Rochalimaea infections. Are they zoonoses?
Explore the source record for details and available documents.
Angiomatosis↗
Biomedical subjects
L S Tompkins
Publications and source records attributed to L S Tompkins.
New technology in the clinical microbiology laboratory: what you always wanted to know but were afraid to ask.
Explore the source record for details and available documents.
Drug Resistance, Microbial↗
Effects of an isogenic Zn-metalloprotease-deficient mutant of Legionella pneumophila in a guinea-pig pneumonia model.
To determine the effects, if any, of the Zn-metalloprotease on virulence of Legionella pneumophila infection, an isogenic mutant deficient in protease (encoded by the proA gene) was tested in an Acanthamoeba cell model, in guinea-pig macrophages, and in a guinea-pig pneumonia model. The cloned proA gene was completely inactivated by insertion of a kanamycin-resistance cassette into the protease gene of L. pneumophila AA100. This mutated gene was then introduced into the L. pneumophila chromosome by allelic exchange to form the isogenic ProA- mutant AA200. AA200 showed no difference in its ability to enter, survive, or grow in Acanthamoeba and explanted guinea-pig macrophages; neither light nor electron microscopy revealed morphological differences in the eukaryotic cells infected with the protease mutant or the wild-type strains. The proA gene was found to be expressed in L. pneumophila during intracellular growth in amoebae by measuring the light produced from a truncated luxC gene fusion with the proA promoter. Virulence of the protease mutant was attenuated when tested in a guinea-pig model of infection employing the intratracheal inoculation method. AA200 was slower to cause death, grew to lower numbers in the lungs, resulted in less necrotic debris and a larger macrophage infiltrate, and was more likely to be found in association with macrophage vacuoles than the parent strain. Although deletion of the protease was not sufficient to completely abolish virulence in a guinea-pig model, the mutation caused a delay in the lethal effects of L. pneumophila and attenuated the infection.
Acanthamoeba↗
Further molecular characterization of the cloned Legionella pneumophila zinc metalloprotease.
On the basis of DNA sequence similarities to other Zn metalloproteases, further studies of the synthesis, processing, and enzymatic structure of the cloned Legionella protease gene, proA, were initiated. TnphoA fusions indicated that the entire proA open reading frame was transcribed and translated, including the 5' leader sequence. The results also suggested that the entire polypeptide was exported to the periplasm before cleavage to produce the mature protease. A site-directed mutation in the putative active site, changing glutamate 378 to asparagine, abolished proteolytic activity and cytotoxicity.
Animals↗
Molecular analysis of urease genes from a newly identified uncultured species of Helicobacter.
"Gastrospirillum hominis" is an uncultured gastric spiral bacterium that has recently been shown by 16S rDNA sequence analysis to be a newly recognized species of Helicobacter that infects humans, and it has been provisionally designated "Helicobacter heilmannii." We used PCR to directly amplify the urease structural genes of "H. heilmannii" from infected gastric tissue. DNA sequence analysis identified two open reading frames, ureA and ureB, which code for polypeptides with predicted molecular weights of 25,729 and 61,831, respectively. The urease subunit genes from "H. heilmannii" were cloned and expressed in Escherichia coli. Western blot (immunoblot) analysis showed that antiserum directed against the ureA and ureB gene products from H. pylori was cross-reactive with the corresponding polypeptides from "H. heilmannii." Analysis of the derived amino acid sequences of "H. heilmannii" UreA and UreB demonstrated that "H. heilmannii" urease is more highly related to the urease from H. felis (found in the stomachs of cats and dogs) than to the urease from H. pylori. These data are consistent with 16S rDNA sequence analysis and suggest that "H. heilmannii" is phylogenetically most closely related to H. felis.
Amino Acid Sequence↗
Growth of Legionella pneumophila in Acanthamoeba castellanii enhances invasion.
Legionella pneumophila is considered to be a facultative intracellular parasite. Therefore, the ability of these bacteria to enter, i.e., invade, eukaryotic cells is expected to be a key pathogenic determinant. We compared the invasive ability of bacteria grown under standard laboratory conditions with that of bacteria grown in Acanthamoeba castellanii, one of the protozoan species that serves as a natural host for L. pneumophila in the environment. Amoeba-grown L. pneumophila cells were found to be at least 100-fold more invasive for epithelial cells and 10-fold more invasive for macrophages and A. castellanii than were L. pneumophila cells grown on agar. Comparison of agar- and amoeba-grown L. pneumophila cells by light and electron microscopy demonstrated dramatic differences in the morphology and structure of the bacteria. Analyses of protein expression in the two strains of bacteria suggest that these phenotypic differences may be due to the expression of new proteins in amoeba-grown L. pneumophila cells. In addition, the amoeba-grown bacteria were found to enter macrophages via coiling phagocytosis at a higher frequency than agar-grown bacteria did. Replication of L. pneumophila in protozoans present in domestic water supplies may be necessary to produce bacteria that are competent to enter mammalian cells and produce human disease.
Acanthamoeba↗
Nosocomial legionellosis: a review of pulmonary and extrapulmonary syndromes.
Surgical patients appear to be at highest risk for acquisition of nosocomial Legionella pneumonia; most appear to become infected during respiratory tract manipulation and mechanical ventilation. Although the lungs are the most common site of nosocomial Legionella infection, an important subset of patients have infection at extrapulmonary sites. We describe 22 cases of extrapulmonary legionellosis reported in the literature. Most of these patients were surgical patients; more than half did not have serious underlying illnesses, and only five (23%) were receiving immunosuppressive agents. A total of 13 extrapulmonary sites of infection were reported, many in the absence of clinical pneumonia; these infections included sinusitis, hip wound infection, and prosthetic valve endocarditis. Five patients (23%) had fatal infections; in four of these cases diagnosis of Legionella infection was made after death, underscoring the need for a high index of clinical suspicion. A large percentage of extrapulmonary Legionella infections may result from direct topical exposure of susceptible tissue to contaminated tap water. Use of tap water must be carefully monitored, particularly in dressing changes and bathing of surgical patients.
Adult↗
An uncultured gastric spiral organism is a newly identified Helicobacter in humans.
"Gastrospirillum hominis" is an uncultivated spiral bacterium in human gastric mucosa that is larger and more tightly coiled than Helicobacter pylori. In an attempt to determine if this organism is a new species of Helicobacter, its 16S rRNA gene was cloned and sequenced. Gastric mucosa from 2 patients infected with "Gastrospirillum hominis" was fed to specific pathogen-free mice. Electron microscopy of gastric tissue confirmed that the mice became colonized with "Gastrospirillum hominis." The 16S rRNA gene from bacterial target sequences was amplified directly from mouse stomach tissue by the polymerase chain reaction (PCR), cloned into Escherichia coli, and sequenced. Both fragments were 16S rRNA genes from the Helicobacter genus that were most closely related to Helicobacter felis. "Gastrospirillum hominis" is probably a newly recognized Helicobacter infection in humans. Because this is the only Helicobacter organism that infects both humans and small animals, it may be particularly suited for studies of pathogenesis.
Adult↗
Role of flagella in adherence, internalization, and translocation of Campylobacter jejuni in nonpolarized and polarized epithelial cell cultures.
Previous studies of Campylobacter jejuni have suggested that flagellin is an adhesin for epithelial cells and that motility is a virulence factor of this bacterium. The role of flagella in the interactions of C. jejuni with nonpolarized and polarized epithelial cells was examined with flagellar mutants. Flagellated, nonmotile (flaA flaB+ Mot-) and nonflagellated, nonmotile (flaA flaB Mot-) mutants of C. jejuni were constructed by in vivo homologous recombination and gene replacement techniques. Both classes of mutants were found to adhere to cells of human epithelial origin (INT 407) equally well; however, on the basis of the percentage of the inoculum internalized, internalization of the flaA flaB Mot- mutants was decreased by factors ranging from approximately 30 to 40 compared with the parent. The flaA flaB+ Mot- mutant was internalized by the INT 407 cells at levels six- to sevenfold higher than the flaA flaB Mot- mutants. Both classes of mutants, unlike the parent, were unable to translocate across polarized Caco-2 monolayers. These results indicate that flagella are not involved in C. jejuni adherence to epithelial cells but that they do play a role in internalization. Furthermore, the results suggest that either the motility of C. jejuni or the product of flaA is essential for the bacterium to cross polarized epithelial cell monolayers.
Bacterial Adhesion↗
Molecular strain typing of Mycobacterium tuberculosis to confirm cross-contamination in the mycobacteriology laboratory and modification of procedures to minimize occurrence of false-positive cultures.
Molecular strain typing by restriction fragment length polymorphism analysis was used to demonstrate that two clusters of Mycobacterium tuberculosis cultures involving six patients resulted from cross-contamination in the mycobacteriology laboratory. Contaminated cultures were processed by the decontamination procedure and were read on the BACTEC instrument following acid-fast bacillus smear-positive specimens from patients with active tuberculosis. Investigation of these episodes suggested opportunities for modification of laboratory procedures to minimize cross-contamination and confirmed the adverse medical and public health consequences of false-positive cultures. Strain-typing results were used in decisions regarding patient care, including the curtailment of unnecessary treatment in one patient. Molecular strain typing appears to be a valuable means of identifying false-positive cultures of M. tuberculosis in selected settings.
Bacterial Typing Techniques↗
Identification and characterization of a Helicobacter pylori hemolysin.
Explore the source record for details and available documents.
Antibodies, Monoclonal↗
Transformation of Helicobacter pylori by electroporation.
A highly efficient method of introducing DNA into an important human pathogen is reported here. Electroporation-mediated transformation of a laboratory-passaged isolate of Helicobacter pylori was successfully used to establish genetic mutants at a transformation frequency of > 10(5)/micrograms DNA. This method should be widely applicable to all isolates of H. pylori and may eliminate the variability reported when natural transformation was used on fresh clinical isolates.
DNA, Bacterial↗
The use of molecular methods in infectious diseases.
Explore the source record for details and available documents.
DNA Fingerprinting↗
Potable water as a cause of sporadic cases of community-acquired legionnaires' disease.
BACKGROUND: The environmental sources of sporadic, community-acquired legionnaires' disease are largely unknown, and culturing of water sources after identification of a case is currently not recommended. We conducted a prospective study of sporadic cases of community-acquired legionnaires' disease to determine whether the environmental reservoirs could be identified. METHODS: We cultured samples of potable water obtained from sources to which each of 20 patients with culture-confirmed, community-acquired legionnaires' disease had been exposed during the two weeks before the onset of symptoms. Monoclonal-antibody subtyping and restriction-endonuclease analysis were performed on the legionella isolates recovered from both the patients and the associated environmental cultures. RESULTS: For 8 of the 20 patients, isolates of Legionella pneumophila with identical subtypes were identified in cultures from both the patient and the potable water to which the patient had been exposed. The environmental reservoirs linked to the infections were the water supplies of two private residences, two nursing homes, two hospital outpatient clinics, and an industrial plant. CONCLUSIONS: Potable-water supplies that harbor L. pneumophila are an important source of community-acquired legionnaires' disease. Future studies should include attempts to identify the environmental sources of this infection.
Adult↗
A quantitative model of intracellular growth of Legionella pneumophila in Acanthamoeba castellanii.
A model of intracellular growth for Legionella pneumophila in Acanthamoeba castellanii has been developed and provides a quantitative measure of survival and replication after entry. In this model, Acanthamoeba monolayers were incubated with bacteria in tissue culture plates under nutrient-limiting conditions. Gentamicin was used to kill extracellular bacteria following the period of incubation, and the number of intracellular bacteria was determined following lysis of amebae. Intracellular growth of virulent L. pneumophila and other wild-type Legionella species was observed when the assay was performed at 37 degrees C. At room temperature, none of the Legionella strains tested grew intracellularly, while an avirulent L. pneumophila strain was unable to replicate in this assay at either temperature. The effect of nutrient limitation on A. castellanii during the assay prevented multiplication of the amebae and increased the level of infection by Legionella spp. The level of infection of the amebae was directly proportional to the multiplicity of infection with bacteria; at an inoculum of 1.03 x 10(7) bacteria added to wells containing 1.10 x 10(5) amebae (multiplicity of infection of 100), approximately 4.4% of A. castellanii cells became infected. Cytochalasin D reduced the uptake of bacteria by the amebae primarily by causing amebae to lift off the culture dish, reducing the number of target hosts; methylamine also reduced the level of initial infection, yet neither inhibitor was able to prevent intracellular replication of Legionella spp. Consequently, once the bacteria entered the cell, only lowered temperature could restrict replication. This model of intracellular growth provides a one-step growth curve and should be useful to study the molecular basis of the host-parasite interaction.
Acanthamoeba↗
Characterization of Helicobacter pylori urease mutants.
The association between Helicobacter pylori, gastritis, and peptic ulcer is well established, and the association of infection with gastric cancer has been noted in several developing countries. However, the pathogenic mechanism(s) leading to disease states has not been elucidated. The H. pylori urease is thought to be a determinant of pathogenicity, since the enzyme is produced by all H. pylori clinical isolates. Evidence indicates that some H. pylori strains are more cytotoxic than others, with a correlation between the activity of the urease and the presence of a vacuolating cytotoxin having been made. However, the number of cytotoxins remains unknown at this time. The relationship between the urease and cytotoxicity has previously been examined with chemical inhibitors. To examine the role of the urease and its relationship to cytotoxicity, urease-deficient mutants were produced following ethyl methanesulfonate mutagenesis of H. pylori 87A300. Two mutants (the ure1 and ure5 mutants) which were entirely deficient in urease activity (Ure-) were selected. Characterization of the isolates at the protein level showed that the urease subunits lacked the ability to complex and form the active urease enzyme. The ure1 mutant was shown to be sensitive to the effects of low pH in vitro and exhibited no cytotoxicity to eucaryotic cells, whereas the parental strain (Ure+) produced a cytotoxic effect in the presence of urea. Interaction between the H. pylori Ure+ and Ure- strains and Caco-2 cells appeared to be similar in that both bacterial types elicited pedestal formation and actin condensation. These results indicate that the H. pylori urease may have many functions, among them (i) protecting H. pylori against the acidic environment of the stomach, (ii) acting as a cytotoxin, with human gastric cells especially susceptible to its activity, and (iii) disrupting cell tight junctions in such a manner that the cells remain viable but an ionic flow between the cells occurs.
Bacterial Adhesion↗
Cefamandole-susceptible strains of Legionella pneumophila serogroup 1: implications for diagnosis and utility as an epidemiological marker.
The standard selective Legionella medium that contains cefamandole failed to grow legionella pneumophila serogroup 1, subtype Bellingham, from a sputum sample from a patient with nosocomial Legionnaires' disease; the isolate did grow on a similar selective medium that substitutes vancomycin for cefamandole. Two Bellingham isolates from this patient's hospital environment also failed to grow when tested on the cefamandole medium. We tested 106 additional L. pneumophila serogroup 1 isolates that belonged to nine different monoclonal antibody subtypes and demonstrated that susceptibility to cefamandole was rare (10%) and limited to the Bellingham subtype. The diagnosis of Legionnaires' disease may be missed unless the culture protocol includes both a nonselective medium and a selective medium that does not contain cefamandole. In vitro susceptibility to cefamandole also provided an epidemiologic marker that linked a water source for a patient to nosocomial Legionnaires' disease.
Cefamandole↗
Helicobacter pylori and gastroduodenal disease: pathogenesis and host-parasite interaction.
Helicobacter pylori has been shown to be the cause of chronic active gastritis and the evidence that it is involved in the development of peptic ulcer disease and gastric cancer is compelling. Narrow host range, tissue specificity, and chronic inflammation are hallmarks of infection. The study of virulence determinants has just begun but it seems likely that urease, adhesins, cytotoxins, and mediators of inflammation will prove to be important.
Animals↗