Biomedical subjects

H Monteil

Publications and source records attributed to H Monteil.

At least 37 records · Page 2Linked to original sources

PubMed2000

Variable expressions of Staphylococcus aureus bicomponent leucotoxins semiquantified by competitive reverse transcription-PCR.

A competitive reverse transcription-PCR method was developed for the semiquantitation of the expression of genes encoding bicomponent leucotoxins of Staphylococcus aureus, e.g., Panton-Valentine leucocidin (lukPV), gamma-hemolysin (hlgA and hlgCB), and LukE-LukD (lukED). The optimization procedure included RNA preparation; reverse transcription; the use of various amounts of enzymes, antisense primer, and RNA; and the final amplification chain reaction. Reproducible results were obtained, with sensitivity for detection of cDNA within the range of 1 mRNA/10(4) CFU to 10(2) mRNA/CFU, depending on the gene. Both specific mRNAs were more significantly expressed at the late-exponential phase of growth. Expression was about 100-fold higher in yeast extract-Casamino Acids-pyruvate medium than in heart infusion medium. Expression of the widely distributed gamma-hemolysin locus in the NTCC 8178 strain was around 10-fold diminished compared with that in the ATCC 49775 strain. Because of the lower level of hlgA expression, the corresponding protein, which is generally not abundant in culture supernatant, should be investigated for its contribution to the leucotoxin-associated virulence. The agr, sar, and agr sar mutant strains revealed a great dependence with regard to leucotoxin expression on the global regulatory system in S. aureus, except that expression of hlgA was not affected in the agr mutant.

Bacterial Proteins↗

PubMed2000

Direct molecular typing of Borrelia burgdorferi sensu lato species in synovial samples from patients with lyme arthritis.

Since Lyme arthritis was first described in the United States, it has now been reported in many countries of Europe. However, very few strains of the causative bacterium, Borrelia burgdorferi, have been isolated from synovial samples. For this reason, different molecular direct typing methods were developed recently to assess which species could be involved in Lyme arthritis in Europe. We developed a simple oligonucleotide typing method with PCR fragments from the flagellin gene of B. burgdorferi sensu lato, which is able to differentiate seven different Borrelia species. Among 10 consecutive PCR-positive patients with Lyme arthritis from the northeastern France, two species were identified in synovial samples: B. burgdorferi sensu stricto in 9 cases and B. garinii in 1 case. Conversely, all B. burgdorferi sensu lato species detected in 10 consecutive PCR-positive biopsies from a second set of patients with erythema migrans from the same geographical area were identified as either B. afzelii or B. garinii (P < 0.001). These results indicate that B. burgdorferi sensu stricto is the principal but not the only Borrelia species involved in Lyme arthritis in northeastern France.

Arthritis, Infectious↗

PubMed2000

[Bacterial growth delay in E. coli: demonstration and evaluation of the beta-lactamase post-inhibitor effect on 6 strains with different resistance phenotypes].

Six E. coli, whose phenotypes of resistance were different, were tested in vitro in order to evaluate a regrowth delay, the post beta-lactamases inhibitor effect (PLIE). This PLIE was investigated after a brief incubation in contact with clavulanic acid (CA) alone or associated with amoxicillin (AMX). After removal of the drugs used during the pre-exposure phase, the bacteria were incubated with AMX at different concentrations. The PLIE was shown not to be in association with any other regrowth delay (post-antibiotic effect or effect inherent to the technical procedures used). A PLIE was evaluated on the five intermediary or high-level beta-lactamases-producing strains. Generally, the duration of the PLIE was prolonged after the CA alone pre-exposure phase and could reach values up to 22 hours. The concentrations of AMX added in cultures previously exposed to sufficient CA concentrations were related to an extended PLIE.

Amoxicillin↗

PubMed1999

Discoupling the Ca(2+)-activation from the pore-forming function of the bi-component Panton-Valentine leucocidin in human PMNs.

The consecutive cell activation, including Ca(2+)-channel opening, and pore formation leading to human neutrophil lysis were the two functions of the staphylococcal Panton-Valentine leucocidin attempted to be discoupled by site-directed mutagenesis. In a first approach consisting in deletions of the cytoplasmic extremity of the transmembranous domain, we produced a LukF-PV DeltaSer125-Leu128 with a slightly reduced Ca(2+) induction but with a significantly lowered lytic activity when combined with its synergistic protein LukS-PV. The second approach consisted in the modification of charges and/or introduction of a steric hindrance inside the pore, which also led to interesting mutated proteins: LukF-PV G131D, G131W and G130D. The latter had an intact Ca(2+) induction ability while the lytic one was 20-fold diminished. Binding properties and intrinsic pore diameters of these discoupled toxins remained comparable to the wild-type protein. The mutated proteins promoted interleukin-8 secretion, but they were rather inactive in an experimental model. New insights are brought concerning the role of the two functions in the virulence of this bi-component leucotoxin.

Amino Acid Substitution↗

PubMed1999

The structure of a Staphylococcus aureus leucocidin component (LukF-PV) reveals the fold of the water-soluble species of a family of transmembrane pore-forming toxins.

BACKGROUND: Leucocidins and gamma-hemolysins are bi-component toxins secreted by Staphylococcus aureus. These toxins activate responses of specific cells and form lethal transmembrane pores. Their leucotoxic and hemolytic activities involve the sequential binding and the synergistic association of a class S and a class F component, which form hetero-oligomeric complexes. The components of each protein class are produced as non-associated, water-soluble proteins that undergo conformational changes and oligomerization after recognition of their cell targets. RESULTS: The crystal structure of the monomeric water-soluble form of the F component of Panton-Valentine leucocidin (LukF-PV) has been solved by the multiwavelength anomalous dispersion (MAD) method and refined at 2.0 A resolution. The core of this three-domain protein is similar to that of alpha-hemolysin, but significant differences occur in regions that may be involved in the mechanism of pore formation. The glycine-rich stem, which undergoes a major rearrangement in this process, forms an additional domain in LukF-PV. The fold of this domain is similar to that of the neurotoxins and cardiotoxins from snake venom. CONCLUSIONS: The structure analysis and a multiple sequence alignment of all toxic components, suggest that LukF-PV represents the fold of any water-soluble secreted protein in this family of transmembrane pore-forming toxins. The comparison of the structures of LukF-PV and alpha-hemolysin provides some insights into the mechanism of transmembrane pore formation for the bi-component toxins, which may diverge from that of the alpha-hemolysin heptamer.

Amino Acid Sequence↗

PubMed1999

Leukocidal toxins of staphylococci.

Leukocidal toxins (synergohymenotropic toxin) are cytotoxins produced by staphylococci (S. aureus and S. intermedius) and consist of two separate components. The toxic effect depends on the synergistic action of two proteins. One of them belongs to class F (e.g. LukF-PV, LukF-R, LukF-I, LukM, HlgB) and the other, to class S (e.g. LukS-PV, LukS-R, LukS-I, HlgA, HlgC). Best known are the toxins produced by S. aureus: gamma-haemolysins, HlgA/HlgB and HlgC/HlgB and leukocidin Panton-Valentine, LukS-PV/LukF-PV (Luk-PV, PVL). Very few data are available concerning the relationship between the production of these toxins and the pathology of staphylococcal infections, because little is known about local and general effects of these leukocidal products in vivo. Frequent isolations of staphylococcal strains producing leukocidal toxins from necrotic skin lesions and furuncles suggest a role of these toxins in the virulence of staphylococci, at least in cutaneous infections. Recent data on mechanisms of cytotoxic effects of staphylococcal leukocidal toxins in vitro as well as effects of leukocidal toxins in vitro are discussed. Cell membranes appear to be a primary target for triggering the lysis of phagocytic cells caused by staphylococcal leukocidal toxins.

Animals↗

PubMed1999

CD14 expression by human mononuclear phagocytes is modulated by Clostridium difficile toxin B.

Toxin B, an exotoxin produced by the anaerobic Gram-positive bacteria Clostridium difficile, is responsible for pseudomembranous colitis in humans. It deeply modifies morphology of cultured cells and enhances their membrane surface area, which suggests a possible alteration of membrane receptor distribution. Since toxin B and bacterial lipopolysaccharide can act synergistically on TNF-alpha production by mononuclear phagocytes, the effect of toxin B on CD14 expression was investigated using flow cytometric analysis. It was shown that monocytes overexpressed CD14 after 5 h of treatment with toxin B. In contrast, after 24 h of treatment, the percentage of CD14 monocytes decreased, although, most frequently, the remaining positive cells expressed high levels of CD14 compared with untreated cells. Macrophages treated for 5 h with toxin B overexpressed CD14, but this effect persisted for at least 24 h. Both the percentage of positive macrophages and the mean level of CD14 per cell were increased. Thus toxin B can modulate expression of CD14 and its modulation depends on the differentiation status and maybe on the activation state, since some individual variations were observed in monocyte response to toxin.

Bacterial Proteins↗

PubMed1999

Bartonella alsatica sp. nov., a new Bartonella species isolated from the blood of wild rabbits.

Bartonella species are considered as emerging human pathogens, with at least six different species pathogenic or possibly pathogenic for humans. However, little is known about Bartonella distribution, species polymorphism and pathogenicity in mammalian species. The objective of this work was to determine the presence, the frequency and the distribution of Bartonella species in wild rabbits (Oryctolagus cuniculus) caught in warrens in Alsace, France. Humans may come into contact with wild rabbits when hunting, especially when they are picked up with bare hands and at time of evisceration. Of 30 blood samples collected and cultured from wild rabbits, nine (30%) were positive for organisms morphologically similar to Bartonella spp. The bacteria appeared as small, fastidious, aerobic, oxidase-negative, Gram-negative rods which could be localized within erythrocytes. Their biochemical properties were similar to those of the genus Bartonella. The sequence of the 16S rRNA gene obtained from the rabbit isolates was highly related to the sequences of the different Bartonella species (97.8-99.3% similarity). The high DNA hybridization rate (81-90% similarity) between the three strains isolated from rabbit blood confirmed that they belong to the same bacterial species. Hybridization values, obtained with the nuclease-TCA method, when testing type strains of recognized Bartonella species (9-14% similarity), support the creation of a new species for the rabbit isolates. The name Bartonella alsatica is proposed for these strains isolated from the blood of wild rabbits. The type strain is IBS 382T (= CIP 105477T).

Animals↗

PubMed1999

Predominant Staphylococcus aureus isolated from antibiotic-associated diarrhea is clinically relevant and produces enterotoxin A and the bicomponent toxin LukE-lukD.

Staphylococcus aureus was isolated as the predominant or only isolate from cultures of stools of 60 patients over 2 years in a university hospital, leading to the collection of 114 isolates. Diarrhea was observed in 90% of the patients. Ninety-eight percent of the patients had received antibiotics in the month before the diarrhea. Ninety-two percent of the S. aureus isolates were methicillin resistant. S. aureus was encountered with antibiotic-associated diarrhea among 47 quite elderly patients affected or not affected by a gastrointestinal disease. Among the antimicrobial treatments, cessation of the previous therapy when possible or rapid application of oral vancomycin therapy was the most appropriate. Analysis of total DNA by pulsed-field gel electrophoresis revealed 27 different SmaI pulsotypes distributed in 15 clusters. The pulsotypes never differed for related isolates from a single patient, even if they originated from patients with bacteremia. S. aureus was not isolated as the predominant isolate in cultures of stools of 57 patients who received an antimicrobial treatment for more than 5 days without diarrhea. Occurence of production of both enterotoxin A and the bicomponent leucotoxin LukE-LukD by the S. aureus isolates was significantly different from that by random isolates. The results strongly suggest that when predominant in stool samples, S. aureus should be considered a possible etiologic agent for some cases of antibiotic-associated diarrhea.

Adolescent↗

PubMed1999

Analysis with a combination of macrorestriction endonucleases reveals a high degree of polymorphism among Bordetella pertussis isolates in eastern France.

From 1990 to 1996, routine screening for whooping cough identified 399 patients with a calmodulin-dependent adenylate cyclase-positive test result and yielded 69 Bordetella pertussis isolates. None of the patients were fully vaccinated, and most were less than 6 months old. Analysis of total DNA by pulsed-field gel electrophoresis (PFGE) after XbaI, SpeI, or DraI macrorestriction yielded 19, 15, and 5 different patterns, respectively, whereas ribotyping failed to demonstrate any strain polymorphism. Discrimination among the isolates was improved by combining the PFGE profiles. Some patterns were more frequent, but the corresponding patients were not clearly epidemiologically related. The patterns for two strains obtained during a 3-month period from patients who were neighbors differed by the length of a single DNA fragment. These data strongly suggest that one type of isolate is widely spread throughout the world and is carried by individuals other than patients who develop a true illness.

Bacterial Typing Techniques↗

PubMed1999

Oxacillin susceptibility testing of staphylococci directly from Bactec Plus blood cultures by the BBL Crystal MRSA ID system.

The BBL Crystal MRSA ID test (Becton Dickinson) was applied directly to blood culture vials containing clusters of gram-positive cocci. The sensitivity and specificity of the test were 84 and 100% and 54 and 100% for vials containing Staphylococcus aureus and coagulase-negative staphylococci, respectively. This test is a reliable method for direct detection of methicillin resistance in positive blood culture vials when S. aureus is identified in parallel by rapid identification procedures.

Blood↗

PubMed1999

One-step reverse transcriptase PCR method for detection of Borrelia burgdorferi mRNA in mouse Lyme arthritis tissue samples.

A one-step reverse transcriptase PCR (RT-PCR) method for detection of Borrelia burgdorferi mRNA in infected C3H mice is described. This simple procedure, less prone to nucleic acid cross-contamination than the standard method, was found to be 10-fold more sensitive than a classical two-step RT-PCR assay. By using one-step RT-PCR, flagellin mRNAs were detected in synovial and heart tissues from all seven infected mice tested.

Animals↗

PubMed1999

[In vitro demonstration of a true post-beta-lactamases inhibitor effect (PLIE) of clavulanic acid on Klebsiella pneumoniae and Haemophilus influenzae].

In vivo, serum concentrations of beta-lactamase inhibitors measured during the last part of the dosing interval are below the levels associated with in vitro activity. Nevertheless, beta-lactam plus beta-lactamase inhibitor combinations remain active in vivo throughout the dosing interval. One of the many reasons for this contradiction may be the PLIE. The PLIE can be evaluated only in the light of the postantibiotic effect (PAE). Also, accurate determination of the PLIE requires a careful investigation of all bacterial regrowth delays (BRDs) inherent to the technical procedures used. The purpose of the study reported herein was to determine the true in vitro PLIE of clavulanic acid (CA) against two beta-lactamase-producing strains, a Klebsiella pneumoniae strain (amoxicillin [AMX] MIC > 256 mg/L; CA MIC = 64 mg/L; and AMX + CA MIX = 4 mg/L) and a Haemophilus influenzae strain (AMX MIC = 32 mg/L; CA > 32 mg/L; AMX-CA = 1 mg/L). For each strain, a stationary phase inoculum of 10(7) was preexposed for 2 h to either CA alone or CA + AMX in various concentrations. Dilution to 10(-2) or 10(-3) was performed to eliminate the CA and/or AMX after the preexposition phase. Hourly bacterial counts were done between 0 and 8 h and after 24 h. Control cultures exposed to AMX after dilution showed a growth delay possibly ascribable to the time needed for bacteria to produce a large enough amount of beta-lactamases. Control experiments were done to unequivocally differentiate PLIE from PAE and BRD. The true PLIE values thus obtained ranged from 0 to 4.5 h for K. pneumoniae and from 0 to 15 h for H. influenzae. For both strains, a PLIE was demonstrated after exposure to CA alone.

Amoxicillin↗

PubMed1999

[Evaluation of Hemofast MRSA for identification of Staphylococcus aureus and detection of methicillin-resistance staphylococci, directly in blood culture broths].

The newly developed Hemofast MRSA system for Staphylococcus aureus identification and methicillin-resistant staphylococci (MRS) detection in blood culture broths was evaluated in 106 Bactec broths containing grapelike clusters of Gram-positive cocci. All 26 S. aureus positive broths were correctly identified by Hemofast MRSA within two hours, and 81% were identified within one hour. Sensitivity and specificity were both 100%. Accuracy of MRS detection was 100%, i.e., no discrepancies versus the agar diffusion method were found. When the 28 broths containing coagulase negative staphylococci (CNS) were tested using an inoculum ten fold larger than for S. aureus testing, a number of discrepancy were recorded. For 49 other broths containing CNS, accuracy was 98.5% when the test was interpreted based on the growth rate of the strain, according to the manufacturer's instructions. One broth containing S. epidermidis strain susceptible yielded a false result with Hemofast MRSA, indicating that it probably contained a contaminant. The other discrepancies occurred with specimens containing mixed populations with CNS or a Micrococcus strain. Most (85%) results were available within 4 hrs 30 min, irrespective of the S. aureus or CNS strains. Avoiding the isolation step on agar, Hemofast MRSA saves 24 to 48 hours, thus allowing earlier antistaphylococcal treatment.

Bacteriological Techniques↗

PubMed1998

Characterization of a novel structural member, LukE-LukD, of the bi-component staphylococcal leucotoxins family.

A new member of the staphylococcal bi-component leucotoxins family, LukE (32 kDa) and LukD (34.3 kDa) has been characterized from Staphylococcus aureus strain Newman. LukE was 58-68% identical with the class S proteins, whereas LukD was 71-77% identical with the class F proteins of the family. A partial immunoreactivity with the various affinity-purified antibodies specific for the other proteins was observed. Immunoprecipitation assay and gene probing confirmed a 30% frequency among human clinical isolates, differing from the distribution of the other known leucotoxins (P<0.005). LukE+LukD was as effective as the Panton-Valentine leucocidin for inducing dermonecrosis when injected in the rabbit skin, but not hemolytic and poorly leucotoxic compared to other leucotoxins expressed by Staphylococcus aureus.

Amino Acid Sequence↗

PubMed1998

Analysis of recent antimicrobial agents in human biological fluids by high-performance liquid chromatography.

Our previous review on the liquid chromatographic (LC) analysis of anti-bacterial agents was published in 1990 in a special issue of the Journal of Chromatography. Eight years later, some new agents have been registered and numerous other are under clinical experiment. In spite of therapeutic problems encountered with certain bacterial pathogens, the development of novel drug candidates has slowed partially due to the need for identification of new bacterial targets and the cost of the research. The present overview updates the LC methods for the quantitations of recent antimicrobial agents (marketed and in clinical development) in human biological fluids. Consideration has been given to procedures permitting the determination of isomers and metabolites as well as methods regarding tissue extracts or liquid sampled from physiological sanctuaries. LC methods are available for the quantitation of almost all registered or investigated recent anti-infective drugs and some are applicable in routine practice. Nevertheless, few techniques have been validated for the determination in tissue extracts limiting the development of penetration studies.

Anti-Bacterial Agents↗

PubMed1998

Synthesis and antimicrobial activity of coumarin 7-substituted cephalosporins and sulfones.

Some coumarin 7-substituted cephalosporins and related sulfones were prepared and an antimicrobial assay was performed. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) carried out on cephalosporins showed a potential activity of some of the synthesized compounds against Gram-positive microorganisms. The tests performed on the corresponding sulfones showed no significant activity, neither as antimicrobial agents nor as inhibitors of beta-lactamase. An association of sulfone 6a with ampicillin was observed to inhibit Gram-positive microorganisms with a lower MIC than for ampicillin alone.

Cephalosporins↗

PubMed1998

The staphylococcal pore-forming leukotoxins open Ca2+ channels in the membrane of human polymorphonuclear neutrophils.

The ability of leukotoxins secreted by Staphylococcus aureus to modify the permeability of the membrane of human polymorphonuclear neutrophils has been studied by spectrofluorometry and appropriate fluorescent probes. This family of bicomponent leukotoxins is constituted by, at least, three pairs of proteins: LukS-PV/LukF-PV, HlgA/HlgB, HlgC/HlgB. After binding of both components to the membrane, each pair induces influxes of divalent cations and ethidium in polymorphonuclear neutrophils, although with different intensities. The influx of divalent cations appears sooner than the influx of ethidium. The pathway for divalent cations is not permeable to monovalent cations (Na+, K+, ethidium+) and is blocked by Ca2+ channel inhibitors that do not block the fluxes of ethidium and monovalent cations. It is concluded that the leukotoxins bind to a receptor linked to a divalent cation-selective channel or to the channel itself which is activated. Then, the leukotoxins open a second pathway by insertion into the membrane and subsequent formation of aspecific pores allowing an influx of ethidium.

Bacterial Proteins↗