Penicillin Binding Proteins as Danger Signals: Meningococcal Penicillin Binding Protein 2 Activates Dendritic Cells through Toll-Like Receptor 4

Neisseria meningitidis is a human pathogen responsible for life-threatening inflammatory diseases. Meningococcal penicillin-binding proteins (PBPs) and particularly PBP2 are involved in bacterial resistance to β-lactams. Here we describe a novel function for PBP2 that activates human and mouse dendritic cells (DC) in a time and dose-dependent manner. PBP2 induces MHC II (LOGEC50 = 4.7 µg/ml±0.1), CD80 (LOGEC50 = 4.88 µg/ml±0.15) and CD86 (LOGEC50 = 5.36 µg/ml±0.1). This effect was abolished when DCs were co-treated with anti-PBP2 antibodies. PBP2-treated DCs displayed enhanced immunogenic properties in vitro and in vivo. Furthermore, proteins co-purified with PBP2 showed no effect on DC maturation. We show through different in vivo and in vitro approaches that this effect is not due to endotoxin contamination. At the mechanistic level, PBP2 induces nuclear localization of p65 NF-kB of 70.7±5.1% cells versus 12±2.6% in untreated DCs and needs TLR4 expression to mature DCs. Immunoprecipitation and blocking experiments showed tha

Experimental Meningococcal Sepsis in Congenic Transgenic Mice Expressing Human Transferrin

Severe meningococcal sepsis is still of high morbidity and mortality. Its management may be improved by an experimental model allowing better understanding of its pathophysiology. We developed an animal model of meningococcal sepsis in transgenic BALB/c mice expressing human transferrin. We studied experimental meningococcal sepsis in congenic transgenic BALB/c mice expressing human transferrin by transcriptional profiling using microarray analysis of blood and brain samples. Genes encoding acute phase proteins, chemokines and cytokines constituted the largest strongly regulated groups. Dynamic bioluminescence imaging further showed high blood bacterial loads that were further enhanced after a primary viral infection by influenza A virus. Moreover, IL-1 receptor–associated kinase–3 (IRAK-3) was induced in infected mice. IRAK-3 is a negative regulator of Toll-dependant signaling and its induction may impair innate immunity and hence result in an immunocompromised state allowing bacterial survival and systemic spread during sepsis. This new approach should enable detailed analysis of the pathophysiology of meningococcal sepsis and its relationships with flu infection

Evaluation of CHROMagar STEC and STEC O104 chromogenic agar media for detection of Shiga toxin-producing Escherichia coli in stool

The performance of CHROMagar STEC and CHROMagar STEC O104 (CHROMagar Microbiology, Paris, France) media for the detection of Shiga toxin-producing Escherichia coli (STEC) was assessed with 329 stool specimens collected over 14 months from patients with suspected STEC infections (June 2011 to August 2012). The CHROMagar STEC medium, after an enrichment broth step, allowed the recovery of the STEC strain from 32 of the 39 (82.1%) Shiga toxin-positive stool specimens, whereas the standard procedure involving Drigalski agar allowed the recovery of only three additional STEC strains. The isolates that grew on CHROMagar STEC medium belonged to 15 serotypes, including the prevalent non-sorbitol-fermenting (NSF) O157:H7, O26: H11, and O104:H4 serotypes. The sensitivity, specificity, and positive and negative predictive values for the CHROMagar STEC medium were between 89.1% and 91.4%, 83.7% and 86.7%, 40% and 51.3%, and 98% and 98.8%, respectively, depending on whether or not stx-negative eae-positive E. coli was considered atypical enteropathogenic E. coli (EPEC) or STEC that had lost Shiga toxin genes during infection. In conclusion, the good performance of CHROMagar STEC agar medium, in particular, the high negative predictive value, and its capacity to identify NSF O157:H7 as well as common non-O157 STEC may be useful for clinical bacteriology, public health, and reference laboratories; it could be used in addition to a method targeting Shiga toxins (detection of stx genes by PCR, immunodetection of Shiga toxins in stool specimens, or Vero cell cytotoxicity assay) as an alternative to O157 culture medium. This combined approach should allow rapid visualization of both putative O157 and non-O157 STEC colonies for subsequent characterization, essential for real-time surveillance of STEC infections and investigations of outbreaks. C ertain strains of Shiga toxin-producing Escherichia coli (STEC) are important causes of food-borne disease in industrialized countries. The clinical manifestations of STEC infections range from mild diarrhea to severe and specific complications, such as hemolytic-uremic syndrome (HUS), which occurs primarily in young children (1, 2). These STEC strains associated with human infections are also called enterohemorrhagic E. coli (EHEC). Animals, and especially cattle, serve as reservoirs for STEC. Transmission occurs via ingestion of contaminated food or water, person-to-person contact, direct animal contact, and exposure to the environment. STEC strains are characterized by their ability to produce toxins related to those of Shigella dysenteriae type 1 (3): two types have been described among STEC isolates, Shiga toxin 1 and Shiga toxin 2, respectively, encoded by the stx 1 and stx 2 genes carried on temperate bacteriophages (4, 5). Most STEC isolates also carry the chromosomally located locus of enterocyte effacement (LEE), a pathogenicity island, first described in enteropathogenic E. coli (EPEC). LEE promotes the development of attaching-and-effacing lesions in the host intestinal mucosa cells (6). One of the LEE genes, eae (for EPEC attaching and effacing), encodes intimin, an outer membrane adhesin essential for the intimate attachment of the bacteria to enterocytes. Other adherence and colonization factors, such as adhesins and pili, are present in LEE-negative STEC strains. The STEC O104:H4 strain responsible for a large outbreak of HUS in Germany and other European countries in 2011 displays a characteristic aggregative adhesion (AA) pattern caused by an enteroaggregative E. coli (EAEC) genetic background (7-11). The laboratory identification of STEC requires screening for Shiga toxin genes or proteins in stool specimens, followed by culture, serotyping, and confirmation of the presence of the virulence genes (at least stx 1 and stx 2 and then eae and aggR) in isolated colonies. Since the first reported STEC outbreak in 1982 (12), various methods for the detection of STEC, especially E. coli O157: H7, which is the most prevalent group of STEC, have been developed (13). STEC O157:H7 had been found to be non-sorbitol fermenting (NSF), and consequently, culture media containing sorbitol have been marketed and widely used. However, the identification of sorbitol-fermenting (SF) STEC O157:H7 (SF O157) strains, mainly in Germany (14-16), and the general increase of non-O157 STEC (generally SF) strains in clinical practic

Emergence of New Virulent Neisseria meningitidis Serogroup C Sequence Type 11 Isolates in France.

International audienceIn France, there have been variations in the incidence of invasive meningococcal infection due to serogroup C isolates. Infection peaks were observed in 1992 and 2003 that involved isolates of phenotypes C:2a:P1.5,2 and/or C:2a:P1.5, which belong to the sequence type 11 (ST-11) clonal complex. We report an emergence of isolates belonging to the ST-11 clonal complex since 2003. These isolates displayed a new phenotype, C:2a:P1.7,1, caused infections that occurred as clusters, and were associated with increased infection severity and high virulence in mice. These isolates may be responsible for a peak in the incidence of serogroup C meningococcal infection in France, for which there is no routine vaccination to date

Rapid emergence of extensively drug-resistant Shigella sonnei in France

International audienceShigella sonnei, the main cause of bacillary dysentery in high-income countries,has become increasingly resistant to antibiotics. We monitored the anti-microbial susceptibility of 7121 S. sonnei isolates collected in France between2005 and 2021. We detected a dramatic increase in the proportion of isolatessimultaneously resistant to ciprofloxacin (CIP), third-generation cephalos-porins (3GCs) and azithromycin (AZM) from 2015. Our genomic analysis of164 such extensively drug-resistant (XDR) isolates identified 13 differentclusters within CIP-resistant sublineage 3.6.1, which was selected in South Asia∼15 years ago. AZM resistance was subsequently acquired, principally throughIncFII (pKSR100-like) plasmids. The last step in the development of theXDR phenotype involved various extended-spectrum beta-lactamase genes(blaCTX-M-3, blaCTX-M-15, blaCTX-M-27, blaCTX-M-55, and blaCTX-M-134) carried bydifferent plasmids (IncFII, IncI1, IncB/O/K/Z) or even integrated into thechromosome, and encoding resistance to 3GCs. This rapid emergence of XDRS. sonnei, including an international epidemic strain, is alarming, and goodlaboratory-based surveillance of shigellosis will be crucial for informeddecision-making and appropriate public health action

Population structure analysis and laboratory monitoring of Shigella by core-genome multilocus sequence typing

International audienceThe laboratory surveillance of bacillary dysentery is based on a standardised Shigella typing scheme that classifies Shigella strains into four serogroups and more than 50 serotypes on the basis of biochemical tests and lipopolysaccharide O-antigen serotyping. Real-time genomic surveillance of Shigella infections has been implemented in several countries, but without the use of a standardised typing scheme. Here, we study over 4000 reference strains and clinical isolates of Shigella, covering all serotypes, with both the current serotyping scheme and the standardised EnteroBase core-genome multilocus sequence typing scheme (cgMLST). The Shigella genomes are grouped into eight phylogenetically distinct clusters, within the E. coli species. The cgMLST hierarchical clustering (HC) analysis at different levels of resolution (HC2000 to HC400) recognises the natural population structure of Shigella. By contrast, the serotyping scheme is affected by horizontal gene transfer, leading to a conflation of genetically unrelated Shigella strains and a separation of genetically related strains. The use of this cgMLST scheme will facilitate the transition from traditional phenotypic typing to routine whole-genome sequencing for the laboratory surveillance of Shigella infections

Dissemination of <i>N. meningitidis</i> in BALB/c congenic mice that were infected by intraperitoneal injection of 5*10⁶ CFU of <i>N. meningitidis</i> (Nm) strain NM0804 expressing the luciferase.

<p>Mice were then analyzed for bioluminescence at the indicated times. Images depict photographs overlaid with colour representations of luminescence intensity, measured in photons/second and indicated on the scales, where red is most intense and blue is least intense. (Top row) (A) Ventral views of a three transgenic mice expressing human transferrin (hTf/+) and two wild type mice (+/+) at the indicated times. The framed region of the first transgenic mice is shown below for more details of the bioluminescence imaging in the skull. (B) Dorsal view of the skull of one transgenic mouse expressing human transferrin and one wild type mouse. The substrate of firefly luciferase was injected directly into the lateral ventricle after 6 h of bacterial intraperitoneal challenge. (C) The luminescence of the hTf/+ and (+/+) mice from A and D was quantified and expressed as means ± SD from each category at the indicated times by defining specific representative region of interest encompassing the entire animal.(D) Ventral views of a three transgenic mice expressing human transferrin (hTf/+) and two wild type mice (+/+) at the indicated times. Mice were first infected by influenza A virus (IAV) by intranasal inhalation. Mice were then infected after 7 days by <i>N. meningitidis</i> (Nm) strain NM0804 by intraperitoneal injection. The framed region of the first transgenic mice is shown below for more details of the bioluminescence imaging in the skull.</p

Primers used for RT-PCR analysis.

<p>Primers used for RT-PCR analysis.</p