A polymorphism in the major immediate-early gene delineates groups among cytomegalovirus clinical isolates.

Major immediate-early gene exon 4 sequences were determined at codons 161-241 and 254-397 in 25 cytomegalovirus clinical strains and compared with those of reference strains AD169 and Towne. The nucleotide sequences at codon 161-241 segregated into three groups which could be determined by restriction mapping of a 247-nucleotide amplified target. AD169 and Towne belonged to the same group. Clustered variations and group-specific amino-acid motifs found in the deduced peptide sequence of the two immediate-early (IE) exon 4 regions raised a question is to the effects of polymorphism on IE1 function and/or immunogenicity. On the basis of restriction analysis of polymerase chain reaction (PCR) products, virus isolates were also classified into four glycoprotein B (gB) genotypes. Strain distribution in IE1 and gB genotypes showed a lack of concordance of the two grouping methods, and no preferential association was observed between the clinical context or kind of specimen and IE1 or gB groups. These data lead up to further prospective studies which could provide important information on the implication of the MIE gene region in virus pathogenesis and indicate whether linkage unbalance exists in particular clinical contexts between IE1 and gB loci.

Cloning of Campylobacter jejuni genes required for leucine biosynthesis, and construction of leu-negative mutant of C. jejuni by shuttle transposon mutagenesis.

Campylobacter jejuni is a Gram-negative pathogen responsible for diarrhoeal diseases in humans. To date, very little is known about the genetic organization and molecular biology of this microorganism. The cosmid vector pHC79 was used to construct a genomic library from the total genomic DNA of C. jejuni strain C31 in Escherichia coli and recombinant cosmids capable of complementing the auxotrophic defect in leucine biosynthesis of E. coli HB101 were identified. Three of 400 clones tested were found to be capable of complementing the nutritional defect of E. coli HB101 as well as those of independent leuB mutants of E. coli strains. These results indicated that the cloned genes responsible for leucine complementation encoded an enzyme analogous to the beta-isopropylmalate dehydrogenase specified by the leuB gene in E. coli strains. The sizes of the recombinant cosmids which became stabilized in E. coli cells ranged from 12.9 to 15.4 kb compared to the expected, originally packaged, 45- to 50-kb molecules, attesting to major rearrangements occurring in this background. The recombinant plasmid pILL547 was shown to carry genes that were analogous to the leuB gene and also to the leuC and leuD genes of E. coli. The gene required for leuB complementation was subcloned on a 1.6-kb restriction fragment and was mapped more precisely by insertional mutagenesis using as transposon a newly constructed (MiniTn3-Km) element engineered to mutagenize Campylobacter genes. The leuB gene of C. jejuni was shown to be expressed from its own promoter in E. coli cells. In E. coli minicells, the cloned insert encoded a polypeptide with an apparent molecular weight of 40 kDa. A leucine auxotrophic mutant of C. jejuni strain C31 was constructed in vitro by allelic exchange, replacing the original copy of the leucine gene by an allele mutated by the insertion of the kanamycin transposable element.

Genotyping of Helicobacter pylori isolates by sequencing of PCR products and comparison with the RAPD technique.

Two genotyping methods were performed on bacterial suspensions of the human pathogen Helicobacter pylori. A total of 29 clinical isolates were analysed by sequencing of a 294-bp PCR-derived internal segment of the essential ureC/glmM gene of H. pylori, and by random amplified polymorphic DNA (RAPD) using a single 11-bp oligonucleotide made up of an arbitrary nucleotide sequence. Each isolate exhibited a distinct sequence over a 210-bp stretch of the ureC/glmM gene. Similarly, the isolates bore different profiles when tested by RAPD fingerprinting. Successive strains arising from patients who relapsed following antibiotic treatment and strains isolated from two patients institutionalized in the same care centre had identical ureC/glmM gene sequences and RAPD profiles. Both methods were found to be discriminatory. However, PCR sequencing of the ureC/glmM gene appeared to be more reproducible and more reliable for distinguishing between strains than the RAPD technique.

[Development of genetic and molecular approaches for the diagnosis and study of the pathogenicity of Helicobacter pylori, agent of gastric inflammatory diseases]. / Développement d'approches génétiques et moléculaires pour le diagnostic et l'étude du pouvoir pathogène de Helicobacter pylori, agent des maladies inflammatoires gastriques.

Helicobacter pylori (H. pylori) is a small gram-negative bacillus, recently discovered, found in the stomach of patients with active chronic gastritis and duodenal ulcers. Production of a potent urease has been described as a trait common to all H. pylori so far isolated. To clarify the role of urease in the pathogenic process, as well as to engineer genetic tools useful for the diagnosis of H. pylori, we cloned the genes responsible for urease activity. A genomic library was constructed in Escherichia coli (E. coli) from the chromosomal DNA of the H. pylori strain 85P using a shuttle cosmind vector that we constructed in vitro capable of replicating both in E. coli and Campylobacter jejuni (C. jejuni). The genes responsible for the urease biosynthesis were cloned into E. coli host, then mobilized into C. jejuni where they were expressed. At least six different genes were shown to be required for the expression of the synthesis of an active enzyme; these genes belong to the same cluster and are regulated at the transcriptional level. The two genes encoding the two subunits of the urease enzyme were identified and sequenced; the products of these genes were compared to the other bacterial ureases. The genetic approach allowed to determine the amino-acid sequence of the most immunogenic antigens of H. pylori. In addition, it provides us with genetic tools: a 294-base pairs (bp) DNA fragment internal to one of the urease genes, was shown to be specific of H. pylori strains. This fragment was selectively amplified by polymerase chain reaction (P.C.R.) using two primers designed to target the urease region of all H. pylori isolates present in biological specimen. In addition, P.C.R. followed by direct DNA sequencing of the 294-bp amplified product was shown to be useful to identify and to distinguish between different H. pylori isolates.

Inter-individual retronasal aroma release variability during cheese consumption: Role of food oral processing.

The aim of our study was to explain inter-individual differences on in vivo aroma release during cheese consumption by oral physiological parameters. To reach this objective, 34 subjects were recruited. Their salivary flow, oral volume and velum opening were determined. Six cheddar-based melted cheeses with different fat levels and firmness were flavoured with nonan-2-one (NO) and ethyl propanoate (EP). During their consumption (free protocol), in vivo retro nasal aroma release was followed by Atmospheric Pressure Chemical Ionisation-Mass Spectrometry (APCI-MS). Chewing activity was evaluated by electromyography recordings. Bolus saliva content, mouth-coating, and bolus rheology were also determined. Based on the quantity of aroma released before and after swallowing, subjects can be clustered into three groups: the first one (HRG) is characterized by a large quantity of aroma release whatever the aroma compound; the second one (MRG) showed a large release for EP and a lower one for NO; the third group (LRG) was characterized by a low quantity of aroma release whatever the compound. Whatever the group of subjects, fat and firmness effects differed according to the aroma compound. EP release increased with firmness and fat content, whereas NO release was not affected by firmness and decreased when fat content increased. Physiological parameters which better differentiated the three groups of subjects according to their release behaviour were chewing activity, mouth coating and frequency of velum opening. Subjects from HRG were differentiated from LRG subjects by a higher chewing activity, and more frequent velum opening. Subjects from MRG presented a lower mouth coating explaining their lower release of NO, the more hydrophobic compound. This study shows that the total amount of aroma released in the nasal cavity during food consumption depends not only on the characteristics of the product but also on the oral physiology of the subjects and on their food oral processing.

Influence of pH, temperature and initial yeast:bacteria ratio on the stimulation of Lactobacillus hilgardii by Saccharomyces florentinus isolated from sugary kefir grains.

The effects of pH, temperature and initial yeast:bacteria ratio on Lactobacillus hilgardii and Saccharomyces florentinus cultivated either in pure or mixed culture were studied. Quadratic polynomial as a function of factors was proposed to express the lactic acid production at different sampling times, and the percentage increase in lactic acid production by Lact. hilgardii in mixed culture compared with pure culture. Temperature was the factor which had the main influence on lactic acid production in mixed culture, whereas stimulation of bacteria depended greatly on pH value. In the range 0.1-20%, the initial yeast-bacteria ratio had no effect on these responses, but presence of the yeast was absolutely necessary to obtain high production of lactic acid. Optimum culture conditions were determined to maximize these characteristics.

[Development of genetic and molecular approaches for the diagnosis and the study of the pathogenic power of Helicobacter pylori, agent of inflammatory gastric diseases]. / Développement d'approches génétiques et moléculaires pour le diagnostic et l'étude du pouvoir pathogène de Helicobacter pylori, agent des maladies inflammatoires gastriques.

Helicobacter pylori (H. pylori) is a small Gram negative bacillus, recently discovered, found in the stomach of patients with active chronic gastritis and duodenal ulcers. Production of a potent urease has been described as a trait common to all H. pylori so far isolated. To clarify the role of urease in the pathogenic process, as well as to engineer genetic tools useful for the diagnosis of H. pylori, we cloned the genes responsible for urease activity. A genomic library was constructed in Escherichia coli (E. coli) from the chromosomal DNA of the H. pylori stain 85P using a shuttle cosmid vector that we constructed in vitro capable of replicating both in E. coli and Campylobacter jejuni (C. jejuni). The genes responsible for the urease biosynthesis were cloned into E. coli host, then mobilized into C. jejuni where they were expressed. At least six different genes were shown to be required for the expression of the synthesis of an active enzyme: these genes belong to the same cluster and are regulated at the transcriptional level. The two genes encoding the two subunits of the urease enzyme were identified and sequenced; the products of these genes were compared to be other bacterial ureases. The genetic approach allowed to determine the amino-acid sequence of the most immunogenic antigens of H. pylori. In addition, it provides us with genetic tools: a 294-base pairs (bp) DNA fragment internal to one of the urease genes, was shown to be specific of H. pylori strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Shuttle cloning and nucleotide sequences of Helicobacter pylori genes responsible for urease activity.

Production of a potent urease has been described as a trait common to all Helicobacter pylori so far isolated from humans with gastritis as well as peptic ulceration. The detection of urease activity from genes cloned from H. pylori was made possible by use of a shuttle cosmid vector, allowing replication and movement of cloned DNA sequences in either Escherichia coli or Campylobacter jejuni. With this approach, we cloned a 44-kb portion of H. pylori chromosomal DNA which did not lead to urease activity when introduced into E. coli but permitted, although temporarily, biosynthesis of the urease when transferred by conjugation to C. jejuni. The recombinant cosmid (pILL585) expressing the urease phenotype was mapped and used to subclone an 8.1-kb fragment (pILL590) able to confer the same property to C. jejuni recipient strains. By a series of deletions and subclonings, the urease genes were localized to a 4.2-kb region of DNA and were sequenced by the dideoxy method. Four open reading frames were found, encoding polypeptides with predicted molecular weights of 26,500 (ureA), 61,600 (ureB), 49,200 (ureC), and 15,000 (ureD). The predicted UreA and UreB polypeptides correspond to the two structural subunits of the urease enzyme; they exhibit a high degree of homology with the three structural subunits of Proteus mirabilis (56% exact matches) as well as with the unique structural subunit of jack bean urease (55.5% exact matches). Although the UreD-predicted polypeptide has domains relevant to transmembrane proteins, no precise role could be attributed to this polypeptide or to the UreC polypeptide, which both mapped to a DNA sequence shown to be required to confer urease activity to a C. jejuni recipient strain.

Detection by molecular hybridization of pap, afa, and sfa adherence systems in Escherichia coli strains associated with urinary and enteral infections.

The genetic determinants responsible for the adherence of Escherichia coli to uroepithelial cells have been identified in recent years by genetic and molecular methods. Specific DNA probes for each of the three operons which have been cloned so far (pap, afa, sfa/foc operons) have been used in colony hybridization experiments to detect the presence of each of these operons in the chromosomal DNA of 443 strains of E. coli; 186 strains were from patients with urinary tract infections (pyelonephritis, 106 strains; cystitis, 59; asymptomatic bacteriuria, 21) and 257 were strains from the stools of healthy subjects (61) or from patients with various enteral infections (196). E. coli strains harbouring the pap operon were found more frequently in the urine of patients with pyelonephritis (p less than 0.001) and cystitis (p less than 0.01) than in control stools. The presence of two operons (pap + afa) or (pap + sfa/foc) was only observed in uropathogenic strains (p less than 0.02). Pap and sfa/foc operons were never found in strains causing enteral infection; however, the afa operon was found in 7.6% of the enteropathogenic E. coli.

Gene disruption and replacement as a feasible approach for mutagenesis of Campylobacter jejuni.

Campylobacter jejuni and Campylobacter coli are important causes of human enteric infections. Several determinants of pathogenicity have been proposed based on the clinical features of diarrheal disease and on the phenotypic properties of Campylobacter strains. To facilitate an understanding of the genetic determinants of Campylobacter virulence, we have developed a method for constructing C. jejuni mutants by shuttle mutagenesis. In the example described here, a kanamycin resistance gene was inserted into Campylobacter DNA fragments encoding 16S rRNA cloned in Escherichia coli. These disrupted, modified sequences were returned to C. jejuni via conjugation. Through the apparent process of homologous recombination, the kanamycin resistance-encoding sequences were rescued by chromosomal integration, resulting in the simultaneous gene replacement of one of the 16S sequences of C. jejuni and the loss of the vector. We propose that Campylobacter isogenic mutants could be developed by using this system of shuttle mutagenesis.

High hydrostatic pressure inactivation of Salmonella typhimurium: effects of pressure, duration, pH and temperature studied by analysis of variance.

High hydrostatic pressure treatments are regarded as possible alternative methods for food preservation. One of the primary considerations for industrial applications is the ability of these methods to eradicate pathogenic microorganisms. This study subjected S. typhimurium suspensions, first in a phosphate buffer (pH 7.0) and then in a citrate phosphate buffer (pH 5.6), to high hydrostatic pressure treatments relative to the following variables: pressure (200-400 MPa), duration (3, 10 and 20 min), temperature (4, 20 and 40 degrees C) and the pH of the suspension medium (5.6 and 7.0). An optimal design of 40 runs was obtained using the Fedorov algorithm, and responses were studied by analysis of variance in terms of cell survival on plate count agar. Efficiency was determined by Log10 comparisons of the numbers of live cells before and after treatment. A statistically significant relationship was found between the four variables considered (pressure, pH, duration and temperature), their interactions (duration x pressure, pH x temperature, pH x pressure) and the inactivation of S. typhimurium. R-squared statistical analysis indicated that the linear model used accounted for more than 98% of the variability in the inactivation of S. typhimurium.

Construction of isogenic urease-negative mutants of Helicobacter pylori by allelic exchange.

Isogenic urease-negative mutants of Helicobacter pylori were constructed by allelic replacement. A region of cloned H. pylori DNA containing the structural urease genes (ureA and ureB) was disrupted by insertion of a mini-Tn3-Km transposon. Electrotransformation of H. pylori cells with kanamycin-ureB-disrupted derivative plasmids resulted in isolation of kanamycin-resistant H. pylori transformants. Competence for electrotransformation appeared to be restricted to certain wild-type H. pylori isolates; only 1 isolate (of 10 tested) was consistently transformed. Two of the kanamycin-resistant H. pylori transformants were further studied and shown to be urease negative. Southern hybridization analyses demonstrated that the urease-negative mutants had been constructed by allelic exchange involving simultaneous replacement of the ureB gene with the kanamycin-ureB-disrupted copy and loss of the vector. Immunoblot studies of whole-cell extracts of the isogenic ureB mutants with anti-H. pylori sera indicated the absence of a polypeptide with an apparent molecular mass of 61 kDa; thus, the mutants no longer synthesized the UreB product. Generation of stable, genetically engineered urease mutants of H. pylori will be useful for addressing the role of urease in the pathogenesis of H. pylori infection.

Enzymatic hydrolysis of conjugated steroid metabolites: search for optimum conditions using response surface methodology.

The optimum conditions for hydrolysing conjugated metabolites of steroid hormones in bovine urine were performed with Helix pomatia juice, beta-glucuronidase from bovine liver and preparations of limpets and abalone entrails using response surface methodology. The experimental design and empirical modelling used allowed us to assess the main effects of factors (time, temperature, pH and enzyme quantity) and to predict the optimum conditions for each enzyme preparation. Confirmatory experiments were applied to check the predicted values and to validate the model. The comparison of the enzyme preparation efficiency for various conjugate steroids and the study of possible by-product synthesis led us to select abalone entrails to hydrolyse natural dehydroepiandrosterone, etiocholanolone, epitestosterone; 17 alpha-estradiol and estrone in bovine urine. The optimum conditions were found to be 20 h at 42 degrees C with the pH adjusted to 5.2 and using 12,000 units of enzyme preparation.

Phenotypic and genotypic assays for the detection and identification of adhesins from pyelonephritic Escherichia coli.

Four different gene clusters have been characterized so far which encode adhesins involved in the specific binding of pathogenic Escherichia coli to epithelial cells of the urinary tractus: the pap, sfa, afa and bma operons. The ability to adhere to uroepithelial cells and to interact with one or several of the specific receptors identified for each of the 4 adhesins, has been studied for 102 E. coli strains isolated from patients with pyelonephritis. These receptor-binding assays are referred to as phenotypic assays. Isolates which adhered to uroepithelial cells 68.6% produced at least 1 of the previously described adhesins. In addition, we used DNA probes to detect homologous sequences of the pap, sfa, and afa operons. Genotypic assays revealed that 87.2% of pyelonephretic E. coli contain DNA sequences related to at least 1 of the 4 operons; 78.4%, 22.5% and 11.8% of the strains harboured sequences related to pap, sfa and afa operons, respectively. The afa- and sfa-adhesion determinants were commonly found associated with the presence of the pap operon (8.8% and 18.6%, respectively). Detection of adhesins using the genotypic approach appears to be reliable (all adhesins detected using the phenotypic approach were also detected with probes). Detection by colony hybridization was significantly higher than by phenotypic assay. Discrepancies may have been due to absence of expression of the detected operons and may have resulted from improper in vitro growth conditions, phase variation, and/or heterogeneity of the genes encoding the adhesins within a family of related sequences.

Genetic diversity and geographical distribution of wild Saccharomyces cerevisiae strains from the wine-producing area of Charentes, France.

Electrophoretic karyotyping, mitochondrial DNA restriction fragment length polymorphism analysis, and PCR amplification of interspersed repeats were used to study the variability, phylogenetic affinities, and biogeographic distribution of wild Saccharomyces cerevisiae enological yeasts. The survey concentrated on 42 individual wine cellars in the Charentes area (Cognac region, France). A limited number (35) of predominant S. cerevisiae strains responsible for the fermentation process have been identified by the above molecular methods of differentiation. One strain (ACI) was found to be distributed over the entire area surveyed. There seemed to be little correlation between geographic location and genetic affinity.

Plasmid-mediated ROB-1 beta-lactamase in Pasteurella multocida from a human specimen.

A Pasteurella multocida human isolate was resistant to beta-lactams because of production of ROB-1 beta-lactamase. The beta-lactamase was encoded by a 4.3-kb plasmid closely related to that of a Pasteurella bovine strain, as shown by Sau3A restriction profile and hybridization with a plasmid probe containing the blaROB-1 gene.

Adhesion to and invasion of HeLa cells by pathogenic Escherichia coli carrying the afa-3 gene cluster are mediated by the AfaE and AfaD proteins, respectively.

The afa-3 gene cluster, expressed by uropathogenic and diarrhea-associated Escherichia coli strains, determines the formation of an afimbrial adhesive sheath composed of the AfaD and AfaE-III adhesins. The adherence to HeLa cells by recombinant HB101 strains producing both or only one of these two adhesins was investigated. Ultrastructural analyses of the interaction and gentamicin protection assays showed adherence to HeLa cells by HB101 producing both the AfaD and AfaE-III proteins and internalization of a subpopulation of the bacteria into the cells. The interactions of HeLa cells either with HB101 mutants producing AfaD or AfaE-III or with polystyrene beads coated with purified His6-tagged AfaD or His6-tagged AfaE-III proteins were studied. These experiments demonstrated that AfaE-III allows binding to HeLa cells and that AfaD mediates the internalization of the adherent bacteria. Ultrastructural analyses of the interaction of His6-AfaD-gold complexes with HeLa cells confirmed that AfaD is able to bind to the HeLa cell surface and indicated that it penetrates the cells via clathrin vesicles. These data demonstrate that the afa gene cluster is unique among bacteria, as alone it encodes both adhesion to and invasion of epithelial cells.

The afimbrial adhesive sheath encoded by the afa-3 gene cluster of pathogenic Escherichia coli is composed of two adhesins.

The afa-3 gene cluster determines the formation of an afimbrial adhesive sheath that is expressed by uropathogenic as well as diarrhoea-associated Escherichia coli strains. It contains six genes (afaA-afaF), among which the afaE3 gene is known to code for the structural AfaE-III adhesin (previously designated AFA-III), whereas no role has yet been identified for the afaD gene product. The afa-3 gene cluster is closely related to the daa operon that codes for an adhesin, the F1845 adhesin, which is highly related to the AfaE-III adhesin; however, unlike the AfaE-III adhesin, F1845 is a fimbrial adhesin. Reported in this work is the construction of chimeras between the afa-3 and daa operons. Analyses of the phenotypes conferred by these afa-3/daa chimeric clusters allowed us to conclude that the biogenesis of a fimbrial or an afimbrial adhesin is fully determined by the amino acid sequence of the AfaE-III and F1845 adhesins. Moreover, the role of the AfaD product in the biosynthesis of the afimbrial sheath was assessed by immunogold and immunofluorescence experiments. The AfaD and the AfaE-III products were purified and used to raise rabbit and mouse antisera. Similar to AfaE-III, AfaD was found to be a surface-exposed protein as well as an adhesin; both AfaD and AfaE-III are concomittantly expressed by the bacterial cell. These results demonstrate, for the first time, that the afimbrial adhesive sheath expressed by pathogenic E. coli is composed of two adhesins.

Characterization of AfaE adhesins produced by extraintestinal and intestinal human Escherichia coli isolates: PCR assays for detection of Afa adhesins that do or do not recognize Dr blood group antigens.

Operons of the afa family are expressed by pathogenic Escherichia coli strains associated with intestinal and extraintestinal infections in humans and animals. The recently demonstrated heterogeneity of these operons (L. Lalioui, M. Jouve, P. Gounon, and C. Le Bouguénec, Infect. Immun. 67:5048-5059, 1999) was used to develop a new PCR assay for detecting all the operons of the afa family with a single genetic tool. This PCR approach was validated by investigating three collections of human E. coli isolates originating from the stools of infants with diarrhea (88 strains), the urine of patients with pyelonephritis (97 strains), and the blood of cancer patients (115 strains). The results obtained with this single test and those previously obtained with several PCR assays were closely correlated. The AfaE adhesins encoded by the afa operons are variable, particularly with respect to the primary sequence encoded by the afaE gene. The receptor binding specificities have not been determined for all of these adhesins; some recognize the Dr blood group antigen (Afa/Dr(+) adhesins) on the human decay-accelerating factor (DAF) as a receptor, and others (Afa/Dr(-) adhesins) do not. Thus, the afa operons detected in this study were characterized by subtyping the afaE gene using specific PCRs. In addition, the DAF-binding capacities of as-yet-uncharacterized AfaE adhesins were tested by various cellular approaches. The afaE8 subtype (Afa/Dr(-) adhesin) was found to predominate in afa-positive isolates from sepsis patients (75%); it was frequent in afa-positive pyelonephritis E. coli (55.5%) and absent from diarrhea-associated strains. In contrast, Afa/Dr(+) strains (regardless of the afaE subtype) were associated with both diarrhea (100%) and extraintestinal infections (44 and 25% in afa-positive pyelonephritis and sepsis strains, respectively). These data suggest that there is an association between the subtype of AfaE adhesin and the physiological site of the infection caused by afa-positive strains.