Francine Grimont

Ribosomal ribonucleic acid gene restriction patterns as potential taxonomic tools

Deoxyribonucleic acid from 41 different bacterial species (including Gram-negative and Gram-positive species) were cleaved by different restriction endonucleases, electrophoresed in agarose and transferred to nylon filters. The DNA fragments carrying rRNA genes (rDNA) were localized by hybridization with a 32P-labelled Escherichia coli 16 + 23S rRNA probe. A pattern of hybridized fragments was obtained for each DNA tested. Within a bacterial species (defined as a DNA hybridization group), one or several rDNA restriction patterns were observed. When DNA hybridization data were available, strains showing identical patterns were highly related with insignificant divergence. In a species, different patterns corresponded to significant divergence, as evaluated by thermal stability studies of DNA/DNA hybrids. Sets of rDNA restriction fragment sizes might constitute useful data for inclusion in species and type strain descriptions. Such data might later prove useful in identification of bacteria when biochemical characteristics are poor or atypical.

Brucella, a Monospecific Genus as Shown by Deoxyribonucleic Acid Hybridization

A total of 51 strains (including type, reference, vaccine, and field strains) representing all species and biovars of Brucella formed a single deoxyribonucleic acid-deoxyribonucleic acid hybridization group (S1 nuclease method). Accordingly, we propose that only one species, Brucella melitensis, be recognized in the genus. We recommend that other specific epithets formerly associated with the generic name Brucella be used in a vernacular form for biovar designation (e.g., Brucella melitensis biovar Abortus 1).

Staphylococcus lugdunensis sp. nov. and Staphylococcus schleiferi sp. nov., Two Species from Human Clinical Specimens

Deoxyribonucleic acid relatedness studies (S1 nuclease method) showed that 23 unidentified Staphylococcus strains form two homogeneous genomic species related 1 to 9% to 24 type strains representing known Staphylococcus species. These new species were named Staphylococcus lugdunensis and Staphylococcus schleiferi. Strains of S. lugdunensis were susceptible to novobiocin, produced a fibrinogen affinity factor, and failed to produce coagulase, heat-stable nuclease, and staphylokinase. S. lugdunensis strains differed from S. hominis (the phenotypically closest species) by production of ornithine decarboxylase and the fibrinogen affinity factor. The guanine-plus-cytosine content of the deoxyribonucleic acid was 32 mol%. The type strain is N860297 (= ATCC 43809). Strains of S. schleiferi were susceptible to novobiocin, produced a heat-stable nuclease and a fibrinogen affinity factor, and failed to produce coagulase and staphylokinase. S. schleiferi strains differed from S. aureus by production of an antigenically different heat-stable nuclease and the lack of pigmentation, free coagulase, protein A, and β-ribitol teichoic acid. The guanine-plus-cytosine content of the deoxyribonucleic acid was 37 mol%. The type strain is N850274 (= ATCC 43808).

Reproducibility and correlation study of three deoxyribonucleic acid hybridization procedures

Reproducibility of relative binding ratios (RBR) was studied with 26 labeled DNA-unlabeled DNA hybridization reactions carried out at two temperatures (60 and 75°C) and following three procedures: the S1-nuclease method with precipitation of S1-resistant DNA by trichloroacetic acid (S1-TCA), the S1-nuclease method with adsorption of S1-resistant DNA onto DE81 filters (S1-DE81), and the hydroxyapatite (HA) method. Analyses of variance indicated that different hybridization methods give different RBR, but similar percent divirgence (ΔTm) results. A curvilinear relationship was found between RBR data obtained with S1-TCA and HA procedures at 60 or 75°C, and between 60°C RBR data obtained with S1-DE81 and HA procedures. At 75°C, RBR obtained with S1-DE81 and HA methods are comparable without any transformation of the data.

Identification of Escherichia coli flagellar types by restriction of the amplified fliC gene.

A total of 182 strains of Escherichia coli (133 reference strains, 22 clinical strains, nine nonmotile strains and 18 strains derived from K-12) were characterized by HhaI restriction of the amplified flagellin gene (fliC). The amplified fliC product was a single band between 0.9 and 2.6 kbp. With the collection of reference strains which represented 48 flagellar types (H-types), a total of 62 patterns (F-types) were observed after HhaI restriction. A single F-type was associated with each of 39 H-types and more than one F-type was associated with the other nine H-types. Antigenically related H-types 12 and 45 gave a single F-type. The determination of HhaI-fliC F-types could allow deduction of all H-types and subdivision of some of these. Application of this identification system to 22 E. coli clinical isolates yielded nine F-patterns and the deduced H-types were confirmed by serotyping in all cases. Nine nonmotile strains were studied and their F-types were also identified. The proposed determination of fliC restriction patterns should be helpful for epidemiological studies.

Universal ribotyping method using a chemically labelled oligonucleotide probe mixture

Some of the present problems in ribotyping are associated with a lack of uniform reactivity of probes when bacterial DNAs are of phylogenetically diverse origins. To overcome these problems, a set of five oligonucleotides (referred to as OligoMix5) was selected to react with conserved sequences located near both extremities of rrs (16S rRNA gene) and near both extremities and the middle of rrl (23S rRNA gene). DNA samples from 13 bacterial species selected to represent various phylogenetic branches within the Eubacteria were cleaved by a restriction endonuclease and electrophoresed in 0.8% agarose, and the fragments were vacuum-transferred to nylon membranes and hybridized with digoxigenin-labelled OligoMix5, plasmid DNA from pKK3535 (cloned rrn operon from Escherichia coli) or pBA2 (cloned rrs from Bacillus subtilis), or acetylamino-fluorene-labelled E. coli 16 + 23S rRNA. The results showed OligoMix5 to visualize patterns in DNA from phylogenetically diverse bacteria with comparable intensity. Banding patterns (not band intensity) obtained with OligoMix5 were identical with those obtained with 16 + 23S rRNA or plasmid pKK3535 for each strain studied and represented complete ribotypes. For DNA from Gram-positive bacteria, complete ribotypes were observed after prolonged enzymatic detection of bands when probes were either E. coli 16 + 23S rRNA or pKK3535. Patterns given by plasmid pBA2 were subsets of the complete ribotypes for 9/13 strains. Each oligonucleotide of the OligoMix5 set was used as a probe to determine its contribution to the complete ribotype. The five oligonucleotide probes, used individually, visualized one to four patterns per DNA sample. Use of DNA from Xenorhabdus sp. CIP 105189 cleaved by EcoRI is suggested to control the quality of the oligonucleotide probes composing OligoMix5. Probe OligoMix5 was found to be an essential tool for ribotyping phylogenetically diverse eubacteria.

DNA relatedness among serovars ofListeria monocytogenes sensu lato

Sixty-six strains ofListeria monocytogenes (as defined in the eighth edition ofBergeys Manual of Determinative Bacteriology) were characterized by DNA relatedness. These strains formed five distinct DNA relatedness groups: (i)L. monocytogenes sensu stricto (30 strains) including the type spain ATCC 15313; (ii) serovar 5 strains (9 strains) corresponding to “L. bulgarica”; (iii) “L. inocua” (11 strains of serovars 6a, 6b, 4ab, and undesignated ones) including the reference strains; (iv) six strains of serovars 6a and 6b; (v) ten strains of various serovars. These five groups were clearly distinct fromL. grayi (4 strains) andL. murrayi (3 strains).

Streptococcus infantarius sp. nov., Streptococcus infantarius subsp. infantarius subsp. nov. and Streptococcus infantarius subsp. coli subsp. nov., isolated from humans and food

Eighteen strains isolated from human specimens or from food products were characterized as atypical variants of mannitol-negative Streptococcus bovis. They were tested for extended biochemical criteria, ribotyping and DNA-DNA hybridization in order to define their taxonomic status. These strains were demonstrated to constitute a DNA relatedness group that includes strains of DNA group 4 of Farrow et al. (1984). Comparative analysis of 16S rRNA sequences demonstrated that these strains represent a new species which belongs to the Streptococcus bovis/Streptococcus equinus complex and which has been provisionally named S. infantarius by Bouvet et al. (1997). Biotyping and ribotyping allowed differentiation of these strains from the aesculin-positive strains of S. bovis belonging to the previously described biotypes I, II.1 and II.2. The results of the ribotyping and hybridization assays demonstrated the presence of two different DNA subgroups within the 18 strains. On the basis of these data, the names S. infantarius subsp. infantarius (aesculin-negative for five strains out of seven, including the type strain HDP 90056T = NCDO 599T) and S. infantarius subsp. coli (aesculin-positive, reference strain HDP 90248 = NCDO 2620) are proposed as the names for these two subspecies within the S. infantarius species.

Multidrug-Resistant Human and Animal Salmonella typhimuvium Isolates in France Belong Predominantly to a DT104 Clone with the Chromosome- and Integron-Encoded β-Lactamase PSE-1

Epidemiologic relationships were investigated in 187 ampicillin-resistant Salmonella typhimurium strains (86 human, 101 animal) from >2000 strains isolated in 1994. Of 23 resistance patterns, the most frequent (ampicillin [Am], chloramphenicol [Cm], tetracycline [Tc], streptomycin and spectinomycin [Sm], and sulfonamides [Su]) was found in 69.5% of human and 64.8% of animal isolates. Four beta-lactamase genes were identified, blaTEM (24%), blaPSE-1 (78%), and blaSHV and oxa-2 (each <3%). blaPSE-1 and the integrase gene, intI1, but not blaTEM, blaSHV or oxa-2, were chromosomeborne and found almost exclusively in the AmCmTcSmSu strains. In these, polymerase chain reaction mapping revealed two distinct integrons carrying blaPSE-1 or aadA2. Lysotypes, plasmid profiles, and restriction fragment length polymorphisms (IS200) were determined for 50 representative isolates and for 3 DT104 strains from the United Kingdom (UK). The phage type of the PSE-1-producing AmCmTcSmSu strains was 12 atypic, indistinguishable from that of the DT104 strains. The combined data indicate that the same multiresistant clone has spread through human and animal ecosystems in the UK and France.

Streptococcus defectivus sp. nov. and Streptococcus adjacens sp. nov., Nutritionally Variant Streptococci from Human Clinical Specimens

Nutritionally variant streptococci (12 strains) formed two deoxyribonucleic acid hybridization groups (as determined by the S1 nuclease method) that were distinct from 39 Streptococcus spp. type and reference strains. These two groups differed by their penicillin-binding protein patterns and biochemical properties. Streptococcus defectivus sp. nov. (with type strain SC10) and Streptococcus adjacens sp. nov. (with type strain GaD) are the names proposed for these species.