PubMed Health⌕ Search

Biomedical subjects

Patrick Berche

Publications and source records attributed to Patrick Berche.

At least 19 recordsLinked to original sources
PubMed2006

ActA is required for crossing of the fetoplacental barrier by Listeria monocytogenes.

The facultative intracellular bacterial pathogen Listeria monocytogenes induces severe fetal infection during pregnancy. Little is known about the molecular mechanisms allowing the maternofetal transmission of bacteria. In this work, we studied fetoplacental invasion by infecting mice with various mutants lacking virulence factors involved in the intracellular life cycle of L. monocytogenes. We found that the placenta was highly susceptible to bacteria, including avirulent bacteria, such as an L. monocytogenes mutant with an hly deletion (DeltaLLO) and a nonpathogenic species, Listeria innocua, suggesting that permissive trophoblastic cells, trapping bacteria, provide a protective niche for bacterial survival. The DeltaLLO mutant, which is unable to escape the phagosomal compartment of infected cells, failed to grow in the trophoblast tissue and to invade the fetus. Mutant bacteria with inlA and inlB deletion (DeltaInlAB) grew in the placenta and fetus as well as did the wild-type virulent stain (EGDwt), indicating that in the murine model, internalins A and B are not involved in fetoplacental invasion by L. monocytogenes. Pregnant mice were then infected with an actA deletion (DeltaActA) strain, a virulence-attenuated mutant that is unable to polymerize actin and to spread from cell to cell. With the DeltaActA mutant, fetal infection occurs, but with a significant delay and restriction, and it requires a placental bacterial load 2 log units higher than that for the wild-type virulent strain. Definitive evidence for the role of ActA was provided by showing that a actA-complemented DeltaActA mutant was restored in its capacity to invade fetuses. ActA-mediated cell-to-cell spreading plays a major role in the vertical transmission of L. monocytogenes to the fetus in the murine model.

Animals↗
PubMed2006

Development of a broad-range 16S rDNA real-time PCR for the diagnosis of septic arthritis in children.

The broad-range PCR has been successfully developed to search for fastidious, slow-growing or uncultured bacteria, and is mostly used when an empirical antibiotic treatment has already been initiated. The technique generally involves standard PCR targeting the gene coding for 16S ribosomal RNA, and includes a post-PCR visualisation step on agarose gel which is a potential source of cross-over contamination. In addition, interpretation of the presence of amplified products on gels can be difficult. We then developed a new SYBR Green-based, universal real-time PCR assay targeting the gene coding for 16S ribosomal RNA, coupled with sequencing of amplified products. The real-time PCR assay was evaluated on 94 articular fluid samples collected from children hospitalised for suspicion of septic arthritis, as compared to the results obtained with bacterial cultures and conventional broad-range PCR. DNA extraction was performed with the automated MagNa Pure system. We could detect DNA from various bacterial pathogens including fastidious bacteria (Kingella kingae, Streptococcus pneumoniae, Streptococcus pyogenes, Salmonella spp, Staphylococcus aureus) from 23% of cases of septic arthritis giving negative culture results. The real-time technique was easier to interpret and allowed to detect four more cases than conventional PCR. PCR based molecular techniques appear to be essential to perform in case of suspicion of septic arthritis, provided the increase of the diagnosed bacterial etiologies. Real-time PCR technique is a sensitive and reliable technique, which can replace conventional PCR for clinical specimens with negative bacterial culture.

Adolescent↗
PubMed2006

Severe osteoarticular infection associated with Panton-Valentine leukocidin-producing Staphylococcus aureus.

We report here a case of osteoarticular infection associated with Panton-Valentine leukocidin-producing Staphylococcus aureus. The 3-year-old child presented osteoarthritis associated with toxic shock syndrome, which was complicated by thrombosis, pneumonia, and diaphysitis, despite appropriate antibiotic treatment. Osteitis associated with toxinogenic S. aureus is rarely described. This case highlights the need of early and aggressive surgical treatment, in addition to appropriate antimicrobial therapy.

Arthritis, Infectious↗
PubMed2006

Microbiological diagnosis of empyema in children: comparative evaluations by culture, polymerase chain reaction, and pneumococcal antigen detection in pleural fluids.

BACKGROUND: Pleural empyema is an increasingly reported complication of pneumonia in children. Microbiological diagnostic tests for empyema by culture frequently have false-negative results due to previous administration of antibiotics. Molecular diagnosis by broad-range 16S ribosomal DNA (rDNA) polymerase chain reaction (PCR) and rapid pneumococcal antigen detection are reliable tools, but their diagnostic value has not been clearly established for pleural fluid samples. Pneumococcal antigen detection has only been validated for urine and cerebrospinal fluid samples. METHODS: Over 4 years, pleural fluid specimens were collected from 78 children with pleural empyema. Standard culture, pneumococcal antigen detection by latex agglutination (Pastorex; Bio-Rad) and immunochromatographic testing (Binax NOW Streptococcus pneumoniae), and 16S rDNA PCR were performed on these specimens. Pneumococcal identification by 16S rDNA PCR and sequencing was confirmed by pneumolysin PCR. RESULTS: Of the 78 cases of pleural empyema, 60 (77%) were microbiologically documented by culture or 16S rDNA PCR. Of the 40 pneumococcal empyema cases, 17 (43%) were only diagnosed by PCR and 23 with PCR and culture. The sensitivity and specificity of the latex antigen detection (with the use of culture and/or PCR as the test standard) were 90% and 95%, respectively. The immunochromatographic test detected pneumococcal antigens in 3 additional specimens for which latex agglutination results were negative, thereby increasing the sensitivity of antigen detection. CONCLUSIONS: Pneumococcal antigen detection in pleural fluid specimens from children provides a rapid and sensitive method of diagnosis of pneumococcal empyema, which can be confirmed by specific pneumolysin PCR when culture results are negative. Broad-range 16S rDNA PCR has value in detecting bacterial agents responsible for culture-negative pleural empyema.

Antigens, Bacterial↗
PubMed2006

[Scientific progress and new biological weapons].

The biological weapons are different from conventional weapons, because living germs hold an extraordinary and predictable potential for multiplication, propagation and genetic variation during their dissemination in a susceptible population. Only natural pathogens (1rst generation weapons) have been used in the past (smallpox virus, plague, anthrax, toxins...). However, new threats are emerging, due to the rapid progress of scientific knowledge and its exponential worldwide diffusion. It is possible to synthesize microorganisms from in silico sequences widely diffused on Internet (poliovirus, influenza...), thus resulting in the accessibility of very dangerous virus confined today in high-security laboratories (virus Ebola...). It is possible also to "improve" pathogens by genetic manipulations, becoming more resistant or virulent (2nd generation weapons). Finally, one can now create de novo new pathogens by molecular breeding (DNA shuffling), potentially highly dangerous for naive populations (3rd generation weapons). Making biological weapons does not require too much technological resources and appears accessible to terrorists, due to low cost and easy use. Although the destructive consequences are difficult to predict, the psychological and social damages should be considerable, because of the highly emotional burden in the population associated to the transgression by man of a taboo of life.

Biological Warfare↗
PubMed2006

Corynebacterium pseudotuberculosis necrotizing lymphadenitis in a twelve-year-old patient.

We report the case of a 12-year-old girl with Corynebacterium pseudotuberculosis necrotizing lymphadenitis. Awareness of this disease is critical for the diagnosis given that corynebacteria are often considered as skin contaminants. The isolate was highly susceptible to antibiotics but only ciprofloxacin, rifampin and gentamicin exerted bactericidal activity.

Adolescent↗
PubMed2006

Invasion of the placenta during murine listeriosis.

Feto-placental infections due to Listeria monocytogenes represent a major threat during pregnancy, and the underlying mechanisms of placental invasion remain poorly understood. Here we used a murine model of listeriosis (pregnant mice, infected at day 14 of gestation) to investigate how this pathogen invades and grows within the placenta to ultimately infect the fetus. When L. monocytogenes is injected intravenously, the invasion of the placenta occurs early after the initial bacteremia, allowing the placental growth of the bacteria, which is an absolute requirement for vertical transmission to the fetus. Kinetically, bacteria first target the cells lining the central arterial canal of the placenta, which stain positively with cytokeratin, demonstrating their fetal trophoblast origin. Bacteria then disseminate rapidly to the other trophoblastic structures, like syncytiotrophoblast cells lining the villous core in the labyrinthine zone of placenta. Additionally, we found that an inflammatory reaction predominantly constituted of polymorphonuclear cells occurs in the villous placenta and participates in the control of infection. Altogether, our results suggest that the infection of murine placenta is dependent, at the early phase, on circulating bacteria and their interaction with endovascular trophoblastic cells. Subsequently, the bacteria spread to the other trophoblastic cells before crossing the placental barrier.

Animals↗
PubMed2006

Value of the chlorhexidine decontamination method for recovery of nontuberculous mycobacteria from sputum samples of patients with cystic fibrosis.

The chlorhexidine method was compared to the N-acetyl-l-cysteine-NaOH-oxalic acid decontamination method currently recommended for the recovery of nontuberculous mycobacteria (NTM) from patients with cystic fibrosis. Sputum samples (n = 827) treated with chlorhexidine yielded twice as many NTM-positive cultures as those treated by the reference method (54 [6.50%] versus 27 [3.25%]; P < 0.0001) despite a higher contamination rate (20% versus 14.2%; P = 0.0017).

Acetylcysteine↗
PubMed2005

Invariant V alpha 14+ NKT cells participate in the early response to enteric Listeria monocytogenes infection.

Invariant Valpha14(+) NKT cells are a specialized CD1-reactive T cell subset implicated in innate and adaptive immunity. We assessed whether Valpha14(+) NKT cells participated in the immune response against enteric Listeria monocytogenes infection in vivo. Using CD1d tetramers loaded with the synthetic lipid alpha-galactosylceramide (CD1d/alphaGC), we found that splenic and hepatic Valpha14(+) NKT cells in C57BL/6 mice were early producers of IFN-gamma (but not IL-4) after L. monocytogenes infection. Adoptive transfer of Valpha14(+) NKT cells derived from TCRalpha degrees Valpha14-Jalpha18 transgenic (TCRalpha degrees Valpha14Tg) mice into alymphoid Rag(null) gamma(c)(null) mice demonstrated that Valpha14(+) NKT cells were capable of providing early protection against enteric L. monocytogenes infection with systemic production of IFN-gamma and reduction of the bacterial burden in the liver and spleen. Rechallenge experiments demonstrated that previously immunized wild-type and Jalpha18null mice, but not TCRalpha(null) or TCRalpha(null) Valpha14Tg mice, were able to mount adaptive responses to L. monocytogenes. These data demonstrate that Valpha14(+) NKT cells are able to participate in the early response against enteric L. monocytogenes through amplification of IFN-gamma production, but are not essential for, nor capable of, mediating memory responses required to sterilize the host.

Animals↗
PubMed2005

Listeria monocytogenes-infected bone marrow myeloid cells promote bacterial invasion of the central nervous system.

Listeria monocytogenes is a facultative intracellular pathogen that is able to invade the central nervous system causing meningoencephalitis and brain abscesses. The mechanisms allowing bacteria to cross the blood-brain barrier are poorly understood. In this work, we used an experimental model of acute listeriosis in the mouse inducing a reproducible invasion of the central nervous system. At the early phase of infection, we find that bacteria invade and rapidly grow in bone marrow cells identified as bone marrow myelomonocytic cells expressing the phenotype CD31pos:Ly-6Cpos:CD11b(pos):LY-6Glow. We demonstrate that central nervous system invasion is facilitated by injecting L. monocytogenes-infected bone marrow cells in comparison with free bacteria or infected spleen cells. In mice transplanted with bone marrow cells from transgenic donor mice expressing the green fluorescent protein (GFP), we show that infected myeloid GFP+ cells adhere to activated brain endothelial cells, accumulate in brain vessels and participate to the pathogenesis of meningoencephalitis and brain abscesses. Our results demonstrate that bone marrow, the main haematopoietic tissue, is a previously unrecognized reservoir of L. monocytogenes-infected myeloid cells, which can play a crucial role in the pathophysiology of meningoencephalitis by releasing infected cells into the circulation that ultimately invade the central nervous system.

Animals↗
PubMed2005

Age-related prevalence and distribution of nontuberculous mycobacterial species among patients with cystic fibrosis.

We studied the prevalence and species distribution of nontuberculous mycobacteria (NTM) in relation to age in 385 patients with cystic fibrosis (CF) (mean age +/- standard deviation [range], 12.0 +/- 6.1 [1 to 24] years; sex ratio, 0.53) attending three Parisian centers. The overall prevalence of NTM in sputum was 8.1% (31 out of 385). The following NTM were isolated (n = 33): Mycobacterium abscessus (n = 13, 39.4%), Mycobacterium avium complex (MAC) (n = 7, 21.2%), Mycobacterium gordonae (n = 6, 18.2%), and other (n = 7, 21.2%). Sixteen patients met the American Thoracic Society microbiological criteria for NTM infection, including 11 patients positive for M. abscessus, 4 for MAC, and 1 for MAC and Mycobacterium kansasii. The overall prevalence of NTM was significantly lower in patients under 15 years old than for patients equal to or more than 15 years old (4.8 versus 14.9%, respectively; P = 0.001). M. abscessus was isolated at all ages, while MAC was not recovered before 15 years (prevalence of 0.0 and 5.2% in patients aged 1 to 14 and 15 to 24, respectively; P = 0.001).

Adolescent↗
PubMed2005

[Pathophysiology and epidemiology of listeriosis].

Listeriosis is a severe infectious disease occurring both in humans and in most other animal species. It is caused by a Gram-positive bacterium, Listeria monocytogenes. This facultative intracellular pathogen is widespread in nature and infects humans through contaminated food. After ingestion, bacteria can cross the gut mucosa and disseminate via the bloodstream. They may then invade the central nervous system and the placenta, causing severe meningoencephalitis and fetoplacental infections, respectively. The mortality rate is about 30%, despite antibiotic therapy. Listeriosis offers an excellent model for studying how bacterial pathogens can cross the blood-brain barrier and placenta. Our understanding of the molecular mechanisms of intracellular parasitism and dissemination to host tissues is better for L. monocytogenes than for any other bacterial pathogen.

Animals↗
PubMed2004

Measurement of immunoglobulin G against Mycobacterial antigen A60 in patients with cystic fibrosis and lung infection due to Mycobacterium abscessus.

BACKGROUND: The diagnosis and prognosis of lung infections due to the emerging nontuberculous mycobacterium (NTM) Mycobacterium abscessus are difficult to establish in children with cystic fibrosis. METHODS: We evaluated the usefulness of an enzyme-linked immunosorbent assay for detecting serum IgG antibodies against the ubiquitous mycobacterial antigen A60. RESULTS: A total of 186 patients with cystic fibrosis (mean age+/-SD, 12.0+/-5.0 years) were studied, including 15 M. abscessus-positive patients who fulfilled American Thoracic Society (ATS) criteria for NTM infection (M. abscessus-infected patients), 7 M. abscessus-positive patients who did not fulfill ATS criteria, 20 patients positive for various other NTM who did not fulfill ATS criteria, and 144 NTM-negative control patients; mean IgG titers (+/-SD) for these 4 groups were 718+/-342 U, 193+/-113 U, 129+/-49 U, and 121+/-53 U, respectively (M. abscessus-infected patients vs. each of the other groups, P<.005). The A60 IgG test was both sensitive (approximately 87%) and specific (approximately 95%) if adapted cutoff values were used (150 U and 250 U for patients aged 10 years, respectively) and correlated well with results of acid-fast bacilli smears. CONCLUSION: Measurement of anti-A60 IgG may be useful for both the diagnosis and assessment of activity of M. abscessus lung infection in persons with cystic fibrosis.

Anti-Bacterial Agents↗
PubMed2004

Immunoproliferative small intestinal disease associated with Campylobacter jejuni.

BACKGROUND: Immunoproliferative small intestinal disease (also known as alpha chain disease) is a form of lymphoma that arises in small intestinal mucosa-associated lymphoid tissue (MALT) and is associated with the expression of a monotypic truncated immunoglobulin alpha heavy chain without an associated light chain. Early-stage disease responds to antibiotics, suggesting a bacterial origin. We attempted to identify a causative agent. METHODS: We performed polymerase chain reaction (PCR), DNA sequencing, fluorescence in situ hybridization, and immunohistochemical studies on intestinal-biopsy specimens from a series of patients with immunoproliferative small intestinal disease. RESULTS: Analysis of frozen intestinal tissue obtained from an index patient with immunoproliferative small intestinal disease who had a dramatic response to antibiotics revealed the presence of Campylobacter jejuni. A follow-up retrospective analysis of archival intestinal-biopsy specimens disclosed campylobacter species in four of six additional patients with immunoproliferative small intestinal disease. CONCLUSIONS: These results indicate that campylobacter and immunoproliferative small intestinal disease are associated and that C. jejuni should be added to the growing list of human pathogens responsible for immunoproliferative states.

Anti-Bacterial Agents↗
PubMed2004

Differential roles of multiple signal peptidases in the virulence of Listeria monocytogenes.

Most bacteria contain one type I signal peptidase (Spase I) for cleavage of signal peptides from exported and secreted proteins. Here, we identified a locus encoding three contiguous Spase I genes in the genome of Listeria monocytogenes. The deduced Sip proteins (denoted SipX, SipY and SipZ) are significantly similar to SipS and SipT, the major SPase I proteins of Bacillus subtilis (38% to 44% peptidic identity). We studied the role of these multiple signal peptidases in bacterial pathogenicity by constructing a series of single- and double-chromosomal knock-out mutants. Inactivation of sipX did not affect intracellular multiplication of L. monocytogenes but significantly reduced bacterial virulence (approximately 100-fold). Inactivation of sipZ impaired the secretion of phospholipase C (PC-PLC) and listeriolysin O (LLO), restricted intracellular multiplication and almost abolished virulence (LD(50) of 10(8.3)), inactivation of sipY had no detectable effects. Most importantly, a mutant expressing only SipX was impaired in intracellular survival and strongly attenuated in the mouse (LD(50) of 10(7.2)), whereas, a mutant expressing only SipZ behaved like wild-type EGD in all the assays performed. The data establish that SipX and SipZ perform distinct functions in bacterial pathogenicity and that SipZ is the major Spase I of L. monocytogenes. This work constitutes the first report on the differential role of multiple Spases I in a pathogenic bacterium and suggests a possible post-translational control mechanism of virulence factors expression.

Amino Acid Sequence↗
PubMed2004

Sequence and binding activity of the autolysin-adhesin Ami from epidemic Listeria monocytogenes 4b.

Ami is an autolytic amidase from Listeria monocytogenes that is targeted to the bacterial surface via its C-terminal cell wall anchoring (CWA) domain. We recently showed that the CWA domain from Ami of L. monocytogenes EGD (serovar 1/2a) (Ami 1/2a) mediated bacterial binding to mammalian cells. Here we studied the sequence and binding properties of Ami from CHUT 82337 (serovar 4b) (Ami 4b). The Ami 4b polypeptide is predicted to be 770 amino acids long (compared with the 917 amino acids of Ami 1/2a from EGD). Ami 1/2a and Ami 4b are almost identical in the N-terminal enzymatic domain (approximately 98% amino acid identity), but the sequence is poorly conserved in the C-terminal CWA domain, with only approximately 54% amino acid identity and eight GW modules in Ami 1/2a compared with six GW modules in Ami 4b. The purified Ami 4b CWA domain efficiently bound serovar 4b bacterial cells and only poorly bound serovar 1/2a bacterial cells. The Ami 4b CWA domain was also significantly less able to bind Hep-G2 human hepatocytic cells than the Ami 1/2a CWA domain. We sequenced the ami regions encoding CWA domains of reference strains belonging to the 12 L. monocytogenes serovars. The phylogenic tree constructed from the sequences yielded a binary division into group I (serovars 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, and 7) and group II (serovars 4a, 4b, 4c, 4d, and 4e). This is the first direct evidence of divergence between serovars 1/2a and 4b in a gene involved in the adhesion of L. monocytogenes to mammalian cells, as well as the first demonstration of allelic polymorphism correlated with the somatic antigen in this species.

Adhesins, Bacterial↗
PubMed2004

Development of a serum-based Taqman real-time PCR assay for diagnosis of invasive aspergillosis.

We compared an Aspergillus fumigatus-specific Taqman real-time PCR assay for the diagnosis of invasive aspergillosis (IA) with the enzyme-linked immunosorbent assay Platelia Aspergillus method. Patients with proven or probable IA had positive results with at least one of the two methods. The PCR method seems to be more specific, and a combination of the two should improve the diagnosis of IA.

Adolescent↗