Biomedical subjects
Publications and source records attributed to B Regnault.
In this work we analyze the physiological state of cells after lethal-UV dose disinfection using independent metabolic markers. Through the detection of some metabolic activities we proved that cell lysis does not immediately follow death in UV-irradiated Escherichia coli K12 cells.
Despite the numerous advantages of fluorescent in situ hybridization (FISH) for identifying a single bacterial cell with 16S rRNA probes, problems are encountered with starving bacteria in natural samples. The original direct viable count procedure (DVC) includes a revivification step in the presence of an antibiotic inhibiting cell division. Cells elongate and accumulate ribosomes. This results in a natural amplification of 16S rRNA molecules (target of FISH). However, it is limited to gram-negative bacteria which are sensitive to nalidixic acid. The objective of this study was to develop a procedure for estimating the number of metabolically active gram-positive Staphylococcus aureus and Enterococcus faecalis cells by the use of a method which combines the number of substrate-responsive cells and their identification by FISH. It was observed that no single published DVC method could apply to taxonomically different gram-positive bacteria. Since cells were not counted, the revivification step in presence of nalidixic acid will be referred to as revivification without cell division. For each species, different low-nutrient media and complex media, different fluoroquinolones and beta-lactam antibiotics, concentrations of antibiotics, combinations of antibiotics, temperature and time were evaluated using bacteria in different physiological states and in natural samples. Enumeration of bacteria by plate counts and direct FISH were compared. The improved procedure should yield information about the physiological state, the taxonomic identity, and the enumeration of viable gram-positive bacteria. The application of DVC to an entire ecosystem is presently still a challenge.
There are several occasions when enumeration of Escherichia coli cells is needed. These include examination of urine specimens and water or food samples. Present methods rely on growth in more or less selective media (colony-forming units on agar or the most probable number method using liquid media). Unfortunately, no really selective medium with 100% efficiency of plating is available for E. coli. A 24-mer oligonucleotide probe (Colinsitu), complementary to a piece of 16S ribosomal ribonucleic acid, has been tested for specifically visualizing E. coli cells by in situ hybridization and epifluorescence microscopy. The fluorescent dye-labeled probe was able to stain cells of E. coli, Shigella spp. and E. fergusonii. Shigella spp. are known to belong to the E. coli genomospecies and E. fergusonii is the nomenspecies closest to E. coli by DNA-DNA hybridization. The probe did not stain any strain of 169 other genomospecies of the family Enterobacteriaceae or of a few other species frequently encountered in the environment. Revivification without cell division allowed the visualization of E. coli cells in contaminated water. In situ hybridization using the Colinsitu probe is a potential tool for the confirmation of (atypical) E. coli in reference centers and the rapid (3-6 h) detection and enumeration of E. coli in urine specimens, contaminated water and food. More work is needed to include in situ hybridization in laboratory routine.
A number of methods have been proposed to assess the viability of cells without culture. Each method is based on criteria that reflect different levels of cellular integrity or functionality. As a consequence, the interpretation of viability is often ambiguous. The purposes of this work were to evaluate the capacity of current viability markers to distinguish between live and dead Escherichia coli K-12 cells. Methods that assess 'viability' by the demonstration of metabolic activities (esterase activity, active electron transport chain, transport of glucose), cellular integrity (membrane integrity, presence of nucleic acids) or the building up of cellular material (cell elongation) have been evaluated in live and UV- or heat-killed cells. With live cells, viability markers detected cells in counts similar to the colony count. However, these so-called viability markers could stain dead cells for some time after the lethal treatment. For the UV-killed cells, residual activities were detected even after 48 h of storage at 20 degrees C. However, for heat-treated cells, these activities disappeared within hours after heat treatment. Only a combination of fluorescence in situ hybridization with rRNA probes and cell elongation in response to nutrients (in the presence of an inhibitor of cell division) had the ability to differentiate live from dead cells. Problems in the definition of a viable but nonculturable state are in part due to the lack of a clear definition of bacterial death. We consider death as an irreversible state where no growth, cell elongation or protein synthesis may occur.
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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.
The random amplification of polymorphic DNA (RAPD) was used for epidemiological typing of Chlamydia trachomatis strains. DNA samples from 39 C. trachomatis, 1 C. pneumoniae and 2 C. psittaci strains were screened by the use of 4 single 10-mer primers. Different and reproducible banding profiles were observed on agarose gel electrophoresis. No common profiles were recorded for strains from different Chlamydia species. All C. trachomatis strains of trachoma biovar were distinguished from lymphogranuloma venereum biovar. Moreover, serotypes A to C were separated from serotypes D to K, and some groups of strains sharing the same serotype D to K were further subdivided by RAPD. Conversely, strains of different serotypes could produce identical patterns of amplification, indicating that RAPD did not reflect serotyping. The patterns of amplified products were compared to the restriction fragment length polymorphism of the omp1 gene after amplification and to DNA fingerprinting by use of ribosomal RNA or randomly cloned DNA probes. RAPD seemed to be an alternative molecular typing procedure for epidemiological study and strain identification in urogenital infections due to serotypes D to K.
DNA fingerprinting of 15 reference strains and 24 clinical isolates of Chlamydia trachomatis, 2 strains of C. psittaci and one strain of C. pneumoniae was studied by use of universal 16 + 23S RNA from Escherichia coli, 16S rDNA-directed oligonucleotide and randomly cloned chlamydial DNA probes. The rRNA-gene restriction patterns (ribotypes) enabled the differentiation of chlamydial species. Following DNA cleavage by restriction endonuclease PvuII, lymphogranuloma venereum and trachoma biovars of C. trachomatis could be differentiated. An oligonucleotide, designed to hybridize the C. trachomatis 16S rDNA, also allowed for both species-specific identification and biovar typing of C. trachomatis human strains. Molecular typing system using 3 lambda clones containing C. trachomatis serotype E random DNA inserts, combined to ribotyping, revealed 12 groups of variable banding patterns within C. trachomatis, and could provide an alternative epidemiological tool.
A total of 67 serovar reference strains and 7 isolates belonging to the genus Leptospira were characterized by ribosomal ribonucleic acid (rRNA) gene restriction patterns. Fifty patterns were observed. Strains belonging to different genomic species always gave different patterns. However, genomic species were subdivided into several patterns. Forty-three serovars gave a specific pattern. Some serovars could not be separated by rRNA gene restriction patterns: strains of serovars icterohaemorrhagiae, copenhageni, lai, pyrogenes and jalna gave pattern 1; serovars birkini, mankarso and wolffi gave pattern 4; serovars canicola, gem, hebdomadis, pomona and hardjo (strain hardjoprajitno) gave pattern 12; serovars valbuzzi and zanoni gave pattern 14; serovars jonsis, malaya and sumneri gave pattern 16; serovars arborea, ballum, castellonis and kenya gave pattern 35; and serovars borincana and shermani gave pattern 43. These data provide the bases for a molecular typing system for the genus Leptospira.
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Nonculturable segmented filamentous bacteria (SFB) have been described in the gut of rats, mice and chickens, and 16S rRNA sequences for these organisms are available. These organisms, peripherically related to Clostridium phylogenetic group I, have been provisionally named 'Candidatus Arthromitus'. This work reports the observation of similar bacteria in the intestinal content of the distal intestine, preferentially, in the adult rainbow trout (Oncorhynchus mykiss) that exhibited episodic acute diarrhea, usually during the summer. Abdominal distension, intestinal fluid-mucus content and epithelium detachment were observed in trout. The demonstration that the observed microorganisms are bacteria and belong in the 'Candidatus Arthromitus' group was achieved by in situ hybridization with, respectively, a eubacterial probe and an oligonucleotide probe designed to react specifically with SFB 16S rRNA (encoded by the rrs gene) sequences. The sequenced rrs gene was compared with published sequences and found to be closely related to (although distinct from) other SFB sequences. Implication of these bacteria in trout diarrheic illness remains hypothetical.