Acta path. rnicrobiol. scand. Sect. B, 83: 129-132, 1975
A FIMBRIATED STRAIN OF P A S T E U R E L L A M U L T O C I D A WITH SPREADING AND CORRODING COLONIES S. D. HEMUKSEN and L. 0. FROHOLM Kaptein W. Wilhelmsen og Frues Bakteriologiske Institutt, Oslo University, Rikshospitalet, Oslo 1, and Statens Institutt for Folkehelse, Oslo 1, Norway
Henriksen, S . D. & Frvholm, L. 0. A fimbriated strain of Pasteurella multocida with spreading and corroding colonies. Acta path. microbiol. scand. Sect. B, 83: 129-132, 1975. A strain identified as Pasteurella multocida (“dog type”) grew on blood agar with spreading and corroding colonies; it was found to be fimbriated. A non-fimbriated variant of the strain which did not produce spreading and corroding colonies was isolated. T h e fimbriated strain regularly formed surface growth by static incubation in broth. The mode of surface translocation of this strain appeared to be twitching. Key words: Pasteurella niultocida; fimbriated strain ; spreading and corroding colonies.
S . D. Henriksen, Bacteriological Institute, Rikshospitalet, Oslo 1, Norway.
Received 9.xi.74
Accepted 9.xi.74
The purpose of this paper is to report the isolation of an unusual strain of P. multocida from a human middle ear. Case history: T h e patient was a 47 years old woman who had suffered from chronic otitis media on the right side; continuous secretion had persisted for 35 years, associated with gradual eczematous changes of the skin. She now visited a n ear-nose-throat specialist, complaining of a n acute exacerbation of the condition and increased secretion. Pus from the right ear yielded abundant growth of the strain to be described and a few colonies of white staphylococci.
METHODS Conventional methods were used for the study of morphological, cultural and biochemical characters of the strain. Electron microscopy was carried out as described previously ( 3 ) . Agar plate microscopy was carried out as described by Henrichsen ( 6 ) . Transforination: T h e methods described by 9 Acta path. mict-obiol. scand. Sect. R. 83, 2
Bsvre ( 1 ), including continuous DNA-exposure, as well as a number of modifications were tried, including the use of different media with and without serum and with and without addition of calcium ions, recipient cultures of different age, different times of DNA-exposure and phenotypic expression, and different concentrations of streptomycin for transformant selection. I n view of the negative results, details d o not seem necessary.
R E S U I, T S
Microscopy: The strain consisted of tiny gram-negative coccoid and coccobacillary cells with a few short rods and occasional thin filaments. Colonies: After about 20 h, colonies were about 1 mm in diameter; they were round, low convex, smooth, shiny, but not mucoid. There was no change of the medium. After 2 days in a humid atmosphere a t 35’ C, the size increased to about 2 mm in diameter. The colonies appeared to have sunk slightly
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Fig. I. Spreading and corroding colonies on blood agar, incubated in a humid atmosphere for 7 days, x 4.
down in the agar, a narrow, shallow groove being in evidence along the margin. Rather deep, irregular depressions became apparent when the colonies were scraped off. After about 3 days, thin zones of spreading growth started to appear around the colonies and, in the following days, the zones increased to considerable size (Fig. 1). Corrosion of the agar under the zones was not noted. From a blood agar culture of the strain, a non-spreading variant was isolated after subcultivation of growth bulging from the margin of a colony on agar base without blood. The colonies of the variant resembled those of the original strain, but did not appear to sink down in the agar; when they were scraped off, only very shallow, smooth, just visible depressions, but no “corrosion”, were seen and, on prolonged incubation, no
Fig. 2. Cells of spreading-corroding variant showing fimbriae (upper), and of non-spreading variant, without fimbriae (lower). Optical differentiation used ( 2 ) , x 60,000.
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spreading zones appeared. Otherwise the two variants appeared to be identical. Growth characteristics: The strains appeared to be somewhat fastidious and failed to grow on some of the simpler fluid media, e.g. Clark & Lub’s medium and nitrate broth. Growth on nutrient agar was fair. No growth occurred at room temperature. The strains were facultative anaerobes. In stationary cultures in fluid media, a marked tendency of the spreading strain to form surface pellicles was noted. Biochemical reactions: The following tests were positive : Catalase, oxidase, acid production (fermentative) from glucose, galactose, xylose (weak) and sucrose, indole production. The following tests were negative: Fermentation of lactose, maltose, rhamnose, arabinose, mannitol, sorbitol, and dulcitol, production of H2S (TSI-medium), urease, phenylalanine deaminase, liquefaction of gelatine and coagulated serum. These reactions appear to be compatible with a classification as P. multocida, the “dog type’’ as described by Frederiksen ( 5 ) . Both strains were sensitive to penicillin and to other commonly used antibiotics. Electron microscopy: About one in ten cells of the corroding and spreading variant was found to be fimbriated, but the cells of the non-corroding, non-spreading variant could not be shown to have fimbriae when several hundred were carefully examined (Fig. 2). Agar microscopy: On media without blood; spreading zones were only irregularly seen ; the small size of the cells and the very narrow dimensions of the spreading zones observed made it difficult to see twitching motility. In some zones, however, particularly around miniature colonies, an arrangement of the cells typical of twitching was seen (Fig. 3 ) . Also the appearance of the spreading zones was strikingly similar to that of zones produced by Moraxella strains, an appearance which is due to twitching. Transformation: All attempts failed to transform any of the two variants, using 9
Fig. 3. Miniature colonies of non-spreading (right) and of spreading variant (left). The arrangement of the cells around the latter colony suggests twitching, x 1,000.
DNA from streptomycin resistant mutants of both variants. DISCUSSION
We have not found any previous description of strains of P. multocida producing spreading and corroding colonies. As in the Moraxella species M . nonliquefaciens, M . bovis and M . kingae (2, 4, 7 ) ) this trait was associated with fimbriation and, also as in the case of Moraxella, it was possible to isolate a nonspreading, non-corroding and unfimbriated variant. The spreading zones produced by the Pasteurella strain were very like those caused by twitching of the mentioned Moraxella species and, although we did not succeed in actually seing twitching cells, we feel that the arrangement of the cells in some spreading zones indicates that they were actually produced by twitching. Contrary to expectation, competence in transformation was not demonstrated in the spreading/corroding strain. This might indicate that fimbriation in this strain is not associated with competence, as in Moraxella species, but it cannot be precluded that the fimbriae may not have been of the right type, or simply that we failed to find conditions suitable for transformation. I t may be mentioned that previous experiments (8) have failed to detect competence
131
in strains of P. multocida, although strains of other Pasteurella species appeared to be transformable. Corroding and spreading was not detected in any of those strains. The isolation of the strain described in this paper shows that the association of spreading/corroding colonies with twitching and fimbriation is not a property of Moraxella species exclusively, but may also be found in P. multocida. I t may be emphasized that such phenomena may remain unobserved unless cultures are incubated for periods of time longer than one or two days. Incubation in a humid atmosphere may also be important, although systematic studies of this point have not been carried out.
REFERENCES 1. Bevre, K.: Studies on transformation in Mo-
raxella and organisms assumed to be related to Moraxella. 1. A method for quantitative transformation in Moraxella and Neisseria, with streptomycin resistance as the genetic marker. Acta path. microbiol. scand. 61: 457-473, 1964.
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2. Bevre, K., Bergan, T . & Froholm, L. 0.: Electron microscopical and serological characteristics associated with colony type in M o raxella nonliquefaciens. Acta path. rnicrobiol. scand. Sect. B, 78: 765-779, 1970. 3. Bovre, K . & Froholm, L. 0.:Variation in colony morphology reflecting fimbriation in Moraxella bovis and two reference strains of M . nonliquefaciens. Acta path. rnicrobiol. scand. Sect. B, 80: 629-640, 1972. 4. Bevre, K . & Froholm, L. 0.: Competence in genetic transformation related to colony type and fimbriation in three species of Moraxellae. Acta path. rnicrobiol. scand. Sect. B, 80: 649659, 1972. 5. Frederiksen, W.: Pasteurella taxonomy and nomenclature. I n Winblad, S. ( E d ) : Contributions to microbiology and immunology. 2. Yersinia, Pasteurella and Francisella. Karger, Base1 1973, pp. 170-176. 6. Henrichsen, 1.: Bacterial surface translocation. A survey and a classification. Bacteriol. Rev. 36: 478-503, 1972. 7. Henrichsen, J., Froholm, L. 0 . & Bevre, K . : Studies on bacterial surface translocation. 11. Twitching motility associated with fimbriation in colony variants of Moraxella nonliquefaciens, M . bovis and M . kingii. Acta path. microbiol. scand. Sect. B, 80: 445-452, 1972. 8. Henriksen, S . D.: Transformation of streptomycin resistance in Pasteurella. Acta path. microbiol. scand. 55: 496, 1962.
A FIMBRIATED STRAIN OF P A S T E U R E L L A M U L T O C I D A WITH SPREADING AND CORRODING COLONIES S. D. HEMUKSEN and L. 0. FROHOLM Kaptein W. Wilhelmsen og Frues Bakteriologiske Institutt, Oslo University, Rikshospitalet, Oslo 1, and Statens Institutt for Folkehelse, Oslo 1, Norway
Henriksen, S . D. & Frvholm, L. 0. A fimbriated strain of Pasteurella multocida with spreading and corroding colonies. Acta path. microbiol. scand. Sect. B, 83: 129-132, 1975. A strain identified as Pasteurella multocida (“dog type”) grew on blood agar with spreading and corroding colonies; it was found to be fimbriated. A non-fimbriated variant of the strain which did not produce spreading and corroding colonies was isolated. T h e fimbriated strain regularly formed surface growth by static incubation in broth. The mode of surface translocation of this strain appeared to be twitching. Key words: Pasteurella niultocida; fimbriated strain ; spreading and corroding colonies.
S . D. Henriksen, Bacteriological Institute, Rikshospitalet, Oslo 1, Norway.
Received 9.xi.74
Accepted 9.xi.74
The purpose of this paper is to report the isolation of an unusual strain of P. multocida from a human middle ear. Case history: T h e patient was a 47 years old woman who had suffered from chronic otitis media on the right side; continuous secretion had persisted for 35 years, associated with gradual eczematous changes of the skin. She now visited a n ear-nose-throat specialist, complaining of a n acute exacerbation of the condition and increased secretion. Pus from the right ear yielded abundant growth of the strain to be described and a few colonies of white staphylococci.
METHODS Conventional methods were used for the study of morphological, cultural and biochemical characters of the strain. Electron microscopy was carried out as described previously ( 3 ) . Agar plate microscopy was carried out as described by Henrichsen ( 6 ) . Transforination: T h e methods described by 9 Acta path. mict-obiol. scand. Sect. R. 83, 2
Bsvre ( 1 ), including continuous DNA-exposure, as well as a number of modifications were tried, including the use of different media with and without serum and with and without addition of calcium ions, recipient cultures of different age, different times of DNA-exposure and phenotypic expression, and different concentrations of streptomycin for transformant selection. I n view of the negative results, details d o not seem necessary.
R E S U I, T S
Microscopy: The strain consisted of tiny gram-negative coccoid and coccobacillary cells with a few short rods and occasional thin filaments. Colonies: After about 20 h, colonies were about 1 mm in diameter; they were round, low convex, smooth, shiny, but not mucoid. There was no change of the medium. After 2 days in a humid atmosphere a t 35’ C, the size increased to about 2 mm in diameter. The colonies appeared to have sunk slightly
129
Fig. I. Spreading and corroding colonies on blood agar, incubated in a humid atmosphere for 7 days, x 4.
down in the agar, a narrow, shallow groove being in evidence along the margin. Rather deep, irregular depressions became apparent when the colonies were scraped off. After about 3 days, thin zones of spreading growth started to appear around the colonies and, in the following days, the zones increased to considerable size (Fig. 1). Corrosion of the agar under the zones was not noted. From a blood agar culture of the strain, a non-spreading variant was isolated after subcultivation of growth bulging from the margin of a colony on agar base without blood. The colonies of the variant resembled those of the original strain, but did not appear to sink down in the agar; when they were scraped off, only very shallow, smooth, just visible depressions, but no “corrosion”, were seen and, on prolonged incubation, no
Fig. 2. Cells of spreading-corroding variant showing fimbriae (upper), and of non-spreading variant, without fimbriae (lower). Optical differentiation used ( 2 ) , x 60,000.
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spreading zones appeared. Otherwise the two variants appeared to be identical. Growth characteristics: The strains appeared to be somewhat fastidious and failed to grow on some of the simpler fluid media, e.g. Clark & Lub’s medium and nitrate broth. Growth on nutrient agar was fair. No growth occurred at room temperature. The strains were facultative anaerobes. In stationary cultures in fluid media, a marked tendency of the spreading strain to form surface pellicles was noted. Biochemical reactions: The following tests were positive : Catalase, oxidase, acid production (fermentative) from glucose, galactose, xylose (weak) and sucrose, indole production. The following tests were negative: Fermentation of lactose, maltose, rhamnose, arabinose, mannitol, sorbitol, and dulcitol, production of H2S (TSI-medium), urease, phenylalanine deaminase, liquefaction of gelatine and coagulated serum. These reactions appear to be compatible with a classification as P. multocida, the “dog type’’ as described by Frederiksen ( 5 ) . Both strains were sensitive to penicillin and to other commonly used antibiotics. Electron microscopy: About one in ten cells of the corroding and spreading variant was found to be fimbriated, but the cells of the non-corroding, non-spreading variant could not be shown to have fimbriae when several hundred were carefully examined (Fig. 2). Agar microscopy: On media without blood; spreading zones were only irregularly seen ; the small size of the cells and the very narrow dimensions of the spreading zones observed made it difficult to see twitching motility. In some zones, however, particularly around miniature colonies, an arrangement of the cells typical of twitching was seen (Fig. 3 ) . Also the appearance of the spreading zones was strikingly similar to that of zones produced by Moraxella strains, an appearance which is due to twitching. Transformation: All attempts failed to transform any of the two variants, using 9
Fig. 3. Miniature colonies of non-spreading (right) and of spreading variant (left). The arrangement of the cells around the latter colony suggests twitching, x 1,000.
DNA from streptomycin resistant mutants of both variants. DISCUSSION
We have not found any previous description of strains of P. multocida producing spreading and corroding colonies. As in the Moraxella species M . nonliquefaciens, M . bovis and M . kingae (2, 4, 7 ) ) this trait was associated with fimbriation and, also as in the case of Moraxella, it was possible to isolate a nonspreading, non-corroding and unfimbriated variant. The spreading zones produced by the Pasteurella strain were very like those caused by twitching of the mentioned Moraxella species and, although we did not succeed in actually seing twitching cells, we feel that the arrangement of the cells in some spreading zones indicates that they were actually produced by twitching. Contrary to expectation, competence in transformation was not demonstrated in the spreading/corroding strain. This might indicate that fimbriation in this strain is not associated with competence, as in Moraxella species, but it cannot be precluded that the fimbriae may not have been of the right type, or simply that we failed to find conditions suitable for transformation. I t may be mentioned that previous experiments (8) have failed to detect competence
131
in strains of P. multocida, although strains of other Pasteurella species appeared to be transformable. Corroding and spreading was not detected in any of those strains. The isolation of the strain described in this paper shows that the association of spreading/corroding colonies with twitching and fimbriation is not a property of Moraxella species exclusively, but may also be found in P. multocida. I t may be emphasized that such phenomena may remain unobserved unless cultures are incubated for periods of time longer than one or two days. Incubation in a humid atmosphere may also be important, although systematic studies of this point have not been carried out.
REFERENCES 1. Bevre, K.: Studies on transformation in Mo-
raxella and organisms assumed to be related to Moraxella. 1. A method for quantitative transformation in Moraxella and Neisseria, with streptomycin resistance as the genetic marker. Acta path. microbiol. scand. 61: 457-473, 1964.
132
2. Bevre, K., Bergan, T . & Froholm, L. 0.: Electron microscopical and serological characteristics associated with colony type in M o raxella nonliquefaciens. Acta path. rnicrobiol. scand. Sect. B, 78: 765-779, 1970. 3. Bovre, K . & Froholm, L. 0.:Variation in colony morphology reflecting fimbriation in Moraxella bovis and two reference strains of M . nonliquefaciens. Acta path. rnicrobiol. scand. Sect. B, 80: 629-640, 1972. 4. Bevre, K . & Froholm, L. 0.: Competence in genetic transformation related to colony type and fimbriation in three species of Moraxellae. Acta path. rnicrobiol. scand. Sect. B, 80: 649659, 1972. 5. Frederiksen, W.: Pasteurella taxonomy and nomenclature. I n Winblad, S. ( E d ) : Contributions to microbiology and immunology. 2. Yersinia, Pasteurella and Francisella. Karger, Base1 1973, pp. 170-176. 6. Henrichsen, 1.: Bacterial surface translocation. A survey and a classification. Bacteriol. Rev. 36: 478-503, 1972. 7. Henrichsen, J., Froholm, L. 0 . & Bevre, K . : Studies on bacterial surface translocation. 11. Twitching motility associated with fimbriation in colony variants of Moraxella nonliquefaciens, M . bovis and M . kingii. Acta path. microbiol. scand. Sect. B, 80: 445-452, 1972. 8. Henriksen, S . D.: Transformation of streptomycin resistance in Pasteurella. Acta path. microbiol. scand. 55: 496, 1962.
