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Biomedical subjects

P Berche

Publications and source records attributed to P Berche.

At least 127 records · Page 7Linked to original sources
PubMed1993

Molecular epidemiology of chronic pulmonary colonisation by Pseudomonas aeruginosa in cystic fibrosis.

The epidemiology of pulmonary colonisation by Pseudomonas aeruginosa was studied in 21 patients with cystic fibrosis (CF) by field inversion gel electrophoresis. DraI-DNA restriction patterns were analysed for 187 P. aeruginosa isolates from these patients. The results revealed that the strains present in individual patients varied during the course of chronic colonisation; the emergence of new strains often was associated with periods of antibiotic therapy. Patients often were colonised by more than one strain (two or three strains were present in 54% of the patients) and the strains obtained from unrelated patients were highly heterogeneous, in contrast to those isolated from a pair of twins. These results demonstrate the heterogeneity and variability of P. aeruginosa isolates in the pulmonary flora of chronically infected CF patients.

Adolescent↗
PubMed1993

Activation of the human complement alternative pathway by Listeria monocytogenes: evidence for direct binding and proteolysis of the C3 component on bacteria.

The capacity of the intracellular pathogen Listeria monocytogenes to activate the alternative pathway of human complement was examined. Incubation of L. monocytogenes with human serum in optimal conditions (20% Mg2+EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid]-chelated serum) consumed (31.3 +/- 3.9)% of C3 hemolytic activity and led to similar amounts of C3 deposition among the 27 strains tested, except for a rough mutant and the penicillin-induced L forms of strain EGD, which bound reduced amounts of C3. The same results were obtained with strains belonging to related species (L. innocua, L. seeligeri, L. welshimeri, and L. ivanovii). Direct evidence is provided that L. monocytogenes induces the deposition of C3b and its cleavage products iC3b and C3d through ester and amide linkages, as demonstrated by the analysis of the released products of radiolabelled purified C3 after treatment with hydroxylamine. Our results clearly demonstrate that L. monocytogenes activates the alternative pathway of human complement, suggesting that bacteria in the blood or in tissues of infected patients are opsonized and targeted to C3 receptor-bearing cells such as macrophages.

Cell Membrane↗
PubMed1993

The zinc metalloprotease of Listeria monocytogenes is required for maturation of phosphatidylcholine phospholipase C: direct evidence obtained by gene complementation.

The maturation of the 33-kDa proenzyme to the 29-kDa phosphatidylcholine phospholipase C (PC-PLC) of Listeria monocytogenes requires the production of the zinc metalloprotease encoded by mpl, the proximal gene of the lecithinase operon. We recently described a low-virulence lecithinase-deficient mutant of L. monocytogenes EGD-SmR, designated JL762, generated by a single insertion of transposon Tn1545 in mpl. This mutant failed to produce the 29-kDa PC-PLC, an exoenzyme probably involved in cell-to-cell spreading. The role of the product of the mpl gene in production of PC-PLC was investigated in trans-complementation experiments. The entire mpl gene was cloned in a plasmid able to replicate in L. monocytogenes. This recombinant plasmid was introduced into JL762 and restored the lecithinase phenotype on egg yolk agar and the production of the active 29-kDa PC-PLC in culture supernatants and partially restored the level of virulence. These results demonstrate that zinc-dependent metalloprotease of L. monocytogenes is involved in the virulence of this bacteria at least through its action on PC-PLC.

Bacterial Proteins↗
PubMed1993

Transposon mutagenesis in Actinobacillus pleuropneumoniae with a Tn10 derivative.

A transposon mutagenesis procedure functional in the gram-negative swine pathogen Actinobacillus pleuropneumoniae was developed for the first time. The technique involved the use of a suicide conjugative plasmid, pLOF/Km, carrying a mini-Tn10 with an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible transposase located outside the mobile element (M. Herrero, V. de Lorenzo, and K. N. Timmis, J. Bacteriol. 172:6557-6567, 1990). The plasmid was mobilized from Escherichia coli to A. pleuropneumoniae through the RP4-mediated broad-host-range conjugal transfer functions provided by the chromosome of the donor strain. When IPTG was present in the mating medium, A. pleuropneumoniae CM5 transposon mutants were obtained at a frequency of 10(-5), while no mutants were detected in the absence of IPTG. Since the frequency of conjugal transfer of the RP4 plasmid from E. coli to A. pleuropneumoniae CM5 was found to be as low as 10(-4), the above result indicated that the expression level of the transposase was a critical factor for obtaining a workable efficiency of transposon mutagenesis. The transposon insertions occurred at random, as determined by Southern blotting of chromosomal DNA of randomly selected mutants and by the ability to generate mutants defective for the selected phenotypes. Almost all the mutants analyzed resulted from a single insertion of the Tn10 element. About 1.2% of the mutants resulted from the cointegration of pLOF/Km into the A. pleuropneumoniae chromosome. The applicability of this transposon mutagenesis system was verified on other A. pleuropneumoniae strains of different serotypes. The usefulness of this transposon mutagenesis system in genetic studies of A. pleuropneumoniae is discussed.

Actinobacillus pleuropneumoniae↗
PubMed1993

Kinetics of antibody production against listeriolysin O in sheep with listeriosis.

The kinetics of antibody production against listeriolysin O (LLO), a major virulence factor of the intracellular bacterial pathogen Listeria monocytogenes, was studied by dot blot analysis with highly purified LLO during oral infection of sheep. Specific antibodies appeared as soon as day 9 of an oral infection and peaked by day 20 of infection; specific antibody levels then remained almost stable for at least 4 months. A subclinical infecting dose (approximately 10(6) viable bacteria) was capable of eliciting a significant antibody response to LLO, almost at the same level as that observed with a high-dose oral challenge (approximately 10(10)). Antibodies to LLO were mostly constituted by immunoglobulin G (IgG), since an IgA response was not detectable and only a transient and inconstant IgM response was observed between day 9 and day 20 of an oral infection. These results show that antibodies to LLO are constantly produced during oral infection even with a low infecting dose, thus confirming that LLO is highly immunogenic. Detection of antibodies to LLO can therefore be used to detect sheep that have been previously exposed to L. monocytogenes.

Animals↗
PubMed1992

L. monocytogenes-induced actin assembly requires the actA gene product, a surface protein.

The intracellular pathogenic bacterium L. monocytogenes can spread directly from cell to cell without leaving the cytoplasm. The mechanism of this movement, generated through bacterially induced actin polymerization, is not understood. By analyzing an avirulent Tn917-lac mutant defective for actin polymerization, we have identified a bacterial component involved in this process. The transposon had inserted in actA, the second gene of an operon. Gene disruption of downstream genes and transformation of the mutant strain with actA showed that the actA gene encodes a surface protein necessary for bacterially induced actin assembly. Our results indicate that it is a 610 amino acid protein with an apparent molecular weight of 90 kd.

3T3 Cells↗
PubMed1992

Human platelet aggregation by Yersinia pseudotuberculosis is mediated by invasin.

Plasmid-free strains of Yersinia pseudotuberculosis induce aggregation of human platelets in vitro. It appears that this phenomenon is mediated by invasin (Inv), a 103-kDa outer membrane protein that permits bacteria to penetrate mammalian cells, since (i) an isogenic inv-deficient mutant failed to aggregate platelets compared with the parental strain; (ii) a monoclonal antibody directed against invasin inhibited platelet aggregation; (iii) Inv+ Escherichia coli HB101 promoted platelet aggregation. Platelet receptors for invasin were identified by using a panel of anti-platelet glycoprotein monoclonal antibodies in a bacterial adhesion assay. We found that bacteria bind to platelet membrane glycoproteins Ic and IIa. Electron microscopic study of bacterium-platelet interactions also revealed that bacteria expressing invasin attach to and are phagocytized by thrombocytes, in contrast to inv-deficient bacteria, indicating that these anucleated cells are able to internalize bacteria in vitro after specific interaction with invasin.

Adhesins, Bacterial↗
PubMed1992

Reduced virulence of a Listeria monocytogenes phospholipase-deficient mutant obtained by transposon insertion into the zinc metalloprotease gene.

A phospholipase-deficient mutant, termed JL762, was obtained from a virulent strain of Listeria monocytogenes by screening a bank of 5,000 Tn1545 transposon-induced mutants on 2.5% egg yolk brain heart infusion agar. As previously shown (J. Mengaud, C. Geoffroy, and P. Cossart, Infect. Immun. 59:1043-1049, 1991), the transposon insertion took place inside the gene mpl, which encodes a zinc metalloprotease. By Western blot (immunoblot) analysis, we showed that loss of phospholipase activity was associated with loss of a 29-kDa zinc-dependent phosphatidylcholine-phospholipase C (PC-PLC) in culture supernatant of JL762 and of EGD-SmR incubated with ion chelator. As the parental strain, JL762 still produced in supernatants approximately 33-kDa proteins antigenically closely related to the 29-kDa PC-PLC. These results strongly suggest that the zinc metalloprotease of L. monocytogenes might play a role in the maturation of the 29-kDa PC-PLC. Although the uptake and the intracellular growth of bacteria were not affected in vitro, we found that the virulence of mutant JL762 was strongly impaired in the mouse.

Animals↗
PubMed1992

The cholera epidemic in Ecuador: towards an endemic in Latin America.

We present an epidemiological study of the first wave of cholera in Ecuador in 1991. One month after the 7th cholera pandemic hit the Pacific coast of Peru, the disease reached the coast of Ecuador and spread to the rest of the country within a few weeks. One year later, 46,320 cases have been notified, giving an incidence of 481 cases per 100,000 inhabitants. The overall mortality rate has been low (697 deaths, i.e. 1.50%), although there have been large differences between the various provinces: in the Andes (Sierra), for example, rates above 8% have been recorded. The first wave peaked about eight weeks after the first case, with 3400 new cases per week throughout the country. This was followed by a gradual decrease towards a baseline of 250-500 new cases per week. A resurgence was observed in most coastal and Andean provinces from November 1991 onwards. The kinetics of the epidemic are compatible with an endemic implantation of the disease in Ecuador, as is probably the case in the entire intertropical Pacific coast region of Latin America. From the data presented, the latin-american episode of the 7th pandemic starting on the Pacific coast, is characterized by a very high attack rate and a low mortality rate as compared to Africa, and the unexpected involvement of populations living on the high Andean plateaux. It is probable from the results collected in Ecuador that cholera will become endemic in Latin America.

Cholera↗
PubMed1992

[Bacterial epidemiology of acute otitis media].

Since the middle of the 1980's, Haemophilus influenzae is the most common bacterial species responsible for acute otitis media in France, followed by Streptococcus pneumoniae. Haemophilus influenzae has maintained its leading position since 1985, but the rate of pneumococcal acute otitis media is increasing fairly steadily. The other recent change with regard to the distribution of bacterial agents is the emergence, four years later than in North America, of Branhamella catarrhalis which currently accounts for 10% of cases of acute otitis media. Modifications in the behavior of microorganisms in relation to antimicrobials usually given to treat upper respiratory tract infections have occurred. Staphylococcus aureus and Branhamella catarrhalis have always exhibited marked resistance to ampicillin due to the production of beta-lactamases. Resistance of Haemophilus influenzae to ampicillin has been increasing gradually since 1985, again with a lag of a few years as compared with the United States; at present, this form of resistance is seen in approximately 35% of strains. More recently in France, 20% of pneumococcal strains recovered from middle ear pus have been shown to have abnormal susceptibility to penicillin. These changes in the behavior of the two main pathogens, i.e., Haemophilus influenzae and Streptococcus pneumoniae, require implementation of rigorous multiregional epidemiologic surveillance programs and may justify reappraisal of current therapeutic approaches.

Acute Disease↗
PubMed1991

Entry of L. monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from gram-positive cocci.

We report the identification of a previously unknown gene, inlA, which is necessary for the gram-positive intracellular pathogen Listeria monocytogenes to invade cultured epithelial cells. The inlA region was localized by transposon mutagenesis, cloned, and sequenced. inlA was introduced into Listeria innocua and shown to confer on this normally noninvasive species the ability to enter cells. Sequencing of inlA predicts an 80 kd protein, internalin. Two-thirds of internalin is made up of two regions of repeats, region A and region B, and the C-terminus of the molecule is similar to that of surface proteins from gram-positive cocci. Internalin has a high content of threonine and serine residues, and the repeat motif of region A has regularly spaced leucine residues. As evidenced by Southern blot analysis, inlA is part of a gene family. One of them is the gene situated directly downstream of inlA, called inlB, which also encodes a leucine-rich repeat protein.

Amino Acid Sequence↗
PubMed1991

Contribution of individual toxin components to virulence of Bacillus anthracis.

Three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF; a calmodulin-dependent adenylate cyclase), compose the lethal (PA + LF) and edema (PA + EF) toxins secreted by Bacillus anthracis. Mutant strains, each deficient in the production of one toxin component, were constructed, and their virulence was then studied. A kanamycin resistance cassette was inserted in each cya (encoding EF) and lef (encoding LF) gene, and the constructs were separately introduced into B. anthracis Sterne on a mobilizable shuttle plasmid. An EF- strain and an LF- strain were then isolated after homologous recombination with the resident toxin-encoding plasmid, pXO1. Spores from these mutants and from a previously constructed PA- mutant were used to inoculate mice, and the lethality and local edema formation were monitored. LF- or PA- mutants were not lethal even at high inocula, whereas the EF- mutant induced lethal infections. This indicates that LF in combination with PA is a key virulence factor required for lethality. Skin edema formation was observed with the LF- mutant, which produces only the combination of PA and EF. However, EF- and LF- mutants were significantly less efficient at inducing, respectively, lethality and edema than was the parental Sterne strain. These results suggest that the three toxin components might act synergistically in vivo to cause lethality and edema formation.

Animals↗
PubMed1991

Intramacrophage growth of Mycobacterium avium during infection of mice.

Growth of the virulent Mycobacterium avium strain TMC 724 in host tissues during persistent infection of mice was studied. Following intravenous infection of C57BL/6 mice, the kinetics of bacterial growth was biphasic in the spleen and liver, with a significant reduction of the multiplication rate after day 21 to 28 of infection. An electron-microscopic study of the liver and spleen of infected mice showed that the bacteria were strictly intracellular. They were observed within inflammatory macrophages populating granulomas disseminated in host tissues. The bacteria were confined to the phagosome compartment, and they were encapsulated. Phagosome-lysosome fusions were encountered, but the bacteria showed no visible signs of degradation and continued to multiply. These results are the first in vivo evidence that virulent M. avium multiplies exclusively intracellularly and that encapsulated bacteria resist the microbicidal mechanisms of macrophages inside the phagosomal compartment.

Animals↗
PubMed1991

Purification and characterization of an extracellular 29-kilodalton phospholipase C from Listeria monocytogenes.

We purified and characterized an extracellular phospholipase produced by Listeria monocytogenes. This enzyme was separated as a homogeneous protein of 29 kDa by chromatography on DEAE-52 cellulose and Bio-Gel P100 columns. It is a zinc-dependent phospholipase C (PLC) that is mainly active at pH 6 to 7 and expresses lecithinase activity and a weaker sphingomyelinase activity. The exoenzyme also hydrolyzed phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin but not phosphatidylinositol. It was distinct from the 36-kDa phosphatidylinositol PLC produced by L. monocytogenes and from the L. ivanovii sphingomyelinase. The pure protein expressed a weak, calcium-independent hemolytic activity and was not toxic in mice. Western immunoblot analysis using a rabbit immune serum raised against the enzyme showed that all virulent strains of L. monocytogenes tested produced in the culture supernatant a 29-kDa PLC. In contrast, no proteins antigenically related to the 29-kDa PLC were detected in supernatants of L. ivanovii, L. seeligeri, L. innocua, or L. welshimeri. The role in virulence of the 29-kDa PLC specifically produced by L. monocytogenes remains to be established.

Amino Acid Sequence↗
PubMed1991

Outbreak of gut colonization by Pseudomonas aeruginosa in immunocompromised children undergoing total digestive decontamination: analysis by pulsed-field electrophoresis.

We analyzed an outbreak of gut colonization by Pseudomonas aeruginosa occurring in an intensive care hematology unit by using conventional typing methods and pulsed-field electrophoresis. In October and November 1989, the feces of four immunocompromised children undergoing total digestive decontamination were colonized by P. aeruginosa. Ten isolates were obtained from the gut flora in pure culture. Retrospective investigations found that one P. aeruginosa isolate from stools of one of the patients was already present at high counts 6 months before the outbreak. This patient had been discharged from the unit in May 1989 and had been readmitted concomitantly with the outbreak. Only pulsed-field electrophoresis could demonstrate that a single epidemic strain was present in the fecal flora of the children. This strain had probably been brought into the unit by the patient with chronic fecal carriage.

Anti-Bacterial Agents↗
PubMed1991

Clinical efficacy and tolerability of cefixime in the treatment of acute sinusitis.

In a noncomparative trial, 73 adults with acute sinusitis confirmed by x-ray received cefixime 400mg once daily for approximately 10 days. At the end of treatment, 60 patients (82%) were cured, 2 (2.7%) had improved and 7 (9.6%) had failed therapy; 4 patients were not evaluable. No relapses were observed at follow-up. Haemophilus influenzae, Streptococcus pneumoniae and Branhamella (Moraxella) catarrhalis were the main pretreatment pathogens, accounting for 65% of all bacterial isolates. Overall, 84% of pathogens were eradicated after treatment. Cefixime was well tolerated, moderate gastrointestinal disturbances being the most frequent adverse effects noted (3 of 4 patients with adverse effects). These results are comparable to those obtained with cefixime 400mg administered orally in 2 divided doses.

Acute Disease↗