A cluster of legionella sternal-wound infections due to postoperative topical exposure to contaminated tap water.
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Adult↗
Biomedical subjects
L S Tompkins
Publications and source records attributed to L S Tompkins.
Restriction enzyme and Southern hybridization analyses of Pseudomonas aeruginosa strains from patients with cystic fibrosis.
Conventional typing schemes for Pseudomonas aeruginosa may not discriminate strains. P. aeruginosa isolates from patients with cystic fibrosis were examined by restriction enzyme analysis and Southern hybridization. There was marked diversity of restriction enzyme analysis patterns among P. aeruginosa DNAs from cystic fibrosis isolates; however, sequential isolates obtained from individual patients showed very little variation over an 8-year period. DNA fragments from the alginate biosynthesis gene complex, the exotoxin A gene, and the elastase gene of P. aeruginosa were used in Southern hybridization analysis. The patterns of hybridization to the elastase and algD gene probes were highly conserved in all isolates, therefore, these DNA fragments are not useful in discriminating strains, in contrast to the exotoxin A gene probe.
Alginates↗
Role conflict and role ambiguity among respiratory care managers.
UNLABELLED: The health occupations and management literature does not specifically address role stress among technical directors of respiratory care departments. We undertook an analysis of role conflict, role ambiguity, and job satisfaction among technical directors of respiratory care departments in Texas. METHODS & MATERIALS: We distributed a questionnaire designed to measure role conflict and ambiguity and a questionnaire to elicit demographic and organization data to 283 technical directors in all Texas hospitals with more than 75 beds. Organization characteristics and demographic factors were studied as moderators. RESULTS: Analysis of the 199 responses received revealed that both role conflict (mean [SD] 3.86 [0.97] on 7.0 scale) and role ambiguity (2.64 [0.93] on 7.0 scale) scores were low compared to the neutral point of measure. Role overload, a component of role conflict, was found to be above the neutral point (4.64 [1.85]). One-way analysis of variance revealed no significant differences between the role conflict or the role ambiguity measures based on age, race, gender, number of employees supervised, size of institution, and position to which the respondent reported. Role conflict and role overload were each found to have significant negative correlations with job satisfaction (p less than 0.01). CONCLUSION: We are encouraged by the low role-conflict and ambiguity scores observed but concerned about the elevated role-overload scores. We believe that an in-depth study of role overload among respiratory care managers is warranted.
Adult↗
The agent of bacillary angiomatosis. An approach to the identification of uncultured pathogens.
BACKGROUND: Bacillary angiomatosis is an infectious disease causing proliferation of small blood vessels in the skin and visceral organs of patients with human immunodeficiency virus infection and other immunocompromised hosts. The agent is often visualized in tissue sections of lesions with Warthin-Starry staining, but the bacillus has not been successfully cultured or identified. This bacillus may also cause cat scratch disease. METHODS: In attempting to identify this organism, we used the polymerase chain reaction. We used oligonucleotide primers complementary to the 16S ribosomal RNA genes of eubacteria to amplify 16S ribosomal gene fragments directly from tissue samples of bacillary angiomatosis. The DNA sequence of these fragments was determined and analyzed for phylogenetic relatedness to other known organisms. Normal tissues were studied in parallel. RESULTS: Tissue from three unrelated patients with bacillary angiomatosis yielded a unique 16S gene sequence. A sequence obtained from a fourth patient with bacillary angiomatosis differed from the sequence found in the other three patients at only 4 of 241 base positions. No related 16S gene fragment was detected in the normal tissues. These 16S sequences associated with bacillary angiomatosis belong to a previously uncharacterized microorganism, most closely related to Rochalimaea quintana. CONCLUSIONS: The cause of bacillary angiomatosis is a previously uncharacterized rickettsia-like organism, closely related to R. quintana. This method for the identification of an uncultured pathogen may be applicable to other infectious diseases of unknown cause.
Acquired Immunodeficiency Syndrome↗
Visceral bacillary epithelioid angiomatosis: possible manifestations of disseminated cat scratch disease in the immunocompromised host: a report of two cases.
Opportunistic infection with the causative agent of cat scratch disease may be responsible for an unusual vascular proliferative lesion, referred to as bacillary epithelioid angiomatosis, previously described only in human immunodeficiency virus (HIV)-infected patients. We present a case of an HIV-infected patient with bacillary epithelioid angiomatosis involving the liver and bone marrow causing progressive hepatic failure. We also report a case of a cardiac transplant recipient with hepatic and splenic bacillary epithelioid angiomatosis manifesting as a fever of unknown origin, a previously unreported event in a non-HIV-infected patient. These cases represent the first documentation of bacillary epithelioid angiomatosis with visualization of cat scratch-like organisms involving internal organs.
Adult↗
Legionella pneumophila zinc metalloprotease is structurally and functionally homologous to Pseudomonas aeruginosa elastase.
The sequence of the structural gene encoding the Legionella pneumophila extracellular zinc metalloprotease has been determined and was found to possess a single large open reading frame (ORF) of 1,629 nucleotides (nt). This ORF was preceded by consensus promoter (TTAACT . . . 17 nt . . . TATAAC) and ribosome-binding (TAAGGAG) sequences. The deduced polypeptide contained a putative signal sequence and a total of 543 amino acid residues with a computed molecular size of 60,775 daltons, substantially larger than the observed 38,000 daltons of the native and recombinant proteins. A homology search revealed extensive amino acid identity with Pseudomonas aeruginosa elastase, a protein that is also encoded by an ORF substantially larger than that predicted for the mature size of the protein. The structural identity between the L. pneumophila protease and P. aeruginosa elastase was most pronounced in the regions forming the enzymatic active site of elastase. Amino acid residues constituting the active-site cleft of elastase were greater than 75% conserved. Elastase residues that interact with and mediate proteolysis of substrate peptides were 100% conserved. Competitive inhibitors of elastase and the structurally and functionally related thermolysin (phosphoramidon and a phosphoramidate analog, Z-GlyP(O)Leu-Ala), were shown to be equally potent at inhibiting the proteolytic activity of the L. pneumophila protease. These inhibitor studies along with the amino acid sequence similarities provide strong evidence that the L. pneumophila protease and P. aeruginosa elastase share a similar molecular mechanism of proteolysis.
Amino Acid Sequence↗
Predicting academic success in a nontraditional program.
Predictors and estimators of success in institutions of higher education have primarily been developed for traditional students in campus-based programs. With the rapidly increasing number of nontraditional allied health programs and students, there is a need to evaluate variables that may be used as predictors specifically for these students. This study examined the admission criteria and measures of success for 40 nontraditional respiratory care students. The subjects included 6 males and 34 females, who ranged from 17 to 50 years of age. The findings revealed a prediction model for three indicators of success: program average, clinical performance rating, and credentialing exam scores. Graduates' records were reviewed to assess which student selection criteria correlated well with these indicators. All models were significant at the 0.05 alpha level. These results may be useful in the selection of students for nontraditional respiratory care programs as well as in the selection of students for other allied health programs.
Adolescent↗
Analysis of a cloned sequence of Legionella pneumophila encoding a 38 kD metalloprotease possessing haemolytic and cytotoxic activities.
The DNA encoding the zinc metalloprotease of Legionella pneumophila Philadelphia 1 has been isolated and expressed in Escherichia coli. This protein, which is 38,000 Daltons in size, possesses immunological and biochemical properties identical to those previously described for the purified L. pneumophila protease. Periplasmic extracts of E. coli clones expressing the recombinant protease are also capable of causing the haemolysis of canine erythrocytes and the cytotoxic destruction of CHO cells. Using transposon mutagenesis, it was determined that a maximum of 1.2 kb of DNA encoded all three biological activities. Inactivation of proteolytic activity by transposon insertion occurred concomitantly with losses of the haemolytic and cytotoxic phenotypes. A putative regulatory sequence approximately 200-500 bp upstream of the gene's coding region was identified. A 4.0 kb fragment encoding these activities hybridized to the chromosomal DNA of the parent strain of L. pneumophila Philadelphia 1 as well as clinical isolates of L. pneumophila.
Blotting, Southern↗
Genetic, immunological, and cytotoxic comparisons of Legionella proteolytic activities.
Several strains of Legionella pneumophila and other species of Legionella with proteolytic activities were compared by assays, including Southern hybridizations and Western immunoblots, to determine their proteolytic, hemolytic, and cytotoxic activities. Only proteases from strains of L. pneumophila were both hemolytic and cytotoxic, and proteolytic activities extracted from other species of Legionella possessed only hemolytic activity. A 4.0-kilobase DNA sequence encoding the 38-kilodalton metalloprotease from L. pneumophila Philadelphia 1 that we showed previously was responsible for the observed hemolytic and cytotoxic phenotypes (F. D. Quinn and L. S. Tompkins, Mol. Microbiol., 3:797-805, 1989) was used in Southern hybridizations to probe chromosomal DNA from several strains of L. pneumophila and other Legionella species. The probe hybridized to the chromosomal DNA of all serogroups of L. pneumophila but not to any strains of L. dumoffii, L. micdadei, L. feeleii, or L. jordanis that we examined. Additionally, Western immunoblots done with rabbit antisera made to the cloned L. pneumophila protease demonstrated cross-reactions among 38-kilodalton proteins from strains of L. pneumophila, but no reactions were observed with proteins from other species of Legionella. Similarly, the cloned protease from L. pneumophila reacted with convalescent-phase sera from patients infected with L. pneumophila, but not with antisera isolated from patients infected with other Legionella species. Thus, despite some similarities among the proteolytic activities of members of the genus Legionella, including proteolytic and hemolytic phenotypes, metal requirements for zinc or iron, sensitivity to EDTA, and temperature and pH optima, we documented distinct genetic, immunological, and cytotoxicity differences among the proteolytic activities produced by Legionella species.
Blotting, Southern↗
Species-specific detection of Legionella pneumophila in water by DNA amplification and hybridization.
A sensitive detection system specific for Legionella pneumophila in water was developed. This system is based on amplification of a chromosomal DNA sequence from L. pneumophila by the polymerase chain reaction, followed by detection of the amplified product by hybridization of a radiolabeled oligodeoxynucleotide. After 35 cycles of amplification, a water sample which had been seeded with 35 CFU of L. pneumophila contained sufficient amplified DNA to be detected on dot blots. Bacteria of other genera tested did not generate positive signals under these conditions. Application of this technique to environmental water samples may help identify the natural reservoirs of nosocomial and community-acquired L. pneumophila infections.
Blotting, Southern↗
Restriction endonuclease analysis of human and bovine group B streptococci for epidemiologic study.
Group B streptococci, a frequent cause of neonatal sepsis and meningitis, postpartum endometritis, and bovine mastitis, may be acquired by several modes of transmission. Detailed epidemiologic study is hampered by the lack of a sufficiently discriminatory typing system, especially for type III and nontypable strains. We examined 54 epidemiologically well-characterized strains by restriction endonuclease analysis (REA) and compared the results with those obtained by serotyping. REA patterns were inspected without knowledge of the epidemiological or serotyping data. Among 21 type Ia, Ia/c, and Ib/c isolates, we found 10 REA patterns; among 5 type II and IIc isolates, we found 5 REA patterns; among 13 type III isolates, we found 6 REA patterns; and among 15 nontypable human and animal isolates, we found 7 different REA patterns. Double digestion of type III isolates with EcoRI and BglII helped us to distinguish the isolates. In total, 28 REA patterns were found in six serotype groups and one nontypable group. Some geographically and epidemiologically separate isolates had identical REA patterns, suggesting dissemination of a limited number of clones. We conclude that REA is a promising tool for detailed epidemiological study of group B streptococci.
Animals↗
Transformation of Actinobacillus pleuropneumoniae and analysis of R factors by electroporation.
An efficient method for DNA transfer is essential for the genetic manipulation of any organism. Such a capacity will be required for the genetic analysis of Actinobacillus pleuropneumoniae as a swine pathogen, as well as for its manipulation for vaccination purposes. For this reason, the use of electroporation as a means of plasmid DNA introduction into this species was examined. The multiple antibiotic-resistant strain 80-8141 of Actinobacillus pleuropneumoniae harbors 3 plasmids: pYG10, pYG15, and pYG12 of 5.0, 2.7, and 2.5 kb, respectively. Electroporation of A pleuropneumoniae strain 4074 with a plasmid extract of strain 80-8141 showed that pYG10 encodes chloramphenicol resistance and that pYG12 encodes ampicillin resistance. Electrical pulse conditions for efficient electroporation of strain 4074 were examined by use of pYG10 DNA isolated from a 4074 transformant. Efficiency, expressed as transformants per microgram of plasmid DNA, increased directly with pulse amplitude. However, high efficiencies were only observed in a narrow window of pulse duration (tau = 12 to 22 ms at 6.25 kV/cm). Longer pulse durations resulted in cell death. Electroporation efficiencies increased with cell density. Yield of transformants increased directly with DNA concentration. Results indicate that electroporation can be used to efficiently transform A pleuropneumoniae and that pYG10 and pYG12 are suitable plasmid vectors for use in the genetic manipulation of this organism.
Actinobacillus↗
Nucleic acid probes in infectious diseases.
Few of the probes described here are available as practical assays for clinical laboratories, and some have argued that currently available kits have not lived up to their promise to provide increased sensitivity, specificity, and speed of identification and detection at a reasonable cost. While these sentiments are true to some extent now, the rapid advance of techniques to enhance the rate of hybridization, to amplify the target sequences, and to label and detect hybrids nonradioactively will have a substantial impact on the availability of hybridization kits for the clinical laboratory. It is conceivable that probe technology will be simplified to the extent that it can be adapted to small laboratories, clinics, and field sites. Not only can we expect probes to speed detection and identification of established pathogens, we also look forward to perhaps the most exciting future aspect of this technology, the discovery of new agents and new infectious disease syndromes that will be brought about through its application.
Animals↗
Legionella prosthetic-valve endocarditis.
Since 1982 seven patients at Stanford University Medical Center have been shown to have prosthetic-valve endocarditis caused by Legionella pneumophila or L. dumoffii. We studied the clinical features of legionella endocarditis at the time of diagnosis and performed a case-control study to analyze risk factors for the infection. All patients with endocarditis had a chronic course (3 to 19 months after surgery) of fever, night sweats, weight loss, and anemia, but no embolic events or immune-complex deposition disease. Five patients required surgical replacement of their infected prosthetic valves. The case-control study revealed that during the early postoperative period, patients who later contracted legionella endocarditis were more likely to have had symptoms and signs attributable to postcardiomyotomy syndrome than were patients who did not contract endocarditis (P less than 0.013). Examination of the legionella isolates by means of molecular techniques demonstrated that the Stanford L. pneumophila isolates were genotypically identical to isolates from the hospital drinking water. L. dumoffii isolates from patients with endocarditis were derived from a single strain apparently unique to this medical center. We conclude that legionella infection was nosocomially acquired in the perioperative period. These cases demonstrate an expanding spectrum of illness caused by legionella species and emphasize the need to consider legionella as a cause of "culture-negative" endocarditis.
Endocarditis, Bacterial↗
High-voltage electroporation of bacteria: genetic transformation of Campylobacter jejuni with plasmid DNA.
Electroporation permits the uptake of DNA by mammalian cells and plant protoplasts because it induces transient permeability of the cell membrane. We investigated the utility of high-voltage electroporation as a method for genetic transformation of intact bacterial cells by using the enteric pathogen Campylobacter jejuni as a model system. This report demonstrates that the application of high-voltage discharges to bacterial cells permits genetic transformation. Our method involves exposure of a Campylobacter cell suspension to a high-voltage exponential decay discharge (5-13 kV/cm) for a brief period of time (resistance-capacitance time constant = 2.4-26 msec) in the presence of plasmid DNA. Electrical transformation of C. jejuni results in frequencies as high as 1.2 x 10(6) transformants per microgram of DNA. We have investigated the effects of pulse amplitude and duration, cell growth conditions, divalent cations, and DNA concentration on the efficiency of transformation. Transformants of C. jejuni obtained by electroporation contained structurally intact plasmid molecules. In addition, evidence is presented that indicates that C. jejuni possesses DNA restriction and modification systems. The use of electroporation as a method for transforming other bacterial species and guidelines for its implementation are also discussed.
Bacteriological Techniques↗
Infant with two relapses of group B streptococcal sepsis documented by DNA restriction enzyme analysis.
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DNA Restriction Enzymes↗
Molecular epidemiology of Legionella species by restriction endonuclease and alloenzyme analysis.
As part of an ongoing investigation into nosocomial Legionella infections at Stanford University Medical Center (SUMC), we applied the technique of restriction endonuclease analysis (REA) to determine strain differences among three species, including Legionella pneumophila, Legionella dumoffii, and Legionella micdadei. A total of 26 human and environmental water isolates from SUMC were selected for REA and compared with control strains that were not epidemiologically linked to SUMC. REA results were compared with results of alloenzyme typing, typing by monoclonal antibodies, and plasmid fingerprinting in all but L. micdadei strains. REA and alloenzyme typing showed that SUMC patient isolates were derived from distinct strains of three species. L. pneumophila strains from SUMC patients were genotypically identical to those isolated from potable water. REA was especially useful in proving that SUMC L. dumoffii patient isolates were derived from a single strain and that patients may have been exposed to a common source(s). REA typing correlated well with alloenzyme typing. These methods complement serologic typing of L. pneumophila and provide discriminating capability between strains of other Legionella species such as L. dumoffii, for which serologic types have not been identified. In addition, REA typing is somewhat easier to perform than alloenzyme typing and can be done in clinical laboratories.
DNA Restriction Enzymes↗
Approaches to the detection of enteric pathogens, including Campylobacter, using nucleic acid hybridization.
A previous study of the detection of Campylobacter jejuni in fecal samples spotted directly onto nitrocellulose filters before hybridization revealed a relatively low sensitivity and some false-positive results. We have investigated two factors that interfere with the detection of Campylobacter jejuni in fecal samples: interfering substances that create false-positive background signals and nonspecificity of the probe. Heterologous deoxyribonucleic acid probes bound nonspecifically to partially extracted fecal samples, indicating that the major basis for false-positive background was not due to homologous sequences. The problem of false-positive results could be reduced, but not eliminated, by extensive deoxyribonucleic acid extraction procedures applied to the clinical sample. The relative concentration of protein in each sample may be an important contributor to nonspecific binding. Other measures, such as sonication, glass bead fragmentation, and column separation, were not helpful. Development of a species-specific deoxyribonucleic acid probe derived from sequences encoding 16S ribonucleic acid is underway.
Campylobacter Infections↗