Shinella fusca sp. nov., isolated from domestic waste compost.

A bacterium, designated strain DC-196(T), isolated from kitchen refuse compost was analysed by using a polyphasic approach. Strain DC-196(T) was characterized as a Gram-negative short rod that was catalase- and oxidase-positive, and able to grow at 10-40 degrees C, pH 6-9 and in NaCl concentrations as high as 3 %. Chemotaxonomically, C(18 : 1) was observed to be the predominant cellular fatty acid and ubiquinone 10 (Q10) was the predominant respiratory quinone. The G+C content of the genomic DNA was determined to be 66 mol%. On the basis of the genotypic, phenotypic and chemotaxonomic characteristics, strain DC-196(T) was assigned to the genus Shinella, although with distinctive features. At the time of writing, 16S rRNA gene sequence similarities of 97.6-96.8 % and the low DNA-DNA hybridization values of 38.2-32.2 % with the type strains of the three recognized Shinella species confirmed that strain DC-196(T) represents a novel species of the genus, for which the name Shinella fusca sp. nov. is proposed (type strain DC-196(T)=CCUG 55808(T)=LMG 24714(T)).

Induction of apoptosis/necrosis in various human cell lineages by Haemophilus ducreyi cytolethal distending toxin.

We investigated the impact of highly purified Haemophilus ducreyi cytolethal distending toxin (HdCDT) on the apoptosis and necrosis of various human cells; including myeloid cells, epithelial cells, keratinocytes, and primary fibroblasts. The levels of apoptosis and necrosis induced in these cells were compared to those induced by HdCDT in human T cells and in the Jurkat T cell line. Levels of caspase-3 activity were measured, and membrane changes like phosphatidylserine (PS) translocation was evaluated after double-staining with the fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide (PI) using flow cytometry. HdCDT induced various degrees of apoptosis and necrosis in dose- and time-dependent manners in cells of various lineages. Early and late apoptosis (annexin V-stained cells) were induced in more than 90% of T cells and monocytes after treatment with 100 ng/ml HdCDT for 24 and 48 h, respectively. The corresponding numbers for epithelial cells, keratinocytes, and fibroblasts were 26-32% after treatment with 100 ng/ml HdCDT for 48 h. HdCDT appears to eliminate effectively by inducing apoptosis those cells that are involved in immune responses. Epithelial cells, keratinocytes and fibroblasts, which are important for the healing of chancroid ulcers, are eliminated by apoptosis or necrosis after contact with HdCDT, albeit slower and to a lesser extent than T cells.

Etiology of genital ulcer disease and association with human immunodeficiency virus infection in two tanzanian cities.

BACKGROUND: The etiological agent is usually not established in cases of genital ulcer disease (GUD) in Tanzania, since diagnosis and treatment of this disease are based mainly on clinical rather than microbiologic parameters. GUD increases the risk of infection with HIV. However, the association between specific GUD infections and HIV infection has not been fully investigated. GOAL: The goal was to determine the etiology of GUD and the prevalence of HIV infection in patients with GUD in urban areas of Tanzania. STUDY DESIGN: A total of 102 clinical specimens were collected from 52 and 50 patients with GUD in Dar es Salaam and Mbeya, respectively, and from 93 patients with genital discharge in a cross-sectional study. Two polymerase chain reaction (PCR) assays were used to identify either a single target DNA or all three DNAs of the major causes of GUD: Haemophilus ducreyi, Treponema palladum and herpes simplex virus type 2 (HSV-2). The sera from all patients were tested for antibodies to HIV and T palladum. RESULTS: In Dar es Salaam, DNA from HSV-2, and was detected in 63%, 13%, and 2%, respectively, of the 52 genital ulcer specimens. The corresponding figures in Mbeya were 34%, 10%, and 0% of 50 specimens. Overall, 9% of the 102 patients with GUD were infected with both HSV-2 and, and 39/102 genital ulcer specimens (38%) were negative for the DNA of all three pathogens. The HIV infection rates among GUD patients were 46% and 52% in Dar es Salaam and Mbeya, respectively; among the non-GUD patients, the corresponding rates were 35% and 45%, respectively. The HIV infection rate in Dar es Salaam was significantly higher among women (11/14; 78%) than among men (13/38; 34%) (P = 0.004). Among the HIV-seropositive GUD patients, 71% and 46% (P < 0.003) were coinfected with HSV-2 in Dar es Salaam and Mbeya, respectively. Furthermore, women with HSV-2 in Dar es Salaam were significantly more likely to be HIV-infected than men (60% versus 39%; P

Toxicity and immunogenicity of purified Haemophilus ducreyi cytolethal distending toxin in a rabbit model.

The cytolethal distending toxin of Haemophilus ducreyi (HdCDT) is a three-component toxin that induces the arrest of the mammalian cell cycle in the G2 phase. All of the individual gene products, CdtA, CdtB and CdtC, are required for toxic activity on cultured mammalian cells. The CdtB component alone exerts nuclease activity. The individual HdCDT components were purified by affinity chromatography or ion-exchange chromatography followed by gel-filtration. HdCDT was reconstituted and purified by the immobilization of a GST-CdtB fusion on a GSTrap column and the subsequent addition of cell sonicates from Escherichia coli recombinants that produced CdtA and CdtC. The purified HdCDT preparation contained all three CDT proteins, as detected by immuno-blotting, and had high cytotoxic activity (10(6)CPU/ml). Immunization of rabbits with the HdCDT complex and with the individual CdtA, CdtB and CdtC proteins elicited high titres of antibodies, as detected by ELISA. All of the immune sera had toxin-neutralizing activities. The pathological effects of the HdCDT complex were investigated in rabbits, since the proliferation of two rabbit cell lines, SIRC and RK-13, was inhibited by HdCDT. Intradermal injection of HdCDT (1, 10, 50 and 100microg protein) into naive rabbits resulted in dose-dependent skin reactions (erythema) about 24h after injection. Similar effects were not observed when the individual HdCDT proteins were injected. HdCDT injection into immune rabbits resulted in dose-dependent skin responses that were characterized by both erythema and oedema. Histological evaluation of the 24-h lesions in naive rabbits that were injected with HdCDT, revealed moderate levels of inflammatory cells, which were mainly granulocytes and macrophages, and dilatation of blood vessels. The skin reactions in HdCDT-injected immunized rabbits showed pronounced vascular changes and extensive infiltration of inflammatory cells, including eosinophils. All of the pathological changes healed after 3 days. In conclusion, purified HdCDT holotoxin is a complex of all three CDT proteins and all three components induce neutralizing antibodies when injected in rabbits. HdCDT causes dose-dependent pathologic skin reactions in both naive and immune rabbits, which is characterized by increased inflammatory responsiveness after each immunization.

The cytolethal distending toxin of Haemophilus ducreyi inhibits endothelial cell proliferation.

Haemophilus ducreyi, the etiologic agent of the sexually transmitted disease chancroid, produces a cytolethal distending toxin (HdCDT) that inhibits mammalian cell proliferation. We investigated the effects of HdCDT on normal human endothelial cells and on tubule formation in an in vitro model of angiogenesis. Endothelial cells were arrested in the G2 phase of the cell cycle, and tubule formation was inhibited in a dose-dependent manner. The antiproliferative activities of HdCDT on endothelial cells might contribute to the characteristic slow healing and persistence of chancroid ulcers.

In vitro and in vivo interactions of Haemophilus ducreyi with host phagocytes.

We investigated the phagocytosis of Haemophilus ducreyi both in vitro and in vivo. Human granulocyte and monocyte phagocytosis of opsonized and nonopsonized, fluorescence-labeled H. ducreyi was assessed by flow cytometry. Both Escherichia coli and noncapsulated H. influenzae were included as controls. The maximal percentage of granulocytes taken up by H. ducreyi was 35% after 90 min. In contrast, 95% of H. influenzae bacteria were phagocytosed by granulocytes after 30 min. These results indicated that H. ducreyi phagocytosis was slow and inefficient. Bacterial opsonization by using specific antibodies increased the percentage of granulocytes phagocytosing H. ducreyi from 24 to 49%. The nonphagocytosed bacteria were completely resistant to phagocytosis even when reexposed to granulocytes, indicating that the H. ducreyi culture comprised a mixture of phenotypes. The intracellular survival of H. ducreyi in granulocytes, in monocytes/macrophages, and in a monocyte cell line (THP-1) was quantified after application of gentamicin treatment to kill extracellular bacteria. H. ducreyi survival within phagocytes was poor; approximately 11 and <0.1% of the added bacteria survived intracellularly after 2 and 20 h of incubation, respectively, while no intracellular H. influenzae bacteria were recovered after 2 h of incubation with phagocytes. The role of phagocytes in the development of skin lesions due to H. ducreyi was also studied in vivo. Mice that were depleted of granulocytes and/or monocytes and SCID mice, which lacked T and B cells, were injected intradermally with approximately 10(6) CFU of H. ducreyi. Within 4 days of inoculation, the granulocyte-depleted mice developed lesions that persisted throughout the experimental period. This result reinforces the importance of granulocytes in the early innate defense against H. ducreyi infection. In conclusion, H. ducreyi is insufficiently phagocytosed to achieve complete eradication of the bacteria. Indeed, H. ducreyi has the ability to survive intracellularly for short periods within phagocytic cells in vitro. Since granulocytes play a major role in the innate defense against H. ducreyi infection in vivo, bacterial resistance to phagocytosis probably plays a crucial role in the pathogenesis of chancroid.