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Volume 52,
Issue 10,
2003
Volume 52, Issue 10, 2003
- Pathogenicity And Virulence
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Characterization of the O antigen gene cluster and structural analysis of the O antigen of Francisella tularensis subsp. tularensis
A gene cluster encoding enzymes involved in LPS O antigen biosynthesis was identified from the partial genome sequence of Francisella tularensis subsp. tularensis Schu S4. All of the genes within the cluster were assigned putative functions based on sequence similarity with genes from O antigen biosynthetic clusters from other bacteria. Ten pairs of overlapping primers were designed to amplify the O antigen biosynthetic cluster by PCR from nine strains of F. tularensis. Although the gene cluster was present in all strains, there was a size difference in one of the PCR products between subsp. tularensis strains and subsp. holarctica strains. LPS was purified from F. tularensis subsp. tularensis Schu S4 and the O antigen was shown by mass spectrometry to have a structure similar to that of F. tularensis subsp. holarctica strain 15. When LPS from F. tularensis subsp. tularensis Schu S4 was used to immunize mice that were then challenged with F. tularensis subsp. tularensis Schu S4, an extended time to death was observed.
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Comparison of Moraxella catarrhalis isolates from children and adults for growth on modified New York City medium and potential virulence factors
Initial studies found that Moraxella catarrhalis isolates from adults that grew on modified New York City medium (MNYC+) that contained antibiotics selective for pathogenic neisseriae differed from strains that did not grow on this medium (MNYC−) in their potential virulence properties. It was predicted that higher usage of antibiotics to treat respiratory illness in children might result in higher proportions of MNYC+ isolates if antibiotics were an important selective pressure for this phenotype. Two of 100 adult isolates (2 %) were MNYC+, compared to 88 of 88 isolates (100 %) from children (P = 0.000). MNYC+ strains were serum-resistant and bound in higher numbers to HEp-2 cells that were infected with respiratory syncytial virus (RSV). Endotoxin from an MNYC+ isolate induced significantly higher pro-inflammatory response levels than endotoxin from an MNYC− strain. MNYC− adult isolates expressed haemagglutinins and bound in lower numbers to RSV-infected cells, but serum resistance was variable. All isolates from children were MNYC+, serum-resistant and bound in greater numbers to RSV-infected cells. These results indicate that both RSV infection and antibiotic usage select for the MNYC+ phenotype.
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- Host Response
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Cytoskeletal rearrangements in gastric epithelial cells in response to Helicobacter pylori infection
More LessHelicobacter pylori causes host epithelial cell cytoskeletal rearrangements mediated by the translocation and tyrosine phosphorylation of an outer-membrane protein, CagA, and by the vacuolating cytotoxin, VacA. However, the mechanisms by which H. pylori mediates cytoskeletal rearrangements in infected host cells need to be more clearly defined. The aim of this study was to determine the effects of H. pylori isolates from children on the architecture of host gastric epithelial cells. Gastric epithelial (AGS) cells were infected with type I (cagE +, cagA +, VacA+) H. pylori, a type II H. pylori strain (cagE −, cagA −, VacA−) or a cagE isogenic mutant. Double-labelled immune fluorescence was used to detect adherent H. pylori and the distribution of F-actin, α-actinin and Arp3. Both type I and type II H. pylori strains induced stress fibres in gastric epithelial cells that were not observed in uninfected cells. Type I H. pylori also induced cell elongation (hummingbird phenotype) after 4 h of infection, whereas the type II H. pylori strain did not. Less elongation occurred when AGS cells were exposed to a cagE isogenic mutant, compared with the parental strain. Confocal microscopy showed Arp3 accumulation in AGS cells infected with wild-type H. pylori, but not in response to infection with the cagE mutant. These findings indicate that type I H. pylori induce a stress fibre-like phenotype in infected gastric epithelia by a mechanism that is different from the induction of host-cell elongation. In addition to CagA and VacA, cagE also impacts on the morphology of infected gastric epithelial cells.
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Virulent Toxoplasma gondii strain RH promotes T-cell-independent overproduction of proinflammatory cytokines IL12 and γ-interferon
The aim of this study was the analysis of the cytokine response in BALB/c mice infected with the highly virulent RH or the weakly virulent Beverley strains of Toxoplasma gondii. Analysis of cytokine messages showed increased expression of IL12, IFN-γ and TNF-α, but not IL4 mRNAs in spleen cells after infection with the T. gondii strains RH and Beverley. High levels of circulating IL12 and IFN-γ were detected in the serum of mice infected with strain RH, although TNF-α levels remained low. In contrast, the same cytokines were detected at only low levels in the serum of mice infected with the Beverley strain. Administration of antibody against IL12 or IFN-γ significantly delayed time to death of mice infected with strain RH compared to controls. T-Cell-deficient as well as normal mice were equally infected by strain RH, suggesting that T lymphocytes do not contribute to the response. Depletion of natural killer cells from the splenocyte population abolished the in vitro production of IFN-γ. Together, our data suggest that the virulent strain RH induces in BALB/c mice a type 1 cytokine pattern with T-cell-independent overproduction of IL12 and IFN-γ that may be involved in the pathogenesis of this micro-organism.
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Viable Mycobacterium avium is required for the majority of human immunodeficiency virus-induced upregulation in monocytoid cells
More LessThe Mycobacterium avium complex (MAC), an intracellular pathogen of cells of the macrophage lineage, often clinically coexists with human immunodeficiency virus type 1 (HIV). It was shown previously that coinfection of the monocytoid cell line U937 with HIV and MAC results in the enhancement of HIV replication. To determine whether MAC-mediated HIV upregulation is due to the exposure of intact organisms to HIV-infected cells or if actual infection with viable organisms is required for the effect, U937 cells were coinfected simultaneously with HIV and live or heat-killed MAC. Live MAC (infection) consistently increased HIV reverse transcriptase (RT) activity by more than 3-fold. Heat-killed MAC, however, failed to enhance RT activity significantly. Further investigation showed that infection of U38 cells [a U937-derived cell line containing regions of the HIV-1 long terminal repeat (LTR) linked to chloramphenicol acetyl transferase (CAT)] with live or heat-killed MAC resulted in a similar enhancement of HIV LTR-CAT transcription. In addition, transient transfection of U937 cells with a full-length wild-type HIV LTR-CAT construct revealed that heat-killed MAC stimulated LTR-mediated CAT activity to levels comparable to those of viable MAC. Finally, both live and heat-killed MAC mediated similar enhancement of NF-κB DNA-binding activity. Taken together, these observations confirm previous findings that MAC-induced NF-κB-dependent LTR-CAT activity is not a major factor in upregulating HIV expression in a coinfection model. It also indicates that MAC infection plays a significant role in the enhancement of HIV replication and suggests that viable MAC either contains or induces the production of an as-yet-unidentified factor(s) that mediates the enhancement of HIV replication.
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- Diagnostics, Typing And Identification
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Fluorescence polarization assay for diagnosis of human brucellosis
More LessFluorescence polarization immunoassay (FPA) uses molecular rotational properties to measure antibody binding to antigen directly. The potential use of this method was assessed in comparison to a competitive enzyme immunoassay (CELISA) and conventional serological tests for the diagnosis of brucellosis on a total of 587 human sera. Based on 340 sera from asymptomatic blood donors with no evidence of brucellosis, the specificity of the FPA was 97.9 % using a cut-off value of 72 mP. Sera from Brucella-infected patients (11 Brucella melitensis, 32 Brucella abortus, 32 Brucella suis and one Brucella sp.) yielded a sensitivity estimate of 96.1 %. In tests on 84 sera from suspected brucellosis patients, the FPA detected 80 cases. Of 87 sera from patients with probable infection, 15 were detected by both CELISA and FPA, three by CELISA only and four by FPA only. The discrepancies in both groups involved sera with low, declining titres. The FPA uses a sample of 40 μl serum, takes about 5 min to complete and has been demonstrated to be accurate for the detection of antibodies to B. abortus, B. melitensis and B. suis and for identifying patients suffering relapses. Because of the ease of the procedure, it could be readily adopted for use in clinical laboratories and blood banks.
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Diagnostic accuracy of serological kits for Helicobacter pylori infection with the same assay system but different antigens in a Japanese patient population
More LessHelicobacter pylori infection is thought to be a causal risk factor for gastric carcinoma. Recently, diagnostic accuracy of serological kits for H. pylori infection that were made in Western countries has been reported to be lower when used among Oriental populations. Diagnostic accuracy of two serological kits [HM-CAP and HM-CAP with antigens extracted from clinically isolated Japanese H. pylori strains (J-HM-CAP)] was investigated in 440 samples from a Japanese patient population by using the 13C-urea breath test as gold standard. According to the original optimal cut-off value, HM-CAP provided 87.5 % sensitivity and 84.8 % specificity with 86.8 % accuracy and J-HM-CAP provided 95.5 % sensitivity and 81.9 % specificity with 92.3 % accuracy. This study suggests that antigens from HM-CAP are satisfactory for examining a Japanese patient population, but that using local antigens improves accuracy.
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Real-time PCR to improve the diagnosis of respiratory syncytial virus infection
R. Mentel, U. Wegner, R. Bruns and L. GürtlerRespiratory syncytial virus (RSV) is one of the most important virus respiratory pathogens in infants and young children. A rapid and sensitive diagnosis is essential to focus any outbreak due to this virus. A real-time RT-PCR method was designed using a primer/probe pair from the F gene. Simultaneously with nested RT-PCR and antigen ELISA, 71 consecutive specimens from hospitalized children with clinical symptoms of acute respiratory distress were evaluated to confirm the incidence of RSV infection. RSV was detected in 25 (35.2 %) specimens by real-time RT-PCR and in 19 (26.7 %) by nested RT-PCR. The assay was specific for RSV. The procedure offers a rapid and sensitive alternative to conventional RT-PCR. Closed-tube detection eliminates the risk of contamination.
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Field application of Lepto lateral flow for rapid diagnosis of leptospirosis
More LessThe Lepto lateral flow assay for leptospirosis was evaluated at a primary health centre in the Andaman Islands, where leptospirosis is endemic. One hundred and seventeen suspected patients were included in the study; acute serum samples were collected from all of them and convalescent samples from 104. The standard criteria for diagnosis of leptospirosis were: (i) isolation of leptospires from blood, (ii) seroconversion in microscopic agglutination test (MAT) with a minimum titre of 100, (iii) a fourfold rise in titre in MAT or (iv) a MAT titre of 400 or more if only a single sample was available. The results of the lateral flow test were compared with these criteria. Lepto lateral flow had sensitivity of 52.9 % (37/70) in the first week of illness and 86 % (49/57) during weeks 2–4. The corresponding specificities were respectively 93.6 % (44/47) and 89.4 % (42/47). The sensitivity was 34.3 % (12/35) on days 2–3 of the illness, 63.3 % (14/22) on days 4–5 and 84.6 % (11/13) at the end of the first week. The test had a positive predictive value of 92.5 % (37/40) during the first week and 90.7 % (49/54) subsequently. Corresponding negative predictive values were respectively 57.1 % (44/77) and 84 % (42/50). Agreement of the results with the standard criteria was low during the first week, but high during weeks 2–4, with a κ value of 0.7491. The positivity rates of the tests showed a logarithmic relationship with the MAT titres of samples (r 2 = 0.9271). All indices of validity and utility of lateral flow were similar to those of IgM ELISA and Lepto dipstick. The test can be performed at the bedside of the patient, as whole blood can also be used for testing.
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- Epidemiology
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Geographical difference of disease association in Streptococcus bovis bacteraemia
From 1996 to 2001, 48 Streptococcus bovis strains were isolated from blood cultures of 37 patients in one hospital. Median patient age was 68 years (range: 1 day–88 years). The male : female ratio was 23 : 14. Most patients (97 %) had underlying diseases, including biliary tract disease in 14 (38 %), diabetes mellitus in 12 (32 %), liver parenchymal disease in seven (19 %), carcinoma of the colon in four (11 %) and other malignancies in four (11 %). No infective foci (indicative of primary bacteraemia) were identified in 15 patients (40 %) and 14 (38 %) had acute cholangitis/cholecystitis, but only four (11 %) had infective endocarditis. Two (5 %), three (8 %) and 32 (87 %) patients had S. bovis of biotypes I, II/1 and II/2, respectively, and three (8 %), two (5 %) and 32 (87 %) patients had S. bovis of genotypes 1, 2a and 2b, respectively. All isolates were sensitive to penicillin, cephalothin and vancomycin, 24 (65 %) were resistant to erythromycin and 15 (41 %) were resistant to clindamycin (these strains were also resistant to erythromycin). Thirteen isolates that were erythromycin- and clindamycin-resistant possessed the ermB gene, 10 possessed the ermT gene and one possessed both the ermB and ermT genes. Overall, seven patients (19 %) died. In contrast to most other reports from western countries, where carcinoma of the colon and infective endocarditis were the major underlying disease and infective focus associated with S. bovis bacteraemia, biliary tract disease and acute cholangitis and/or cholecystitis were the major underlying diseases associated with S. bovis bacteraemia in our locality.
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Antigenic and genetic relatedness of Leptospira strains isolated from the Andaman Islands in 1929 and 2001
More LessLeptospirosis is a major public health problem in Andaman Islands. Several strains of Leptospira have been isolated from the Andamans over the years. Leptospires isolated recently from human cases were compared with one of the earliest available isolates from these islands, dating back to 1929, to study their serological and genetic relatedness. Randomly amplified polymorphic DNA (RAPD) fingerprints of the isolates, generated with a primer used previously to differentiate between Leptospira species and serovars, revealed that some of the recent isolates were genetically identical to the 1929 isolate. The antigenic properties of these strains, as revealed by microscopic agglutination tests with group-specific rabbit antisera and mAbs, were also similar. These findings suggest that a Leptospira strain originally isolated in 1929 has possibly persisted in these islands for over 70 years and continues to cause acute leptospirosis in humans.
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- Clinical Microbiology And Virology
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Leptospira interrogans serovar Valbuzzi: a cause of severe pulmonary haemorrhages in the Andaman Islands
More LessOutbreaks of leptospirosis that present with predominant pulmonary signs and symptoms have been occurring in the Andaman Islands since the late 1980s. Before this, pulmonary haemorrhage had not been observed as a common complication of leptospirosis in India. During an outbreak on North Andaman in 1997, four leptospire isolates were obtained from blood of a fatal case and three other patients who recovered. These isolates were characterized using serological and molecular techniques. Cross-agglutination absorption tests and microscopic agglutination tests using mAbs were used for serological characterization. Genetic typing was done using DNA sequencing of PCR products. Serologically, the isolates were closely related to strain Valbuzzi serovar Valbuzzi of serogroup Grippotyphosa. The sequences of PCR products from these isolates were compared with those of 45 strains belonging to seven species. The isolates showed 97.5–100 % sequence similarity to reference strains belonging to Leptospira interrogans, indicating that the isolates belong to L. interrogans. Serogroups Icterohaemorrhagiae and Australis have been incriminated as the cause of pulmonary haemorrhage in China, Korea and Australia. The four isolates characterized in the present study were obtained from patients with similar symptoms. However, they belonged to serovar Valbuzzi of serogroup Grippotyphosa, indicating that serogroups other than Icterohaemorrhagiae and Australis can also cause pulmonary haemorrhage.
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Fatal outcome of bacteraemic patients caused by infection with staphylokinase-deficient Staphylococcus aureus strains
Staphylokinase (SAK) is a plasminogen-activator protein produced by Staphylococcus aureus. SAK production was evaluated in vitro in S. aureus isolates from the bloodstream of patients with lethal (n = 56) and non-lethal (n = 57) bacteraemia and from anterior nares of healthy subjects (n = 48). Most isolates (93/161) produced SAK, and 68 % of SAK-producing isolates expressed both surface-bound and secreted types of SAK. SAK production was significantly less common among isolates from patients with lethal bacteraemia (39 %) than isolates from patients with non-lethal bacteraemia (68 %) or nasal carriage isolates (67 %) (P < 0.01). After adjusting for infection with methicillin-resistant S. aureus and APACHE II score, patients infected with SAK-deficient isolates were 4.3 times more likely to have lethal bacteraemia than patients whose infecting isolate produced high levels of SAK (⩾5 μg ml−1), suggesting that in vitro SAK production was inversely associated with clinical outcome among patients with S. aureus bacteraemia. The high frequency of SAK production in nasal isolates and in cases with uncomplicated bacteraemia suggests that SAK may be one of the adaptive mechanisms of S. aureus symbiosis with the host.
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- Human And Animal Microbial Ecology
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Displacement of bacterial pathogens from mucus and Caco-2 cell surface by lactobacilli
More LessCompetition, competitive exclusion and displacement of eight strains of Escherichia coli and Salmonella spp. by Lactobacillus rhamnosus GG and Lactobacillus casei Shirota from adhesion on human intestinal mucus glycoproteins and Caco-2 cell surfaces were studied. Lactobacilli were able to compete with, exclude and displace pathogenic gastrointestinal (GI) bacteria when they were incubated together, but the degree of inhibition of adhesion was bacterial strain-dependent. Competition and exclusion profiles of GI bacteria by lactobacilli were similar. Displacement profiles of GI bacteria were different from those of competition and exclusion and the process was relatively slow: displacement equilibrium took more than 2 h. These findings are important for development, selection and in vitro assessment of target- and function-specific probiotics.
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- Correspondence
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Volumes and issues
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Volume 74 (2025)
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Volume 73 (2024)
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Volume 72 (2023 - 2024)
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Volume 71 (2022)
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Volume 70 (2021)
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Volume 69 (2020)
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Volume 68 (2019)
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Volume 67 (2018)
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Volume 7 (1974)
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Volume 6 (1973)
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Volume 5 (1972)
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Volume 4 (1971)
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Volume 2 (1969)
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Volume 1 (1968)
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