are shown in Table 1. Of the 712 strains, 407 were from biotypes 10, 11, 12, and 13, and are genotypically fimbriate, are able

Transcription

1 JOURNAL OF BACTERIOLOGY, Nov. 1972, p Copyright i 1972 American Society for Microbiology Vol. 112, No. 2 Printed in U.S.A. Temperature-Dependent Utilization of meso- Inositol: a Useful Biotyping Marker in the Genealogy of Salmonella typhimurium DAVID C. OLD Bacteriology Department, University of Dundee, Dundee, Scotland Received for publication 20 June 1972 Salmonella typhimurium strains from natural sources either ferment or do not ferment meso-inositol in peptone water in 24 hr at 37 C. Ninety-five percent of the strains that are designated inositol-nonfermenting on the basis of their phenotype at 37 C ferment inositol when incubated at 25 C. Two classes of temperature-sensitive mutants were detected among the 712 strains of S. typhimurium examined. The occurrence of low-temperature fermentation of inositol among wild-type strains of S. typhimurium from biotypes 10 through 13 and FIRN suggested a genealogical relationship between these two groups, and that FIRN strains (fim-inlt8rla-) might have descended from ancestral types like biotype 10 through 13 strains (fim+inltsrha+). meso-inositol is a useful sugar in the differential diagnosis of Salmonella species and in the separation of the different fermentative types that may exist within a species (9). For example, the ability to ferment inositol in peptone water cultures is one of the ten characters used to differentiate strains of Salmonella typhimurium from natural sources into 21 fermentation types or "biotypes" (7, 8, 10). Strains from 12 of these biotypes-1, 2, 2M, 3, 4, 5, 6, 7, 8, 14, 19, and 20-were characterized by their ability to ferment inositol in peptone water cultures at 37 C within 24 hr and strains from the other 9 biotypes-9, 10, 11, 12, 13, 15, 16, 17, and 18-by the absence of this property. In the course of a biotyping survey of wild-type strains of S. typhimurium, it became apparent that temperature was important in the designation of these strains as inositolfermenting or inositol-nonfermenting. This paper reports on the inositol character of 712 epidemiologically distinct isolates of S. typhimurium and reveals the conservation of a temperature-sensitive inositol function in most wild-type strains that show the mutant phenotype at 37 C. possible, originated from a single epidemic episode. They were mainly from the series examined for fimbriation and biotype by Duguid et al. (6) and a further collection from E. S. Anderson. Their characters are shown in Table 1. Of the 712 strains, 407 were from biotypes 10, 11, 12, and 13, and are genotypically fimbriate, are able to ferment rhamnose in peptone water and utilize it as sole carbon source in minimal medium, but are unable to ferment inositol in 24 hr at 37 C; i.e., fim+inl-rha+. Eight strains from biotype 10, one from biotype 11, and three from biotype 12 were atypical because they were genotypically fim-. Of the 712 strains, 267 were from the four biotypes 15, 16, 17, and 18, designated FIRN (fim-inl-rha-), i.e., they do not produce type-1 fimbriae and are unable to ferment inositol or rhamnose in peptone water or use them as sole carbon source in minimal medium (6, 12). They are closely related genetically (D. C. Old, Ph.D. thesis, University of Edinburgh, 1963). The 674 strains from biotypes 10, 11, 12, 13, and FIRN were not strong fermenters of xylose, and in Bitter's minimal peptone medium (3) they were Bitter xylose negative. Another 22 strains from a new, inositol-nonfermenting biotype reported by Morgenroth and Duguid (12) were Rhaw, i.e., slow (10 to 24 hr) fermentation of rhamnose in peptone water but no utilization of rhamnose in minimal medium. These wild-type Rhaw strains were also fim+, meso-tartrate positive, MATERIALS AND METHODS and Bitter xylose negative, and hence, because they Bacteria. The 712 wild-type strains of S. typhimurium examined had been collected over a considtype 10, were placed in a new provisional biotype seemed like natural Rhaw mutants of strains of bioerable number of years and represented a wide range called 1ORW (J. P. Duguid, personal communication). of sources and places of origin. Each strain, as far as r79

2 780 OLD TABLE 1. Fermentation of inositol at 25 C by strains of Salmonella typhimurium from inositolnonfermenting biotypes No. of strains tested that ferment inositol in peptone Femetoa Frettona Fim- 37Co: Boye water at 25 C (and and class tbrian type of reaction)d Rham- Ino- Xy- In 2 In 14 Not at noseb sitolb losec days days all Non-FIRN (A) 12(B) 3 1ORW (A) 20(B) (A) 1(A) (A) 1(A) (A) 2(A) (A) 1(A) 0 FIRN 15 _ (A) _ (A) 5(A) 0 17 _ (A) 2(A) (A) 0 a The fimbriae were type-1 (5). b In peptone water. c In Bitter's minimal peptone medium (3). d A, The fermentation of inositol was strong with a complete color change throughout culture, and was rapid, usually in 2 days, rarely in 3 to 7 days. B, The fermentation of inositol was weak, often with only a transient color change not at the air interface, and slow, usually in 7 to 14 days, rarely in 3 to 7 days. The remaining 16 strains were inositol-nonfermenting strains from biotype 9. Like strains from biotypes 10 to 13, they were fim+inlrha+ but differed by being Bitter xylose positive. Culture media. Nutrient broth was Oxoid Nutrient Broth No. 2, ph 7.5. Nutrient agar was Oxoid Nutrient Agar, ph 7.4. The basic medium used to determine the fermentation of inositol was Oxoid (L37) peptone water, ph 7.2. meso-inositol was added to 1% (w/v), and the indicator used was bromocresol purple (0.0008%, w/v). The medium was dispensed in 5-ml amounts in cotton wool-stoppered test tubes (1 by 12.5 cm), and the production of an acid ph during incubation of the culture was noted by a change in indicator color to yellow. Minimal medium was that of Davis and Mingioli (4). It contained per liter of water: 7 g of K,HPO4, 3 g of KH2PO4, 1 g of (NH4)2SO4, and 0.1 g of MgSO4 *7H2O, ph 7.0. meso-inositol was added to 0.3% (w/v), and the medium was dispensed in 10-ml amounts in test tubes (1.5 by 15 cm). For growth of auxotrophs (mainly strains from biotypes 11 and 16), this medium was supplemented with the required growth factor at the concentration recommended by Meynell and Meynell (11). Chemicals. The meso-inositol and the bromocresol purple were purchased from The British Drug J. BACTERIOL. Houses Ltd., Poole, England. Inocula. Cultures of S. typhimurium were maintained on Dorset's egg slants at ambient temperature. Each strain was plated on nutrient agar, and after 24 hr of incubation a single colony was inoculated into nutrient broth and incubated aerobically and statically for 24 hr. The test media were given inocula of ca ml of broth culture per 5 ml of medium. The inoculated tubes of fermentation medium and inositol minimal medium were incubated at 25 and 37 C. The results reported represent findings from replicate tests for each strain. RESULTS Strains from biotypes 10 through 18 with mutant (nonfermenting) phenotype at 37 C. All of the 674 S. typhimurium strains from the biotypes 10, 11, 12, 13, and FIRN (15, 16, 17, and 18) failed to ferment inositol in peptone water or to utilize inositol in minimal salts medium at 37 C. When, rarely, after prolonged (>24 hr) incubation, fermentation or utilization of the inositol was observed, this was shown to be due to the origin from the parent strain of spontaneous inositol-fermenting mutants that behaved like wild-type inl+ S. typhimurium strains; i.e., they fermented inositol in peptone water in 24 hr at 37 C and utilized inositol as sole carbon source in minimal medium at 37 C. When the temperature of incubation was 25 C, 630 (93%) of the 674 cultures of strains from biotypes 10 through 18 that had not fermented inositol at 37 C did so within 48 hr (Table 1). Inoculation from a 25 C peptone water culture, in which inositol had been fermented in 48 hr, to fresh inositol peptone water produced a culture that still failed to ferment inositol at 37 C in 14 days. Thus, the low-temperature-fermenting character was a property associated with the strain and not with inl+ mutants that might have appeared in the parent culture at 25 C. Similarly, another 12 strains were found that fermented inositol at the low temperature in from 4 to 7 days (Table 1), and replicate tests on these latefermenting strains showed that the late-fermenting character was a constant property of the strain; no early-fermenting mutants were detected when such late-fermenting cultures were inoculated to fresh inositol peptone water medium and incubated at either 25 or 37 C. All of the 642 low-temperature-fermenting strains grew in inositol minimal salts medium at 25 C but not at 37 C. Strains from biotypes 10 through 18 with mutant (nonfermenting) phenotype at 25 C. This survey also revealed that 32 (ca. 5%) of

3 VOL. 112, 1972 TEMPERATURE-DEPENDENT INOSITOL UTILIZATION the 674 strains from biotypes 10 through 18 were unable to ferment or utilize inositol at either temperature in 14 days of incubation. Twenty-eight of these inositol-negative strains were from biotype 12, and the other four from biotypes 10 (two), 11 (one) and FIRN (one) (Table 1). Strains from biotype 1ORW. The behavior of all but one of the 22 strains from biotype 1ORW was quite different from that described above for the majority of strains from biotypes 10 through 18. It was found that 20 of the strains (isolated from 10 sources in six different countries over a 12-year period) behaved as a homogeneous group with respect to their inositol character. They failed to ferment and to utilize inositol at 37 C, but all fermented it slowly at 25 C in 5 to 14 days in peptone water and generally did not grow in inositol minimal medium even in 14 days. The exact time of this slow fermentation of inositol was difficult to assess because these cultures produced in the indicator a color change that appeared only in the depths of the culture and was often transient. The other two strains were the exceptional ones in this series. One of them showed the temperature-sensitive reaction described as typical for most strains from biotypes 10 through 18 (strong, 48-hr fermentation in peptone water and utilization in minimal medium), and the other failed to ferment or utilize inositol in either medium at either temperature (Table 1). Strains from biotype 9. Biotype 9 strains proved most difficult to analyze. One of the 16 strains fermented and utilized the inositol only at 25 C, and another three were unable to ferment at either temperature. The remaining twelve strains fermented inositol at 25 C, usually in 3 to 5 days, often as late as 9 days, and sometimes grew in inositol minimal medium at 25 C, i.e., the kind of reaction described for the 20 weakly fermenting strains from biotype 1ORW, except that the mean time of this slow fermentation was earlier for biotype 9 strains (4 days) than for biotype 1ORW strains (9 days). There were two problems in working with strains from biotype 9. Firstly, some of them produced abundant amounts of a brown pigment that masked any weak color changes in the fermentation medium, and, secondly, all biotype 9 strains were unstable in their inositol negativity at both temperatures. Some of these strains seemed "leaky." Most strains frequently gave cultures that fermented inositol strongly, and this strong fermentation occurred any time in the day incubation period and was shown to be due to the selection of inositol fermenting mutants (24 hr at 37 C). This ease of isolation of spontaneous strongly fermenting mutants from biotype 9 strains was in marked contrast to the rarity with which it was observed in cultures of strains from biotypes 10 through 18. DISCUSSION Many wild-type strains of S. typhimurium have been shown to exhibit a mutant (nonutilizing) phenotype with respect to inositol utilization when tested at 37 C and no expression of this mutant phenotype when tested at 25 C. Attempts to elucidate the recent evolutionary development of wild-type strains of S. typhimurium have been made using the genetic markers, fimbriation, and rhamnose fermentation, and earlier analyses of transductional crosses among members of the FIRN group had suggested that all FIRN strains have their fim- and rha- mutations at identical or overlapping sites (12; D. C. Old, Ph.D. thesis, University of Edinburgh, 1963). The discovery that the low-temperature utilization of inositol was a function conserved in most strains of S. typhimurium that are inositol nonfermenters at 37 C revealed further relationships among strains within this serotype. If FIRN strains had originated at different times, then it would be most unlikely that these independent mutations in the inositol gene(s) would be expressed as a temperature-sensitive product in all cases, and the finding that all but one of the 267 strains of FIRN examined possessed this temperature-sensitive ability to utilize inositol is a further indication of the genealogical relatedness of FIRN strains. Furthermore, it adds weight to the speculation that all FIRN strains, diversified as they are today in phage type, biotype, and colicin type, have descended from a single ancestral FIRN-type bacterium (12; D. C. Old, Ph.D. thesis, University of Edinburgh, 1963). Morgenroth and Duguid (12) further speculated that this FIRN ancestor had most likely descended from the more commonly occurring fim+inl-rha+ S. typhimurium strains by successive mutations in the fim and rha genes. This survey provides the first real evidence of the proposed relationship between FIRN and fim+inl-rha+ strains, and the finding that 93% of the strains from biotypes 10 through 13 (fim+in1-rha+) show the same temperaturesensitive phenotype as those from FIRN biotypes (fim-inl-rha-) is encouragingly in line with their speculations. Furthermore, since the

4 782 OLD majority of strains from biotypes 10 through 13 show this inositol temperature-sensitive phenotype, it is unlikely that these strains are the progeny of a large number of independent mutants all of which have perchance resulted in this unusual phenotype. It is more satisfactory to think that the mutations in inl in the biotype 10 through 13 strains are identical and expressed as a temperature-sensitive gene product. Preliminary transductional crosses with the 32 inl- strains from biotypes 10, 11, 12, and FIRN and other strains with the init6 genotype suggest that the inl- strains carry the original temperature sensitivity mutation and additional mutation(s) that result in the mutant phenotype even at the low temperature (D. C. Old and P. Rodger, unpublished data). Furthermore, crosses by transduction between strains from biotypes 10 through 13 and FIRN, and within each of these groups, indicate identity of the inlt mutation between, as well as within, the two groups (D. C. Old and P. Rodger, unpublished data). Thus, despite the spectrum of biochemical activities among strains from biotypes 10 through 18, the temperature-sensitive inositol reaction is strong indication of their relatedness and of their common ancestry. Strains from biotype 9 differ from the inositol-nonfermenting strains of all other inl- biotypes in being Bitter xylose positive, i.e., able to ferment xylose in a minimal peptone medium, and they are the only ones examined that differ from the presumed archetypal strains (biotype 1) by a single character (namely inlm). The diversity of phenotypic response experienced within this small group of 16 strains leads to the thought that several biotype 9 lines may have been derived by independent mutations, all of them distinct from the line of descent of the biotype 10 through 18 group. It was not surprising, therefore, to find that the inositol character of biotype 9 strains was different from that of strains from biotypes 10 through 18. Similarly, strains from biotype 1ORW did not behave like the strains from the other Bitter xylose-negative, inositol-nonfermenting biotypes, and phenotypically they seem more closely related to biotype 9 strains. Certainly, their supposed relationship to biotype 10 strains is in question because biotype 1ORW strains are seen to differ from biotype 10 strains in both their rhamnose and inositol fermentations. Transduction crosses have established that the inlt" mutation in the strains from biotypes 9 and 1ORW is different from J. BACTERIOL. that in strains from biotypes 10 through 18. Speculation on the exceptional strains, one in each of the biotypes 9 and 1ORW, that showed the reaction characteristic for strains from biotypes 10 through 18, requires their more detailed analysis, but both are probably mutants derived from true biotype 10 strains (with mutations to Bitter xylose positivity and Rhaw). Apart from the observations by Berman and Magasanik (1, 2) on the initial reactions involved in the metabolism of meso-inositol by Aerobacter aerogenes, little is known of this pathway in other bacteria, and, hence, the actual function affected by the temperature-sensitive alteration is not known and must await the detailed analysis of this pathway. Since wild-type strains have been found that are inl-, the conservation of the inl"8 gene cannot be considered essential to the cell. Furthermore, unless the temperature-sensitive mutation has appeared only recently in the evolutionary time scale, the selective pressures against this mutation cannot be great since the majority of strains with the mutant phenotype at elevated temperatures show wild-type activity at low temperatures. ACKNOWLEDGMET I thank J. P. Duguid and E. S. Anderson for the gift of strains and information about them. LITERATURE CITED 1. Berman, T., and B. Magasanik The pathway of myo-inositol degradation in Aerobacter aerogenes. Dehydrogenation and dehydration. J. Biol. Chem 241: Berman, T., and B. Magasanik The pathway of myo-inositol degradation in Aerobacter aerogenes. Ring scission. J. Biol. Chem. 241: Bitter, L., F. Weigmann, and H. Habs Bestimmung der gebildeten Sauremenge zur Unterscheidung verwandter Bakterien. Muench. Med. Wochenschr. 73: Davis, B. D., and E. S. Mingioli Mutants of Escherichia coli requiring methionine or vitamin B12. J. Bacteriol. 60: Duguid, J. P., and E. S. Anderson Terminology of bacterial fimbriae, or pili, and their types. Nature (London) 215: Duguid, J. P., E. S. Anderson, and I. Campbell Fimbriae and adhesive properties in salmonellae. J. Pathol. Bacteriol. 92: Hansen, A. C Die beim Hausgefluigel in Danemark festgestellten Salmonella-typen. Zentralbl. Bakteriol. Parasitenk. Infektionsk. Abt. 1 Orig. 149: Harhoff, N Gastroenteritis baciller of Salmonellagruppen in Danmark. Nyt Nordisk Forlag-Arnold Busck, Copenhagen. 9. Kauffmann, F The bacteriology of enterobacteriaceae. Munksgaard, Copenhagen.

5 VOL. 112, 1972 TEMPERATURE-DEPENDENIT INOSITOL UTILIZATION Kristensen, M., K. Bojlen, and C. Faarup Bakteriologisk-epidemiologiske erfaringer om infektioner med gastroenteritisbaciller af Paratyphusgruppen. Bibl. Laeger 129: Meynell, G. G., and E. Meynell Theory and practice in experimental bacteriology. University Press, Cambridge. 12. Morgenroth, A., and J. P. Duguid Demonstration of different mutational sites controlling rhamnose fermentation in FIRN and non-firn rha- strains of Salmonella typhimurium: an essay in bacterial archaeology. Genet. Res. 11: