The complete genome sequence of Corynebacterium pseudotuberculosis FRC41 isolated from a 12-year-old girl with necrotizing lymphadenitis reveals insights into gene-regulatory networks contributing to virulence.

BACKGROUND: Corynebacterium pseudotuberculosis is generally regarded as an important animal pathogen that rarely infects humans. Clinical strains are occasionally recovered from human cases of lymphadenitis, such as C. pseudotuberculosis FRC41 that was isolated from the inguinal lymph node of a 12-year-old girl with necrotizing lymphadenitis. To detect potential virulence factors and corresponding gene-regulatory networks in this human isolate, the genome sequence of C. pseudotuberculosis FCR41 was determined by pyrosequencing and functionally annotated. RESULTS: Sequencing and assembly of the C. pseudotuberculosis FRC41 genome yielded a circular chromosome with a size of 2,337,913 bp and a mean G+C content of 52.2%. Specific gene sets associated with iron and zinc homeostasis were detected among the 2,110 predicted protein-coding regions and integrated into a gene-regulatory network that is linked with both the central metabolism and the oxidative stress response of FRC41. Two gene clusters encode proteins involved in the sortase-mediated polymerization of adhesive pili that can probably mediate the adherence to host tissue to facilitate additional ligand-receptor interactions and the delivery of virulence factors. The prominent virulence factors phospholipase D (Pld) and corynebacterial protease CP40 are encoded in the genome of this human isolate. The genome annotation revealed additional serine proteases, neuraminidase H, nitric oxide reductase, an invasion-associated protein, and acyl-CoA carboxylase subunits involved in mycolic acid biosynthesis as potential virulence factors. The cAMP-sensing transcription regulator GlxR plays a key role in controlling the expression of several genes contributing to virulence. CONCLUSION: The functional data deduced from the genome sequencing and the extended knowledge of virulence factors indicate that the human isolate C. pseudotuberculosis FRC41 is equipped with a distinct gene set promoting its survival under unfavorable environmental conditions encountered in the mammalian host.

Comparison of cefoxitin and moxalactam 30 microg disc diffusion methods for detection of methicillin resistance in coagulase-negative staphylococci.

OBJECTIVES: To compare cefoxitin and/or moxalactam 30 microg disc diffusion (DD) methods to detect methicillin resistance in coagulase-negative staphylococci (CoNS) using both high- and low-density (HD/LD) inoculum techniques. METHODS: A challenge set of 192 CoNS was tested. DD test results were compared with PBP2a detection. RESULTS: With the LD inoculum, the sensitivity/specificity of cefoxitin and moxalactam were 94.4%/100% and 100%/92.4%, respectively, using the DD breakpoints of the Comité de l'Antibiogramme de la Société Française de Microbiologie. With the HD inoculum, the sensitivity/specificity of cefoxitin and moxalactam were 93.7%/100% and 100%/96.9%, using the cefoxitin DD breakpoints of the CLSI and a resistant/susceptible breakpoint of < 20 mm/>or=20 mm for moxalactam. Comparison of receiver operating characteristic AUCs did not show significant difference between studied assays, but the overlapping zone where both PBP2a-positive and PBP2a-negative isolates were observed concerned a lower number of strains with moxalactam than with cefoxitin (P < 0.001). Combination of cefoxitin and moxalactam DD methods demonstrated that all isolates with a concordant cefoxitin/moxalactam phenotype were correctly classified. Interestingly, all isolates misclassified by each DD method used alone were cefoxitin-susceptible and moxalactam-resistant. CONCLUSIONS: Although all DD methods studied here performed well for detecting methicillin resistance in CoNS, moxalactam had a higher accuracy than cefoxitin to differentiate heteroresistant isolates from PBP2a-negative strains. Identification of isolates that should be submitted to a confirmatory test to conclude on methicillin resistance can be easily obtained by combining cefoxitin and moxalactam DD methods.

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.

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.

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.

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.