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Volume 54,
Issue 5,
2005
Volume 54, Issue 5, 2005
- Obituary
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- Pathogenicity And Virulence
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Identification of two new Helicobacter pylori surface proteins involved in attachment to epithelial cell lines
More LessHelicobacter pylori causes the development of gastritis, gastric ulcers and adenocarcinomas in humans. The establishment of infection is influenced by adherence to the gastric epithelium, and several bacterial adhesins and host cell receptors have been identified. H. pylori recognize the Lewisb receptor through the BabA adhesin but also readily adhere to epithelia in the absence of the Lewisb epitope, demonstrating the relevance of additional adhesive interactions. This study presents a novel method of identifying bacterial adhesins. Nickel beads were coated with H. pylori-derived, recombinantly expressed ORF proteins, and epithelial cells from the human stomach, intestine or urinary tract were allowed to adhere to those beads. The binding of epithelial cells to the protein-coated nickel beads was confirmed by electron microscopy or flow cytometry using antibodies directed towards the His-tags. Among the five ORFs tested, two new adhesive proteins (HP1188 and HP1430) were identified. Both were expressed on the surface of virulent H. pylori, with the HP1188 protein being most abundant. The purified HP1188 and HP1430 proteins bound more strongly to gastric than to other epithelial cell lines, suggesting that they may be involved in the colonization of the human gastric mucosa. In conclusion, this method facilitates the identification of ORFs of microbial origin involved in cellular interactions such as adherence.
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Antigenic and phenotypic modifications of Yersinia pestis under calcium and glucose concentrations simulating the mammalian bloodstream environment
More LessTo study the possible mechanism of extracellular resistance to phagocytes developed by Yersinia pestis in the early stage of plague infection, the behaviour of two Y. pestis strains, the vaccine EV-76 and fully virulent 231 (LD50, 10 c.f.u.), was studied in-depth after cultivation in vitro at the host temperature in conditions simulating the bloodstream environment of mammals. For this, two standard basal media supplemented with calcium and glucose in appropriate concentrations were employed: Hottinger broth, routinely used for growth of Y. pestis in vitro, and RPMI 1640, simulating human extracellular fluid. Although both media permitted Y. pestis to achieve the resistant state, RPMI enabled significantly higher bacterial proliferation and increased modifications in the production of the principal surface antigens that affect the relevant phenotype characteristics. In general, our results indicate that the Y. pestis bacteria in the resistant state do not produce species-specific antigens, i.e. fraction 1 or F1, ‘murine’ toxin or Ymt, plasminogen activator (Pla) and any surface-specific polysaccharides, resulting in unmasking of the cross-reactive epitopes of lipid A in reduced Y. pestis lipopolysaccharide. This may produce mimicry by Y. pestis of some human tissue and blood cell components, with no immune response and inflammation at the site of infection at the early stage, which enables Y. pestis to survive, extensively multiply and spread into the circulation.
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- Host Response
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Immunogenicity of mycobacterial PPE44 (Rv2770c) in Mycobacterium bovis BCG-infected mice
More LessThe Pro-Pro-Glu (PPE) protein family of Mycobacterium tuberculosis includes 69 glycine-rich proteins with a conserved N-terminal domain. Their role in tuberculosis is unknown, but it has been speculated that they may have an important immunological significance. In this investigation, the immunogenicity of the ppe44 (Rv2770c) gene product in BALB/c mice infected subcutaneously or intravenously with Mycobacterium bovis bacille Calmette–Guérin (BCG) was evaluated. Mice infected subcutaneously developed high titres of anti-PPE44 IgG1 antibodies, while PPE44-specific IgG2a antibodies were absent at all times tested. PPE44-primed cells from draining lymph nodes and spleen produced low levels of IFN-γ, and a moderate degree of delayed-type hypersensitivity was observed following PPE44 intracutaneous challenge. In mice infected intravenously, the anti-PPE44 IgG1 antibody response was markedly higher compared with the subcutaneous infection; anti-PPE44 IgG2a antibodies at titres approximately 0.5–2.0 log10 lower than IgG1 were detected. Interferon (IFN)-γ production in PPE44-stimulated spleen-cell cultures was transient. These results indicate that PPE44 represents a novel mycobacterial antigen expressed during subcutaneous and intravenous infection by M. bovis BCG in BALB/c mice. Both infection models seem to polarize the immune response to PPE44 towards a Th2 phenotype, as testified by the IgG1 isotype being predominant over IgG2a and by the low IFN-γ and delayed-type hypersensitivity responses.
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- Diagnostics, Typing And Identification
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Sensitivity of laser light depolarization analysis for detection of malaria in blood samples
Automated light depolarization analysis could be a useful tool for diagnosing malarial infections. This work discusses the results of a diagnostic efficacy study on 411 samples from patients with suspected malaria infection performed with a Cell-Dyn 4000 analyser. Light dispersed at 90° and depolarized can be used for identifying and counting eosinophils. However, other cell populations with depolarizing capacity occur in malarial samples; these result from leukocytes ingesting haemozoin that is derived from the degradation of the haem group of haemoglobin performed by the parasite. A sensitivity of 72 % and specificity of 98 % were recorded, with positive and negative predictive values of 78 % and 97 %, respectively. Although the sensitivity level of the automated light depolarization analysis is not adequate to replace the existing methods for the diagnosis of parasitic diseases, it could alert clinicians to unsuspected infections by parasites, particularly those from the genus Plasmodium.
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Failure to detect capsule gene bexA in Haemophilus influenzae types e and f by real-time PCR due to sequence variation within probe binding sites
More LessDetection of the conserved capsule gene bexA is used to distinguish capsulate from non-capsulate Haemophilus influenzae. While developing a real-time PCR assay to detect bexA, it was found that bexA probes produced a detectable signal for H. influenzae types a to d, but failed to do so for H. influenzae types e and f. Sequencing revealed differences compared with H. influenzae types a to d within probe binding sites. To prevent misclassification of strains as non-capsulate, assays must detect all capsular types.
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Diagnosis of human brucellosis caused by Brucella canis
More LessThe transmission of Brucella canis to man commonly occurs through contact with infected dogs or their secretions, or through direct laboratory exposure. The disease is underdiagnosed due to a general lack of serological testing facilities and misconceptions concerning its prevalence. This report shows the potential use of an indirect ELISA (IELISA) for the diagnosis of human brucellosis caused by B. canis in a population of patients negative by smooth-Brucella antigen tests but positive by rapid slide agglutination test (RSAT). One hundred and ten sera from asymptomatic people found negative by tests using smooth Brucella abortus antigen and by RSAT showed an IELISA specificity of 100 % when a cut-off value of 27 % positivity (%P) was selected. For 17 sera from patients with positive B. canis culture or in close contact with culture-positive dogs, the IELISA sensitivity was 100 % with the same cut-off value. The positive patients presented clinical symptoms similar to brucellosis caused by other species of Brucella and some of them received antibiotic treatment and made good progress. Using this cut-off value, we studied 35 patients with negative blood cultures but positive RSATs, and IELISA detected 18 as positive; of the 17 IELISA-negative, two were RSAT-positive at dilution 1 : 2 and 15 were weakly positive with pure serum. These samples were probably from patients at an early stage of infection or indicate false-positive results. No cross-reaction was observed among the sera from nine cases with a diagnosis other than brucellosis, but cross-reactivity was evident in sera from patients infected with smooth-Brucella species. Since routine brucellosis diagnosis does not include B. canis investigation, infection with this species may be more widespread than is currently suspected. The RSAT could be a suitable screening test for the diagnosis of B. canis human brucellosis, and a supplementary technique, such as IELISA, performed on all positive RSAT samples that were negative by B. abortus antigen could ensure diagnostic specificity and confirm the diagnosis.
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A novel porA-based real-time PCR for detection of meningococcal carriage
More LessReal-time PCR based on the capsule transfer gene (ctrA) is a significant aid in the diagnosis of meningococcal infection but fails to detect a high proportion (60 %) of non-groupable strains associated with nasopharyngeal carriage. This study aimed to design a novel real-time (TaqMan) PCR that would detect more strains of meningococci and be suitable for large-scale carriage studies. Primer and probe sequences were based on the meningococcal porA gene and designed specifically to exclude the highly related porA pseudogene in Neisseria gonorrhoeae. The specificity of the assay was confirmed by testing strains of N. gonorrhoeae known to contain the porA pseudogene together with commensal strains of Neisseria lactamica and Neisseria sicca. None of these was detected in the assay. Neisseria meningitidis strains representing a wide range of serogroups together with non-groupable strains isolated from the nasopharynx were tested by ctrA assay and the novel porA-based TaqMan PCR. All carriage strains were detected by the porA-based assay including four that gave weak or no reaction with the ctrA assay. Comparison of ctrA and porA assays on 71 throat swabs obtained from university students showed that the porA assay detected meningococcal DNA in all samples that were ctrA positive plus three that were ctrA negative but culture positive. This novel porA-based TaqMan assay provides a highly specific method for detecting meningococcal DNA that is more sensitive than the ctrA assay for detecting meningococcal carriage and is particularly suitable for carriage studies where non-groupable strains and other Neisseria are present.
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Comparison of serotyping, pulsed field gel electrophoresis and amplified fragment length polymorphism for typing of Streptococcus pneumoniae
More LessThe aim of the present study was to compare serotyping, PFGE and AFLP for typing of Streptococcus pneumoniae with regard to discriminatory power, typeability and typing system concordance. Thirty-four isolates from cerobrospinal fluid and 34 time-matched blood culture isolates collected from in-patients at two hospitals in western Norway during the period from January 1994 to May 2002 were included in the study. The discriminatory powers of serotyping, PFGE and AFLP were 0.93, 0.99 and 0.95, respectively. The typeabilities for serotyping, PFGE and AFLP were 1, 1 and 0.99, respectively. A good concordance was shown between all the typing methods. Serotyping would most probably have a higher discriminatory power if further subtyping had been performed. PFGE was more discriminatory than AFLP, and AFLP grouped more-distantly related isolates together. The two typing methods thus provided different information, and therefore both could be useful adjuncts to serotyping for the characterization of S. pneumoniae.
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- Epidemiology
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Spore shedding pattern of Enterocytozoon bieneusi in asymptomatic children
Stool samples from seven human immunodeficiency virus (HIV)-negative and two HIV-positive children with asymptomatic Enterocytozoon bieneusi infections were daily examined to quantify spore shedding using Gram-chromotrope staining under light microscopy. The spore shedding pattern and intensity in these children was variable. Mean spore concentrations in the stool samples from these children ranged from 2.4 × 102 to 1.2 × 105 spores per gram. Light microscopy could detect spores in stool specimens for 9–33 days, while PCR was able to detect E. bieneusi in stool specimens for 3–40 days longer. This suggests that light microscopy may not detect low levels of spore shedding. Considering that the asymptomatic group are a potential source of infection, detection methods with a higher sensitivity should be used.
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Isolation and characterization of provisional serovar Shigella boydii E16553 from diarrhoeal patients in Bangladesh
In previous studies with strains of the Shigella dysenteriae provisional serovars E22383 and E23507 from diarrhoeal stools from patients in Bangladesh, two strains of Shigella species were identified as Shigella boydii provisional serovar E16553 by a reference laboratory. Further tests with an antiserum to an international type strain of the provisional serovar E16553 identified an additional 15 isolates. None of the isolates reacted with antisera to the established Shigella serovars or any other provisional serovars reported so far and all showed biochemical reactions typical of S. boydii. All of the isolates harboured the 140 MDa invasion plasmid, had the ipaH gene and produced keratoconjunctivitis in the guinea pig eye. All isolates were susceptible to ampicillin, sulfamethoxazole-trimethoprim, nalidixic acid, ciprofloxacin and mecillinam but eight strains were resistant to tetracycline. A single PFGE type (type A) was shown for all 17 clinical isolates, indicating a common source of origin. The pulsotype of the Bangladeshi isolates was closely related to that of a Japanese strain but was different from that of the type strain. On the basis of these biochemical, serological and virulence markers, and diverse geographical origin, it is recommended that the provisional status of serovar E16553 be changed and that it be included in the international serotyping classification scheme as S. boydii 19.
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- Clinical Microbiology And Virology
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Activity of Bulgarian propolis against 94 Helicobacter pylori strains in vitro by agar-well diffusion, agar dilution and disc diffusion methods
Propolis exhibits antimicrobial, anti-inflammatory and other biological effects. The aim of this study was to evaluate the activity of 30 % ethanolic extract of Bulgarian propolis against 94 Helicobacter pylori strains by three methods. By the agar-well diffusion method, only 13.8 % of the strains exhibited no inhibition by 30 μl propolis extract (containing 9 mg propolis) and all isolates were inhibited to some extent by 90 μl of the extract (27 mg propolis) per well. The mean diameters of growth inhibition by 30, 60 or 90 μl propolis extract or 30 μl 96 % ethanol per well were 16.8, 19.2, 27.5 and 8.3 mm, respectively. The propolis extract was more active than the ethanol (P < 0.001). With 90 μl propolis extract per well, 69.4 % of the strains exhibited large diameters of growth inhibition (⩾20 mm) versus 26.6 % with 30 μl per well (P < 0.001). With moist propolis discs, inhibition was detected in more strains (92.1 %) than with dried discs (78.2 %, P < 0.05), with mean inhibitory diameters of 18.7 and 13.8 mm, respectively. By the agar dilution method, 100 and 300 μg propolis ml−1 inhibited the growth of 57.1 % and 76.2 %, respectively, of the 21 strains tested. In conclusion, Bulgarian propolis had a strong and dose-dependent activity against most of the H. pylori strains tested. Although the effect of propolis on H. pylori in vitro is promising, further microbiological, pharmacological and clinical trials are required.
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- Veterinary Microbiology
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Colonization of 8-week-old conventionally reared goats by Escherichia coli O157 : H7 after oral inoculation
Enterohaemorrhagic Escherichia coli O157 : H7 infections of man have been associated with consumption of unpasteurized goat's milk and direct contact with kid goats on petting farms, yet little is known about colonization of goats with this organism. To assess the contribution of flagella and intimin of E. coli O157 : H7 in colonization of the goat, 8-week-old conventionally reared goats were inoculated orally in separate experiments with 1×1010 c.f.u. of a non-verotoxigenic strain of E. coli O157 : H7 (strain NCTC 12900 Nalr), an aflagellate derivative (DMB1) and an intimin-deficient derivative (DMB2). At 24 h after inoculation, the three E. coli O157 : H7 strains were shed at approximately 5×104 c.f.u. (g faeces)−1 from all animals. Significantly fewer intimin-deficient bacteria were shed only on days 2 (P = 0.003) and 4 (P = 0.014), whereas from day 7 to 29 there were no differences. Tissues from three animals inoculated with wild-type E. coli O157 : H7 strain NCTC 12900 Nalr were sampled at 24, 48 and 96 h after inoculation and the organism was cultured from the large intestine of all three animals and from the duodenum and ileum of the animal examined at 96 h. Tissues were examined histologically but attaching-effacing (AE) lesions were not observed at any intestinal site of the animals examined at 24 or 48 h. However, the animal examined at 96 h, which had uniquely shed approximately 1×107 E. coli O157 : H7 (g faeces)−1 for the preceding 3 days, showed a heavy, diffuse infection with cryptosporidia and abundant, multifocal AE lesions in the distal colon, rectum and at the recto-anal junction. These AE lesions were confirmed by immunohistochemistry to be associated with E. coli O157 : H7.
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- Oral Microbiology
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DNA identification of the pathogen of candidal aspiration pneumonia induced in the course of oral cancer therapy
More LessAspiration of oropharyngeal bacteria and fungi is occasionally suspected in patients with pneumonia. A patient with oral carcinoma underwent chemoradioimmunotherapy and, about 4 weeks from the start of the therapy, the patient suffered from severe oral mucositis induced by chemoradiotherapy, and candidal pneumonia was subsequently induced. The candidal pneumonia was insufficiently improved by potent antifungal drugs, taking a lethal course. Randomly amplified polymorphic DNA analysis and DNA sequence examination of strains isolated from the oral cavity 1 week before the onset of pneumonia and autopsied lung revealed the identity of both strains as Candida albicans, and the DNA analysis supported aspiration of oral Candida. These results indicate that the pathogen of the pneumonia, C. albicans, was aspirated from the oral cavity and that oral Candida is easily aspirated and becomes the pathogen of pneumonia.
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Concurrence between the gene expression pattern of Actinobacillus actinomycetemcomitans in localized aggressive periodontitis and in human epithelial cells
More LessActinobacillus actinomycetemcomitans is a facultatively intracellular pathogen and the aetiological agent of localized aggressive periodontitis. Screening of the genome of A. actinomycetemcomitans for in vivo-induced antigen determinants previously demonstrated that the proteome of this organism differs in laboratory culture compared with conditions found during active infection. The aim of the present study was to determine whether the bacterial gene expression pattern inferred with in vivo-induced antigen technology (IVIAT) in human infections was consistent with the gene expression pattern occurring upon epithelial cell association. To this end, a real-time PCR method was developed and used to quantify absolute and relative bacterial gene expression of A. actinomycetemcomitans grown extra- and intracellularly in two human epithelial cell lines (HeLa and IHGK). The amount of template used in the assay was normalized using the total count of viable bacteria (c.f.u.) as a reference point and performed in duplicate in at least two independent experiments. Controls for this experiment included 16S rRNA and gapdh. Transcription of all eight ORFs tested increased significantly (P < 0.05) in HeLa and IHGK cells compared with bacteria grown extracellularly. The concurrence of gene expression patterns found in the two models suggests that these epithelial cells are valid in vitro models of infection for the genes tested. IVIAT is an experimental platform that can be used as a validation tool to assess the reliability of animal and other models of infection and is applicable to most pathogens.
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- Case Reports
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Unusual clinical presentation of brucellosis caused by Brucella canis
Brucella canis is considered a rare cause of human brucellosis. The clinical importance of this infection may have been underestimated so far because of difficulties with presumptive diagnosis. The case described here presented symptoms compatible with brucellosis but the routine tests using Brucella abortus antigen were negative. The infection would have remained undiagnosed if culture had not been positive. This case illustrates the potential for a favourable outcome in Brucella canis diagnosis and supports recommendations for the use of B. canis serology. The infection should be suspected in patients with compatible symptoms and negative serology for B. abortus antigen.
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- Correspondence
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Volumes and issues
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Volume 72 (2022 - 2023)
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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 66 (2017)
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Volume 65 (2016)
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Volume 64 (2015)
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Volume 63 (2014)
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Volume 62 (2013)
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Volume 61 (2012)
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Volume 60 (2011)
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Volume 59 (2010)
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Volume 58 (2009)
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Volume 57 (2008)
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Volume 56 (2007)
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Volume 55 (2006)
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Volume 54 (2005)
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Volume 53 (2004)
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Volume 52 (2003)
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Volume 51 (2002)
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Volume 50 (2001)
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Volume 49 (2000)
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Volume 48 (1999)
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Volume 47 (1998)
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Volume 46 (1997)
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Volume 45 (1996)
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Volume 44 (1996)
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Volume 43 (1995)
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Volume 42 (1995)
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Volume 41 (1994)
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Volume 40 (1994)
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Volume 39 (1993)
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Volume 38 (1993)
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Volume 37 (1992)
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Volume 36 (1992)
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Volume 35 (1991)
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Volume 34 (1991)
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Volume 33 (1990)
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Volume 32 (1990)
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Volume 31 (1990)
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Volume 30 (1989)
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Volume 29 (1989)
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Volume 28 (1989)
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Volume 27 (1988)
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Volume 26 (1988)
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Volume 25 (1988)
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Volume 24 (1987)
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Volume 23 (1987)
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Volume 22 (1986)
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Volume 21 (1986)
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Volume 20 (1985)
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Volume 19 (1985)
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Volume 18 (1984)
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Volume 17 (1984)
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Volume 16 (1983)
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Volume 15 (1982)
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Volume 14 (1981)
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Volume 13 (1980)
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Volume 12 (1979)
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Volume 11 (1978)
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Volume 10 (1977)
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Volume 9 (1976)
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Volume 8 (1975)
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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 3 (1970)
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Volume 2 (1969)
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Volume 1 (1968)
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