A Thesis Submitted by for the Degree of in the UNIVERSITY of LONDON
Total Page：16
File Type：pdf, Size：1020Kb
1 THE CLASSIFICATION OF STREPTOCOCCAL STRAINS A thesis submitted by GEOFFREY COLMAN for the degree of DOCTOR OF PHILOSOPHY in the UNIVERSITY OF LONDON Department of Bacteriology, Wright-Fleming Institute, St..Mary's Hospital Medical School, Paddington, London, W.2. February 1970 2 Abstract Three hundred and sixty-four strains of strepto- cocci were examined using standard serological methods with 23 different sera and by more than 40 different cultural and physiological tests. The cell wall constituents of 232 of these strains were determined and the data from 216 strains were examined by numerical methods using three computer programmes that formed clusters in different ways. Some species that have been formed in the past by conventional methods were seen to be satisfactory and these were: Streptococcus pyogenes, S. airalactiae,m S. ecLuisimilis, S. faecalis, S. faecium, S. bovis, S. uberis, the haemolytic i large-colony' strains of Lancefield group G, S. salivarius, S. lactic, S. cremoris, S. pneumoniae and S. mutans. The definitions of S. milleri, S. sanpuis and S. mitior have been amended and many streptococci that in the past would have been classed as I viridans-like' were assigned to one or other of these aggregated species. The validity of the species S. milleri, S. sanuis and S. mitior were tested by performing transformation reactions between 14 strains allotted to one or other of these species. The numbers of recombinants produced in these experiments were greater in intra- specific reactions than in inter-specific reactions. Some taxa, descriptions of which are available in the literature, were not represented among the newly isolated strains. The relationships between some culture collection strains of these taxa and the 16 species listed above was not always clear and this would presumably be a profitable field for future investigation. Acknowled9ments The work described in this thesis was carried out under the direction of Professor R.E.O. Williams. The project was supported by the Medical Research Council and the ultra-sonic disintegrator used in the transformation experiments was supplied by the Smith, Kline and French Foundation. Many of the strains studied were isolated in other laboratories and these laboratories are listed in Table 1, page 13. The workers who generously sent strains for examination made a considerable contribution to the investigation. Technical assistance was provided at different times by Miss Catherine Norton, Mr. John Hayward, Mr. Derek Manson-Smith and Miss Doreen Place. k Contents Page number General introduction 6 Strains studied 11 Methods used in tests of cultural characteristics 14 Methods of study of the bacterial cell walls 34 Serological methods 38 Reproducibility of the methods 41 Techniques in the transformation experiments 47 Description of the computer programmes 54 Attributes common to the streptococci 64 Results of the numerical studies 72 Guide to the properties of the various taxa 82 Streptococcus py212,1enes (Lancefield group A) 87 0S. agalactiae (group B) 105 S. equisimilis and other streptococci of group C 117 S. faecalis, S. faecium, S. bovis and S. eq,uinus 128 S. lentus (group E), group P and group U 147 S. uberis and S. acidominimus 157 S. rnilleri and Lancefield group F 169 Strains of serological group G 197. S. sanFuis and serological group H 206 S. salivarius and serological group K 226 Serological group L 249 Serological group N 252 5 Contents (continued) Page number Strains giving multiple group reactions 258 0a lactis and S. cremoris (both group N) 266 S. mitior and serological group 0 280 S. pneumoniae 298 S. avium (group Q) 308 Serological group R 311 Serological group S 313 Serological group T 315 S. mutans 317 Streptococci pathogenic for fish 326 An unclassified strain 327 Strains excluded from the streptococci 329 References 335 0 General Introduction The investigation described in this thesis was begun at the end of 1961 with the aim of seeing whether the application of techniques such as cell wall analysis or numerical methods of taxonomy, which were then relatively new procedures, would bring order to the viridans-like streptococci. The first step in the investigation was to examine strains, some from culture collections and some freshly isolated, by the conventional cultural and serological procedures. It s,7)(7,n became apparent that the investigation could not be restricted to the viridans-like streptococci because some strains gave anomalous results. Some strains, for instance, that carried the Lancefield A antigen were non-haemolytic and resistant to bacitracin. It was thought that these cultures were not representative of S. pyogenes but clearly the non-haemolytic strains had to be compared with haemolytic strains of the same serological group. Where possible, the bacteria described in this thesis have been assigned to established taxa. Some species among the streptococci, for example, S. pneumoniae, are intuitively satisfying, but the published description of a species such as S. mitior 7 seu viridans is often merely a list of distinguishing properties that are absent from the species. The strains composing some species were seen to have characteristics that were highly correlated within the species but the characteristics rarely occurred together outside the species. The strains of S. agalactiae, for example, were streptococci of Lancefield group B, that grew on 40% bile agar, in --2 sodium chloride, hydrolysed hippurate and gave the CAMP reaction, but did not hydrolyse aesculin, grow at 45°C. or survive 60°C. for 30 minutes. Not one of the properties just listed is unique to S. agalactiae, but these properties are found in this combination only in this species. In her original description of the serological grouping of streptococci Dr. Lancefield (1933) demonstrated that clear correlations existed between serological group, sources of the strains, pathogen- icity and the results of a range of cultural tests. It is unfortunate that later workers used less stringent criteria when they formed additional serol- ogical groups. Some experienced bacteriologists, for example Williamson (1964), believe that serological groups should be the basis of the classification of many of the streptococci. There are however well- documented instances where a classification based solely on serological croup has yielded physiologically heterogeneous taxa. This is true of serological group K (Williams 1956), group M (Skadhauge and Perch 1959) and group D (Deibel 1964). Some pediococci yield precipitates with sera of group D (Coster and White 1964). The classification of streptococci has increased in complexity from the early comprehensive classific- ations, such as that of Brown (1919). Brown divided the genus into three divisions on the basis of changes produced in blood agar, and further sub-divided these categories into 11 named species on the results of testing the 2ermentation of six substrates. Sherman (1937) has convincingly argued that increased precision in the definition of streptococcal species has followed the application of new tests to taxonomic problems. The advances listed by Sherman all followed empirical testing. There is, for example, no biological theory from which a prediction could have been made that S. lactis and S. cremoris could be distinguished by differences in their temperature limits of growth or differences in tolerance of sodium chloride. Sherman (1937) placed the various species of streptococci into one or other of four divisions, and he named these divisions 'pyogenic', 'viridans', 'lactic' and 'enterococcus'. His viridans division contained the species S. salivarius, S. equinus, S. bovis and its varieties, and S. thermophilus. The species in the pyogenic division were distinguished Primarily by their group antigens. The features of the species in Sherman's viridans division were the absence of group antigens, and the inability of the strains to grow at 10°C. or in 0.1 methylene blue milk. The strains in this division did not hydrolyse arginine and, with the exception of some strains of S. salivarius, all grew at 45°C. Swift (1952) extended Sherman's classification by placing additional species in the pyogenic and viridans divisions. Two of the species Swift listed as viridans streptococci were S. uberis and Strepto- coccus sp. MG and strains of these species hydrolysed arginine and did not grow at 45°C Later work made the boundaries between the pyogenic and viridans divisions even less clear. Streptococcus sp. MG, for instance, was found to carry the Lancefield F antigen (Willers, Ottens and Michel 1964) and many strains of S. sanguis carry the H antigen (Porterfield 1950). In the light of such findings 10 it was decided that in this thesis the species would be used, as far as possible, as the basic taxonomic unit, and Sherman's divisions of the genus would not be followed. The experimental findings with each species is prefaced by an account of the evolution of the concept of that species. These historical accounts could, alternatively, have been brought together as a general introduction. The latter arrangement was not used because it was thought that the description of the development of a species would be of more interest in juxtaposition to an account of the strains assigned to that species than if it were one of a series of descriptions. 11 Strains studied This work is based on the study of 386 strains of bacteria assembled in an attempt to make a collection that included representative strains of as many as possible of the recognized species of streptococci as well as a large number of streptococci from man that do not fall into any of the established species. The collection does not therefore represent any natural population. The sources of the strains are shown in Table 1. Most were of human origin; 354 were streptococci that grew readily in air on horse blood agar; 10 were strains isolated from dental caries in man and animals and had a requirement for carbon dioxide.