Daniel Izard

Siderophore Typing, a Powerful Tool for the Identification of Fluorescent and Nonfluorescent Pseudomonads

ABSTRACT A total of 301 strains of fluorescent pseudomonads previously characterized by conventional phenotypic and/or genomic taxonomic methods were analyzed through siderotyping, i.e., by the isoelectrophoretic characterization of their main siderophores and pyoverdines and determination of the pyoverdine-mediated iron uptake specificity of the strains. As a general rule, strains within a well-circumscribed taxonomic group, namely the species Pseudomonas brassicacearum, Pseudomonas fuscovaginae, Pseudomonas jessenii, Pseudomonas mandelii, Pseudomonas monteilii, “Pseudomonas mosselii,” “Pseudomonas palleronii,” Pseudomonas rhodesiae, “Pseudomonas salomonii,” Pseudomonas syringae, Pseudomonas thivervalensis, Pseudomonas tolaasii, and Pseudomonas veronii and the genomospecies FP1, FP2, and FP3 produced an identical pyoverdine which, in addition, was characteristic of the group, since it was structurally different from the pyoverdines produced by the other groups. In contrast, 28 strains belonging to the notoriously heterogeneous Pseudomonas fluorescens species were characterized by great heterogeneity at the pyoverdine level. The study of 23 partially characterized phenotypic clusters demonstrated that siderotyping is very useful in suggesting correlations between clusters and well-defined species and in detecting misclassified individual strains, as verified by DNA-DNA hybridization. The usefulness of siderotyping as a determinative tool was extended to the nonfluorescent species Pseudomonas corrugata, Pseudomonas frederiksbergensis, Pseudomonas graminis, and Pseudomonas plecoglossicida, which were seen to have an identical species-specific siderophore system and thus were easily differentiated from one another. Thus, the fast, accurate, and easy-to-perform siderotyping method compares favorably with the usual phenotypic and genomic methods presently necessary for accurate identification of pseudomonads at the species level.

Phenotypic differentiation of bifidobacteria of human and animal origins

The phenotypes of 153 strains belonging or related to the genus Bifidobacterium were studied. These organisms included 38 collection strains and 115 wild strains (41 strains of human origin, 56 strains of animal origin, and 18 strains obtained from rivers or sewage). Our phenotypic analysis revealed seven main groups that were subdivided into 20 subgroups. Seven subgroups contained no type or collection strain. Among the human strains, the type strains of Bifidobacterium pseudocatenulatum and B. catenulatum fell into group I, which contained the type strains of B. adolescentis (subgroup Ib), B. dentium (subgroup Ic), and B. angulatum (ungrouped). The type strain of B. breve belonged to subgroup IIIa1, and the type strains of B. infantis and B. longum fell into subgroup IIIb1. Group VII comprised only wild strains that were isolated from human infant feces. Among the animal strains, group II consisted mainly of bifidobacteria that were isolated from pig feces and contained the type strains of B. suis (subgroup IIb), B. thermophilum (subgroup IIf), B. choerinum, and B. boum (ungrouped). Wild strains belonging to group V were isolated from pig, calf, cow, and chicken feces; this included the type strains of B. animalis (subgroup Va), B. magnum (subgroup Vb), B. pseudolongum, and B. globosum (subgroup Vc). The strains of human origin (groups I, III, and VII) were well separated from the animal strains (groups II, IV, and V). It was not surprising that the wild strains isolated from surface water or sewage were distributed in the animal groups as well as the human groups. Thus, bifidobacteria can be considered to be successful indicators of human or animal fecal pollution when they are correctly classified. The acidification patterns were not adequate to differentiate Bifidobacterium species, as determined previously by Mitsuoka (Bifidobacteria Microflora 3:11-28, 1984) and Scardovi (p. 1418-1434, in P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt, ed., Bergeys Manual of Systematic Bacteriology, vol. 2, 1986). However, enzymatic tests furnished new taxonomic criteria for the genus.

DNA relatedness among Pseudomonas strains isolated from natural mineral waters and proposal of Pseudomonas veronii sp nov.

The taxonomic position of eight strains isolated from mineral water and previously grouped in the authentic pseudomonads on the basis of a phenotypic analysis (cluster Ib of M. Elomari, L. Coroler, D. Izard, and H. Leclerc [J. Appl. Bacteriol. 78:71-81, 1995]) has been further studied by DNA-DNA hybridizations. Using the S1 nuclease method at 60 degrees C and labeled reference DNA from a representative strain, CFML 92-134, we showed that members of cluster Ib constituted a homogeneous group with a relative binding ratio of greater than 80% and changes in melting temperature of less than 1 degree C. With a total of 67 strains representing known or partially characterized species of the genus Pseudomonas, only 4 to 47% DNA hybridization and changes in melting temperature of between 8 and 20 degrees C were found, the highest hybridization values being measured with members of the saprophytic fluorescent pseudomonads. Since cluster Ib could also be clearly differentiated from members of the latter group and from other phenotypic clusters containing isolates from mineral water, we designated the Ib strains members of a new Pseudomonas species for which the name Pseudomonas veronii sp. nov. has been proposed. Members of this species grew on alpha-aminobutyrate, sucrose, butyrate, isobutyrate, erythritol, L-tryptophan, and trigonelline as sole sources of carbon and energy. The average G+C content of the DNA of the eight strains of P. veronii was 61.5 +/- 0.5 mol%. The type strain is CFML 92-134T (CIP 104663T), with a G+C content of 61 mol%. The clinical significance of P. veronii is unknown.

Pseudomonas monteilii sp. nov., isolated from clinical specimens

We propose the name Pseudomonas monteilii for a new species of gram-negative, rod-shaped, motile bacteria that were nonhemolytic on blood agar and were isolated from clinical sources. The 10 strains of P. monteilii were incapable of liquefing gelatin. They grew at 10 degrees C but not at 41 degrees C, produced fluorescent pigments, catalase, and cytochrome oxidase, and possessed the arginine dihydrolase system. They were capable of respiratory but not fermentative metabolism. They did not hydrolyze esculin or starch and were able to use benzylamine, alpha-aminobutyrate, D-ribose, L-arabinose, butyrate, valerate, isovalerate, isobutyrate, inositol, phenylacetate, D-alanine, and amylamine. They possessed L-phenylalanine arylamidase, L-lysine arylamidase, L-alanine arylamidase, gamma-glutamyl-transferase, glycyl-phenylalanine arylamidase, L-tryptophan arylamidase, glycyl-L-alanine arylamidase, esterase C4, esterase C6, esterase C8, esterase C9, esterase C10, and esterase C18. DNA relatedness studies revealed that P. monteilii strains formed a homogeneous DNA hybridization group. A total of 57 strains representing previously described or partially characterized taxa belonging to the genus Pseudomonas were 6 to 54% related to P. monteilii. The highest hybridization values were obtained with strains belonging to or related to Pseudomonas putida biovar A. The average G+C content of the DNA was 60.5 +/- 0.5 mol% for four of the P. monteilii strains studied. The type strain of P. monteilii is CFML 90-60 (= CIP 104883); it was isolated from bronchial aspirate and has a G+C content of 60 mol%. The clinical significance of these organisms is not known.

Serratia fonticola, a New Species from Water

Twenty strains of Enterobacteriaceae isolated from water are assigned to a new species in the genus Serratia on the basis of phenetic (numerical analysis) and genetic (deoxyribonucleic acid [DNA]-DNA hybridization) evidence. They are closely related to each other (75 to 91% DNA-DNA relatedness) and show 50% DNA relatedness with species of Serratia but only 20 to 30% relatedness with other members of the family Enterobacteriaceae. The strains are acetoin, gelatinase, and deoxyribonuclease negative, but lysine, ornithine decarboxylase, and Tween-esterase positive. They are metabolically very active, fermenting all the sugars and alcohols tested except melezitose, inulin and sorbose. The name Serratia fonticola is proposed for these strains. Strain 11 (= ATCC 29844) is the type strain of the species.

Taxonomic Study of Bacteria Isolated from Natural Mineral Waters: Proposal of Pseudomonas jessenii sp. nov. and Pseudomonas mandelii sp. nov.

The taxonomic position of 23 strains isolated from mineral waters and previously grouped in the authentic pseudomonads on the basis of a phenotypic analysis (cluster IX, subclusters XIIIa and XIIIc of VERHILLE, S., ELOMARI, M., COROLER, L., IZARD, D., LECLERC, H. (Syst. Appl. Microbiol, 20, 137-149, 1997) has been genotypically further studied in the present work. On the basis of hybridization results, these strains were gathered into two new genomic groups for which we propose the names of Pseudomonas jessenii sp. nov. (Type strain CIP 105274) and Pseudomonas mandelii sp. nov. (Type strain CIP 105273). Deoxyribonucleic acid relatedness levels showed homologies ranging from 78 to 100% for Pseudomonas jessenii and from 77 to 100% for Pseudomonas mandelii. Furthermore, hybrization rates with 66 representative well characterized species or only partially characterized species of the genus Pseudomonas were below 53%, with delta Tm values of 7 degrees C and more. The mol% G + C content ranged from 57 to 58. The two new species presented basic morphological characteristics common to all pseudomonads. Various phenotypic features, such as denitrification, growth at 4 degrees C or 41 degrees C, trigonelline assimilation, alpha-L-glutamyl-L-histidine arylarmidase activity, growth on benzoate and meso-tartrate were found to differentiate Pseudomonas jessenii from Pseudomonas mandelii and from other Pseudomonas species. Pseudomonas jessenii encompassed a total of 9 strains from both phenotypic groups IX and XIIIa. Pseudomonas mandelii clustered a total of 13 strains from both phenotypic groups IX and XIIIc. Their clinical significance is unknown. The 16S rDNA of each type strain was sequenced and compared with the known sequences of the representative strains of the genus Pseudomonas. A phylogenetic tree was constructed to determine the intrageneric relationships within the genus Pseudomonas.

Multiplex PCR using 16S rRNA gene-targeted primers for the identification of bifidobacteria from human origin

Three multiplex polymerase chain reactions (PCRs) targeted on Bifidobacterium and related species were designed to identify human species. The selected primers yielded amplified products of various sizes, each specific for a species. Three to four pairs were gathered in one PCR reaction and their specificity under multiplex conditions was confirmed using DNA from 26 reference strains. Using this technique on unidentified faecal strains, B. bifidum, B. longum and B. breve species were commonly recovered in infants while B. adolescentis, B. catenulatum/B. pseudocatenulatum continuum and B. longum species were predominant in adults. Thus, a single PCR can provide the assignment of a strain to one these species, reducing the number of PCR reactions and hands-on time for the identification of human isolates of bifidobacteria. Moreover, this technique is also applicable for the in situ detection of bifidobacteria in DNA extracts from human stools.

Pseudomonas rhodesiae sp. nov., a new species isolated from natural mineral waters

Summary Deoxyribonucleic acid relatedness studies (S1 nuclease method) showed that 7 strains of the cluster IIa previously described by Elomari et al. (J. Appl. Bacteriol 78, 71-81, (1995)) formed a homogenous deoxyribonucleic acid hybridization group. This group of strains forms a new species: Pseudomonas rhodesiae sp. nov. A total of 67 strains representing known or partially characterized sprecies of the genus Pseudomonas had 7 to 48 % DNA hybridization with Pseudomonas rhodesiae. This new species composed of strains isolated from natural mineral waters, were Gram negative, rod shaped, and motile by means of a single polar flagellum. They were not able to accumulate poly-γ-hydroxybutyrate, were capable of respiratory but not fermentative metabolism. They grew at 4°C but not a 41°C, produced fluorescent pigment, catalase, cytochrome oxidase and lecithinase, and possessed arginine dihydrolase system. They did not hydrolyse gelatin and starch, and were able to use glucose, trehalose, 2-ketogluconate, inositol, L-valine and γ-alanine. They possessed L-pyrrolidone arylamidase, L-histidyl-L-serine arylamidase but did not have osidase, esterase = C12, esterase = C14, esterase = C16, and glycyl-L-tryptophane arylamidase. The average guanine + cytosine (G + C) content of the deoxyribonucleic acid was 59 ± 1 mol%. The type strain of P. rhodesiae has been deposited as CIP 1046641. The clinical significance of Pseudomonas rhodesiae is unknown.

Priority of Klebsiella planticola Bagley, Seidler, and Brenner 1982 over Klebsiella trevisanii Ferragut, Izard, Gavini, Kersters, DeLey, and Leclerc 1983

The published descriptions of Klebsiella planticola Bagley, Seidler, and Brenner 1982 and Klebsiella trevisanii Ferragut, Izard, Gavini, Kersters, De Ley, and Leclerc 1983 are very similar. The reactions given by the type strains of K. planticola and K. trevisanii were identical when these strains were submitted to 51 common tests and in 118 of 119 nutritional tests. The level of deoxyribonucleic acid relatedness between the two type strains was 79 to 94% (nitrocellulose filter method) or 70 to 104% (S1 nuclease method) depending on which deoxyribonucleic acid was tritium labeled. Heteroduplexes showed a thermal stability very close to that of homoduplexes. Thus, K. planticola and K. trevisanii are subjective synonyms. Since K. planticola has priority over K. trevisanii, K. planticola should be the only name used to refer to the species formerly designated K. planticola or K. trevisanii.

Numerical Taxonomy of Pseudomonas alcaligenes, P. pseudoalcaligenes, P. mendocina, P. stutzeri, and Related Bacteria

A numerical phenotypic analysis, in which the unweighted pair group average linkage method and Dice similarity coefficient were used, was performed on 155 strains received as Pseudomonas alcaligenes, Pseudomonas pseudoalcaligenes, Pseudomonas mendocina, or Pseudomonas stutzeri. These organisms are the clinically important nonfluorescent species belonging to ribosomal ribonucleic acid group I of Palleroni and co-workers. Six major clusters, which could be further divided into 20 subclusters, were formed. Most strains received as P. alcaligenes fell into three subclusters (subclusters A1, A2, and B1), whereas strains received as P. pseudoalcaligenes were mainly classified in two other subclusters (subclusters C2 and C3). All but two strains (subcluster D1) of organisms received as P. mendocina were grouped in subcluster D2. Most of the 45 strains received as P. stutzeri were contained in a large subcluster, subcluster E2 (39 strains). Strains belonging to fluorescent pseudomonad species (Pseudomonas aeruginosa, Pseudomonas fluorescens, and Pseudomonas putida), which were included in the analysis for control purposes, were contained in one cluster, which comprised seven subclusters.