 7. Gould, F. K., R. Freeman, P. R. Sisson, B. D. Cookson, and N. F. Lighfoot: Inter-strain comparison by pyrolysis mass spectrometry in the investigation of Staphylococcus aureus nosocomial infection.]. Hosp. Inf. 19 (1991) 41-48 8. Verbist, L.: The antimicrobial activity of fusidic acid. J. Antimicrob. Chemother. 25, Supp!. B. (1990) 1-5
Prof. R. Freeman, Public Health Laboratory, Institute of Pathology, Newcastle General Hospital, Westgate Road, Newcastle-upon-Tyne NE4 6BE