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Biomedical subjects

P A Totten

Publications and source records attributed to P A Totten.

At least 37 records · Page 2Linked to original sources
PubMed1990

The rpoN gene product of Pseudomonas aeruginosa is required for expression of diverse genes, including the flagellin gene.

The product of the rpoN gene is an alternative sigma factor of RNA polymerase which is required for transcription of a number of genes in members of the family Enterobacteriaceae, including those that specify enzymes of nitrogen assimilation, amino acid uptake, and degradation of a variety of organic molecules. We have previously shown that transcription of the pilin gene of Pseudomonas aeruginosa also requires RpoN (K. S. Ishimoto and S. Lory, Proc. Natl. Acad. Sci. USA 86:1954-1957, 1989) and have undertaken a more extensive survey of genes under RpoN control. Strains of P. aeruginosa that carry an insertionally inactivated rpoN gene were constructed and shown to be nonmotile because of the inability of these mutants to synthesize flagellin. The mutation in rpoN had no effect on expression of extracellular polypeptides, outer membrane proteins, and the alginate capsule. However, the rpoN mutants were glutamine auxotrophs and were defective in glutamine synthetase, indicating defects in nitrogen assimilation. In addition, the P. aeruginosa rpoN mutants were defective in urease activity. These findings indicate that the sigma factor encoded by the rpoN gene is used by P. aeruginosa for transcription of a diverse set of genes that specify biosynthetic enzymes, degradative enzymes, and surface components. These rpoN-controlled genes include pili and flagella which are required for full virulence of the organism.

Bacterial Outer Membrane Proteins↗
PubMed1990

Characterization of the type a flagellin gene from Pseudomonas aeruginosa PAK.

Flagella in procaryotes are complex structures requiring the coordinate expression of over 50 genes, including flagellin, the major repeating structural protein. We have previously shown that a functional RpoN gene product is required for expression of flagellin in Pseudomonas aeruginosa PAK (P. A. Totten and S. Lory, J. Bacteriol. 172:389-396, 1990) and have now cloned, sequenced, and determined the transcriptional start site of the structural gene for this flagellin. The clones containing this gene produced a protein that reacted on Western immunoblots with polyclonal and four different monoclonal antibodies to purified flagella. However, this flagellin protein in Escherichia coli was slightly smaller (41 kDa) than flagellin protein produced in P. aeruginosa PAK (45 kDa), indicating degradation in E. coli or modification in P. aeruginosa. Comparison of the deduced amino acid sequence of this gene with the amino acid sequences of other flagellins revealed a conservation in the N- and C-terminal domains, suggesting conservation of secretion or assembly signals between these organisms. The sequence 5' of the structural gene contained potential RpoN-specific promoters as well as a promoter sequence recognized by RpoF (sigma 28), the alternative sigma factor required for expression of flagellin genes in E. coli (and Bacillus subtilis). Deletion analysis of the promoter region as well as transcriptional start site mapping implicated the RpoF, and not the RpoN, consensus sequences as the functional promoter for the flagellin gene. Models for the involvement of both RpoN and RpoF in the expression of flagellin in P. aeruginosa are presented.

Amino Acid Sequence↗
PubMed1987

Prevalence and characterization of hippurate-negative Campylobacter jejuni in King County, Washington.

A total of 593 strains of thermophilic Campylobacter species were isolated either from humans with diarrhea or from poultry in King County, Washington. Of these strains, 98 (52 hippurate-positive strains and all 46 of the hippurate-negative strains) were selected for further phenotypic characterization and genetic classification. Hippurate hydrolysis, the test typically used to differentiate Campylobacter jejuni and C. coli, did not always correlate with the genetic classification. All hippurate-positive strains were classified as C. jejuni. Of the hippurate-negative strains, 20% were C. jejuni, 78% were C. coli, and 2% were C. laridis. Assuming that the remaining hippurate-positive strains were all C. jejuni, then hippurate-negative C. jejuni represented a small percentage (9 of 556 or 1.6%) of C. jejuni strains but a significant percentage (9 of 46 or 20%) of hippurate-negative strains. This finding suggests that hippurate hydrolysis should not be used as the sole criterion for differentiating thermophilic Campylobacter species, particularly when describing the disease states associated with these organisms.

Animals↗
PubMed1986

An unusual Neisseria isolated from conjunctival cultures in rural Egypt.

Seven isolates of an unusual Neisseria sp. were obtained from eye cultures of children in two rural Egyptian villages. These Neisseria utilized only glucose, they exhibited a positive reaction when tested with antisera to crude antigen from Neisseria meningitidis and N. gonorrhoeae, and they did not react with the fluorescent antibody tests for N. gonorrhoeae or with the monoclonal antibodies used to serotype gonococci. The Egyptian isolates had colony morphology more typical of meningococci than gonococci and showed opaque and transparent colony variants. On SDS-PAGE, the major outer-membrane proteins had different patterns than those noted for comparable proteins of meningococci and gonococci; heat-modifiable outer-membrane proteins were present. Four of the six isolates examined had cryptic plasmids of 2.8 megadaltons, which were slightly larger than the cryptic plasmid of N. gonorrhoeae. These plasmids were homologous to the gonococcal cryptic plasmid, but had different restriction enzyme fragment patterns. The DNA from the Egyptian isolates, like DNA from N. meningitidis but unlike DNA from N. gonorrhoeae, could be cut with the restriction enzyme HaeIII. The frequency of transformation into a temperature-sensitive mutant of N. gonorrhoeae was 0.2 for the Egyptian isolates and 0.1 for N. meningitidis, a frequency that was 5-10-fold lower than that for the N. gonorrhoeae control isolates. Whole-cell DNA from the Egyptian isolates showed 68%-73% homology with N. gonorrhoeae and 57%-63% with N. meningitidis. On the basis of our observations, the Egyptian isolates are distinct from N. meningitidis and may represent a variant of N. gonorrhoeae. We suggest that the isolates be called Neisseria gonorrhoeae ssp. kochii.

Bacterial Proteins↗
PubMed1986

Two-dimensional thin-layer chromatography for the specific detection of hippurate hydrolysis by microorganisms.

Glycine, one of the end products of hippurate hydrolysis by microorganisms, was detected by a rapid, specific technique utilizing two-dimensional thin-layer chromatography. A loopful of growth of each organism from its suitable agar medium was washed, suspended, and incubated with 0.1% sodium hippurate for 30 min at 37 degrees C. The supernatant of the incubated suspension from each organism was then dansylated, and the dansyl derivatives were separated by two-dimensional thin-layer chromatography on polyamide sheets. Glycine, a product of hippurate hydrolysis, was detected under UV light. This technique does not require prolonged incubation and was found to be more specific and reliable than the standard ninhydrin reaction. In addition, it is inexpensive and can be easily conducted in a clinical microbiological reference laboratory. By this method, 100% (22/22) of Campylobacter jejuni and 0% (0/9) of Campylobacter coli reference strains were positive. In addition, 100% (13/13) of group B streptococci, 100% (24/24) of group D streptococci, and 90% (18/20) of Gardenerella vaginalis clinical isolates were positive for hippurate hydrolysis. This method is useful for the identification to the species level of Campylobacter organisms and the biotyping of Gardnerella organisms and for the detection of hippurate hydrolysis by unknown microorganisms.

Bacteria↗
PubMed1986

Isolation of "Campylobacter hyointestinalis" from a human.

We isolated "Campylobacter hyointestinalis" from the rectal culture of a homosexual man with proctitis. Phenotypic characterization of the isolate was confirmed by DNA hybridization by using the taxonomic spot blot. "C. hyointestinalis" was previously isolated only from animals but should be considered in the identification of Campylobacter species isolated from humans.

Adult↗
PubMed1985

Campylobacter cinaedi (sp. nov.) and Campylobacter fennelliae (sp. nov.): two new Campylobacter species associated with enteric disease in homosexual men.

In a recent study in Seattle, we isolated Campylobacter-like organisms (CLOs) significantly more often from homosexual men with intestinal symptoms than from asymptomatic heterosexual and homosexual control subjects. Biochemical tests, morphology, and guanine-plus-cytosine content of whole-cell DNA showed that these organisms were similar to, but distinguishable from, other species in the genus Campylobacter. DNA homology tests showed that the Campylobacter-like organisms belong to four unique genetic groups, two of which were phenotypically identical. We developed a rapid screening test to genetically group these organisms and to assess the correlation of each genotype with enteric disease in homosexual men. Three of the four CLO groups were isolated only from men with proctitis, proctocolitis, and/or enteritis. The remaining group, isolated from both symptomatic and asymptomatic homosexual men, was significantly correlated with the presence of polymorphonuclear leukocytes in the rectal secretions of asymptomatic men, a finding that suggests subclinical disease. We have tentatively classified two of the CLO groups into species, Campylobacter cinaedi sp. nov. and Campylobacter fennelliae sp. nov.

Campylobacter↗
PubMed1985

Evaluation of a DNA-hybridization method for detection of African and Asian strains of Neisseria gonorrhoeae in men with urethritis.

The 2.6-megadalton (MDa) cryptic plasmid and the 4.4-MDa beta-lactamase plasmid of Neisseria gonorrhoeae were radiolabeled with [32P] nucleotides and used as probes for direct detection of gonococci and beta-lactamase plasmids in urethral exudates from men with urethritis. The sensitivity and specificity of the DNA probes were compared with culture isolation of N. gonorrhoeae and biochemical tests of gonococcal isolates for beta-lactamase production. Of 216 urethral specimens, 180 were positive for N. gonorrhoeae by DNA probe and culture, 27 were negative by both tests, and 9 gave discordant results. Compared with culture and with the chromogenic cephalosporin assay, the sensitivity and the specificity of the DNA probe was 99% and 93% and that of the beta-lactamase probe assay was 91% and 96%, respectively. Electrophoresis of plasmids isolated from 90 gonococcal cultures showed that all contained the 2.6-MDa plasmid, 29 possessed a 3.2-MDa plasmid, 18 a 4.4-MDa beta-lactamase plasmid, and 11 had a 24.5-MDa conjugal plasmid. We conclude that the sensitivity of our DNA probes was comparable to that of culture for diagnosis of gonorrhea and to conventional tests for detection of beta-lactamase.

Africa↗
PubMed1985

Proctitis associated with Neisseria cinerea misidentified as Neisseria gonorrhoeae in a child.

An 8-year-old boy developed proctitis. Rectal swabs yielded a Neisseria sp. that was repeatedly identified by API (Analytab Products, Plainview, N.Y.), Minitek (BBL Microbiology Systems, Cockeysville, Md.), and Bactec (Johnston Laboratories, Towson, Md.) methods as Neisseria gonorrhoeae. Subsequent testing in a reference laboratory yielded an identification of Neisseria cinerea. It is suggested that identification of a Neisseria sp. isolated from genital or rectal sites in a child be confirmed by additional serological, growth, and antibiotic susceptibility tests and, if necessary, by a reference laboratory. The implications of such misidentifications are discussed.

Child↗
PubMed1984

Characterization of Campylobacter-like organisms isolated from homosexual men.

Thirteen Campylobacter-like organisms (CLOs) isolated from rectal cultures from homosexual men were studied. Like catalase-positive Campylobacter species, CLOs were curved gram-negative rods that did not grow aerobically, were motile, were oxidase- and catalase-positive, and did not utilize glucose. However, CLOs could not be classified within any of the Campylobacter species because they grew slowly and had unusual colony morphology; did not grow at 25 C, hydrolyze hippurate, produce H2S in triple sugar-iron agar, or tolerate 2% NaCl; were inhibited by 30-micrograms disks of nalidixic acid; and tolerated 1% glycine and 0.04% triphenyltetrazolium chloride. Three groups of CLOs were identified based on differences in nitrate reduction, growth at 42 C, and sensitivity to cephalothin. By the colony hybridization technique, whole-cell DNA isolated from a strain in each CLO group hybridized with DNA from other strains in the same group, but not with strains in other groups or with reference strains of catalase-positive Campylobacter species.

Campylobacter↗
PubMed1984

Characterization of Neisseria cinerea, a nonpathogenic species isolated on Martin-Lewis medium selective for pathogenic Neisseria spp.

An asaccharolytic, gram-negative, oxidase-positive diplococcus was isolated on Martin-Lewis medium from the cervix of a patient attending an arthritis clinic at Seattle Public Health Hospital, Seattle, Wash. This strain, NRL 32165, did not produce detectable acid from glucose, maltose, sucrose, fructose, mannitol, or lactose in either cystine Trypticase agar (BBL Microbiology Systems, Cockeysville, Md.) or modified oxidation-fermentation medium and was identified presumptively as a glucose-negative Neisseria gonorrhoeae strain, but was identified later as Neisseria cinerea on the basis of its biochemical reactions. Nitrate was not reduced, nitrite (0.001%, wt/vol) was reduced, and polysaccharide was not produced from sucrose. Proline, arginine, and cystine-cysteine were required for growth on defined medium. Strain NRL 32165 did not react with antigonococcal protein I monoclonal antibodies and did not produce immunoglobulin A protease. In DNA:DNA homology studies with N. gonorrhoeae NRL 8038 (F62) and N. cinerea type strain NRL 30003, strain NRL 32165 showed 95% homology relative to N. cinerea and 44% homology relative to N. gonorrhoeae. Thus, the identity of strain NRL 32165 was confirmed as N. cinerea (von Lingelsheim 1906) Murray 1939. Of all Neisseria spp., N. cinerea is most likely to be misidentified as a glucose-negative N. gonorrhoeae strain.

Agglutination Tests↗
PubMed1984

Biotypes of Gardnerella vaginalis.

A simple and reproducible scheme for identifying biotypes of Gardnerella vaginalis has been developed, based on reactions for lipase, hippurate hydrolysis, and beta-galactosidase. Among a total of 359 strains tested, eight biotypes were observed, the most common ones being types 1 (beta-galactosidase positive, lipase positive, hippurate positive), 2 (beta-galactosidase negative, lipase positive, hippurate positive), and 5 (beta-galactosidase negative, lipase negative, hippurate positive). The distribution in biotypes was similar among isolates from Antwerp, Seattle, and Nairobi. There were no differences in biotypes between strains isolated from patients with and without bacterial vaginosis (nonspecific vaginitis). Up to 14% of women with bacterial vaginosis harbored at least two different biotypes of G. vaginalis in the vagina. G. vaginalis strains isolated before and after treatment for bacterial vaginosis belonged to identical biotypes when the time interval between two specimens was less than 1 week. Similarly, G. vaginalis isolates from the vaginas of women with bacterial vaginosis and from the urethras of their male sex partners belonged to identical biotypes when strains were isolated within the same 24-h period from both partners (P less than 0.005).

Female↗
PubMed1984

Bacteremia caused by Campylobacter-like organisms in two male homosexuals.

Bacteremia caused by newly described Campylobacter-like organisms occurred in two immunosuppressed homosexual patients with tuberculosis. Although these organisms grow well in aerobic bottles using a radiometric blood culture system, they are not readily seen in gram-stained smears and are easily missed if routine subculture methods are used. Microscopic examination of wet preparations and subculture to brucella agar base supplemented with 10% sheep blood and incubated in microaerophilic conditions are useful for identification and isolation. The recovery of Campylobacter-like organisms from the blood suggests that these organisms, formerly known only to be associated with proctocolitis or asymptomatic rectal infection in homosexual men, can also cause systemic infection in these patients.

Adult↗
PubMed1983

Nonspecific vaginitis. Diagnostic criteria and microbial and epidemiologic associations.

Numerous previous studies of nonspecific vaginitis have yielded contradictory results regarding its cause and clinical manifestations, due to a lack of uniform case definition and laboratory methods. We studied 397 consecutive unselected female university students and applied sets of well defined criteria to distinguish nonspecific vaginitis from other forms of vaginitis and from normal findings. Using such criteria, we diagnosed nonspecific vaginitis in up to 25 percent of our study population; asymptomatic disease was recognized in more than 50 percent of those with nonspecific vaginitis. A clinical diagnosis of nonspecific vaginitis, based on simple office procedures, was correlated with both the presence and the concentration of Gardnerella vaginalis (Hemophilus vaginalis) in vaginal discharge, and with characteristic biochemical findings in vaginal discharge. Nonspecific vaginitis was also correlated with a history of sexual activity, a history of previous trichomoniasis, current use of nonbarrier contraceptive methods, and, particularly, use of an intrauterine device. G. vaginalis was isolated from 51.3 percent of the total population using a highly selective medium that detected the organism in lower concentration in vaginal discharge than did previously used media. Practical diagnostic criteria for standard clinical use are proposed. Application of such criteria should assist in clinical management of nonspecific vaginitis and in further study of the microbiologic and biochemical correlates and the pathogenesis of this mild but quite prevalent disease.

Adult↗
PubMed1983

DNA hybridization technique for the detection of Neisseria gonorrhoeae in men with urethritis.

A technique to detect Neisseria gonorrhoeae directly in clinical specimens was developed using a modified DNA-hybridization method. It uses the gonococcal cryptic plasmid as the radiolabeled probe, can detect as few as 100 colony-forming units of N gonorrhoeae or as little as 0.1 pg of purified gonococcal plasmid DNA, and is highly specific. This technique for differentiating between gonococcal and nongonococcal urethritis was evaluated in men with symptomatic urethritis in Seattle. Sixty-three (89%) of 71 who had cultures positive for N gonorrhoeae were also positive by DNA hybridization, and all 42 whose cultures were negative were also negative by DNA hybridization. Five of six isolates from patients who were positive by culture but negative by hybridization lacked the gonococcal cryptic plasmid and belonged to a unique auxo-type which requires proline, citrulline, and uracil for growth.

DNA, Bacterial↗
PubMed1983

Gardnerella vaginalis and anaerobic bacteria in the etiology of bacterial (nonspecific) vaginosis.

G. vaginalis was originally described as the etiologic agent of bacterial vaginosis (nonspecific vaginitis) because it was recovered only from women with signs and symptoms of "bacterial vaginitis" and not from normal controls. Recent data have shown that G. vaginalis is present in normal women but at concentrations lower than the limit of sensitivity of the media formerly used. Detection of low concentrations of G. vaginalis in normal controls has been made possible by development of a selective and differential medium (HBT). Anaerobically performed studies of the vaginal flora have indicated that while lactobacilli predominate in the normal vagina with or without G. vaginalis, anaerobic bacteria including Bacteroides spp., Peptococcus spp., Eubacterium spp. and curved rods as well as G. vaginalis predominate in bacterial vaginosis. Anaerobic bacteria and G. vaginalis are decreased after appropriate therapy. After treatment with metronidazole, lactobacilli again predominate. Lactobacilli are less prevalent after treatment with ampicillin or amoxicillin. These data suggest that as in infections at other mucous membrane sites, bacterial vaginosis is a mixed infection involving a finite number of facultative and anaerobic species. The data also suggest an important role for facultative lactobacilli.

Anti-Bacterial Agents↗