Alban Le Monnier

Increasing incidence of listeriosis in France and other European countries

The cause of an increase among persons >60 years of age in France is unknown.

Conjugated action of two species-specific invasion proteins for fetoplacental listeriosis.

The ability to cross host barriers is an essential virulence determinant of invasive microbial pathogens. Listeria monocytogenes is a model microorganism that crosses human intestinal and placental barriers, and causes severe maternofetal infections by an unknown mechanism. Several studies have helped to characterize the bacterial invasion proteins InlA and InlB. However, their respective species specificity has complicated investigations on their in vivo role. Here we describe two novel and complementary animal models for human listeriosis: the gerbil, a natural host for L. monocytogenes, and a knock-in mouse line ubiquitously expressing humanized E-cadherin. Using these two models, we uncover the essential and interdependent roles of InlA and InlB in fetoplacental listeriosis, and thereby decipher the molecular mechanism underlying the ability of a microbe to target and cross the placental barrier.

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.

Human Listeriosis Caused by Listeria ivanovii

Two species of Listeria are pathogenic; L. monocytogenes infects humans and animals, and L. ivanovii has been considered to infect ruminants only. We report L. ivanovii–associated gastroenteritis and bacteremia in a man. This isolate was indistinguishable from prototypic ruminant strains. L. ivanovii is thus an enteric opportunistic human pathogen.

Fusobacterium necrophorum middle ear infections in children and related complications: report of 25 cases and literature review.

Background: Fusobacterium necrophorum is associated with Lemierre syndrome (pharyngitis with septic thrombosis of the internal jugular veins) but it can also be involved in other head and neck infections, including sinusitis, parotitis, dental infections, and otitis media. Methods: This retrospective study analyzes a series of 25 pediatric cases of acute otitis media caused by F. necrophorum and treated in our institution between 1995 and 2006. Results: We observed 3 clinical presentations: (1) uncomplicated otitis media (44%; n = 11); (2) acute mastoiditis (40%; n = 10); and (3) otogenic variant of Lemierre syndrome (16%; n = 4) associating acute mastoiditis, suppurative thrombophlebitis of the lateral and/or cavernous sinuses, meningitis syndrome, and sometimes distant septic metastasis or extensive osteolysis of the temporal bone. Sixty percent of these cases were diagnosed during the last 4 years of the study. Children less than 1 year of age were at increased risk for Lemierre syndrome. Broad range 16S rDNA polymerase chain reaction and sequencing were used to confirm the identification of F. necrophroum and to detect secondary sites of infection. All patients had favorable clinical outcome, but complicated cases (mastoiditis and otogenic variant of Lemierre syndrome) required prolonged hospital stays and duration of treatment. Conclusions: Based on bacteriologic investigation, we recommend systematic culture for anaerobes and that antibiotic treatment of F. necrophorum middle ear infections and subsequent complications includes coverage for anaerobic bacteria.

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

ABSTRACT 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 (ΔLLO) and a nonpathogenic species, Listeria innocua, suggesting that permissive trophoblastic cells, trapping bacteria, provide a protective niche for bacterial survival. The ΔLLO 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 (ΔInlAB) 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 (ΔActA) strain, a virulence-attenuated mutant that is unable to polymerize actin and to spread from cell to cell. With the ΔActA 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 ΔActA 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.

Polyphasic characterization and genetic relatedness of low-virulence and virulent Listeria monocytogenes isolates

BackgroundCurrently, food regulatory authorities consider all Listeria monocytogenes isolates as equally virulent. However, an increasing number of studies demonstrate extensive variations in virulence and pathogenicity of L. monocytogenes strains. Up to now, there is no comprehensive overview of the population genetic structure of L. monocytogenes taking into account virulence level. We have previously demonstrated that different low-virulence strains exhibit the same mutations in virulence genes suggesting that they could have common evolutionary pathways. New low-virulence strains were identified and assigned to phenotypic and genotypic Groups using cluster analysis. Pulsed-field gel electrophoresis, virulence gene sequencing and multi-locus sequence typing analyses were performed to study the genetic relatedness and the population structure between the studied low-virulence isolates and virulent strains.ResultsThese methods showed that low-virulence strains are widely distributed in the two major lineages, but some are also clustered according to their genetic mutations. These analyses showed that low-virulence strains initially grouped according to their lineage, then to their serotypes and after which, they lost their virulence suggesting a relatively recent emergence.ConclusionsLoss of virulence in lineage II strains was related to point mutation in a few virulence genes (prfA, inlA, inlB, plcA). These strains thus form a tightly clustered, monophyletic group with limited diversity. In contrast, low-virulence strains of lineage I were more dispersed among the virulence strains and the origin of their loss of virulence has not been identified yet, even if some strains exhibited different mutations in prfA or inlA.

Invasion of the Placenta during Murine Listeriosis

ABSTRACT 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.

Listeria monocytogenes–Associated Joint and Bone Infections: A Study of 43 Consecutive Cases

BACKGROUND Little is known about Listeria monocytogenes-associated bone and joint infections. Only case reports of this infection have been published. METHODS Retrospective study of culture-proven bone and joint cases reported to the French National Reference Center for Listeria from 1992 to 2010. RESULTS Forty-three patients were studied: 61% were men, and the median age was 72 (range, 16-89); 24 patients exhibited comorbidities (56%). Thirty-six patients (84%) had orthopedic implant devices: prosthetic joints (n = 34) or internal fixation (n = 2); the median time after insertion was 9 years (0.1-22). Subacute infection was more frequent (median, 4 weeks [range, 2-100], 74%) than acute infection (<7 days, 23%), with nonspecific clinical features; 45% of patients had no fever. Blood cultures were positive in 3 of 19 cases. Isolate polymerase chain reaction genogrouping revealed 4 patterns: IVb (21 of 42, 50%), IIa (17 of 42, 40%), IIb (2 of 42, 5%), and IIc (2 of 42, 5%). Five groups of strains with similar pulsotype patterns were identified without an epidemiological link. Antibiotics, primarily amoxicillin (80%) with aminoglycosides (48%), were prescribed for a median duration of 15 weeks (range, 2-88). Eighteen patients (50%) underwent prosthesis replacement; all were successful after median follow-up of 10 months (range, 1-75). Five of 13 patients for whom material was not removed had protracted infection despite prolonged antibiotherapy; 3 of these patients later underwent prosthesis replacement with sustained recovery. CONCLUSIONS Osteoarticular listeriosis primarily involves prosthetic joints and occurs in immunocompromised patients. It requires intensive treatment with antibiotherapy and usually requires implant removal or replacement for cure.

Rapid eradication of Listeria monocytogenes by moxifloxacin in a murine model of central nervous system listeriosis.

ABSTRACT Listeriosis is a rare but life-threatening infection. A favorable outcome is greatly aided by early administration of antibiotics with rapid bactericidal activity against Listeria monocytogenes. Moxifloxacin, a new-generation fluoroquinolone with extended activity against gram-positive bacteria, has proved its effectiveness in vitro against intracellular reservoirs of bacteria. The efficacies of moxifloxacin and amoxicillin were compared in vivo by survival curve assays and by studying the kinetics of bacterial growth in blood and organs in a murine model of central nervous system (CNS) listeriosis. We combined pharmacokinetic and pharmacodynamic approaches to correlate the observed efficacy in vivo with plasma and tissue moxifloxacin concentrations. Death was significantly delayed for animals treated with a single dose of moxifloxacin compared to a single dose of amoxicillin. We observed rapid bacterial clearance from blood and organs of animals treated with moxifloxacin. The decrease in the bacterial counts in blood and brain correlated with plasma and cerebral concentrations of antibiotic. Moxifloxacin peaked in the brain at 1.92 ± 0.32 μg/g 1 hour after intraperitoneal injection. This suggests that moxifloxacin rapidly crosses the blood-brain barrier and diffuses into the cerebral parenchyma. Moreover, no resistant strains were selected during in vivo experiments. Our results indicate that moxifloxacin combines useful pharmacokinetic properties and rapid bactericidal activity and that it may be a valuable alternative for the treatment of CNS listeriosis.