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Biomedical subjects

H L Mobley

Publications and source records attributed to H L Mobley.

At least 109 records · Page 6Linked to original sources
PubMed1988

Characterization of urease from Campylobacter pylori.

Campylobacter pylori, a suspected agent of gastritis and peptic ulceration, rapidly hydrolyzes urea. Because urease serves as the basis of detection of the organism in gastric biopsies and may represent an important virulence factor, biochemical characteristics of the enzyme were determined. C. pylori was isolated from antral biopsies from 10 patients with complaints of abdominal pain or history of peptic ulcer disease. All isolates were urease positive, with an average rate of hydrolysis by cell lysates being 36 +/- 28 mumol of NH3 per min per mg of protein, more than twice that of Proteus mirabilis and 10 times that of other urinary tract isolates. The enzyme had an apparent molecular weight of 625,000 +/- 15,000 by column chromatography, an isoelectric point of 5.9, a Km of 0.8 +/- 0.1 mM urea, an optimal temperature of 45 degrees C, and an optimal pH of 8.2. Ten isolates tested produced ureases with identical electrophoretic mobilities on nondenaturing 5% polyacrylamide activity gels. Acetohydroxamic acid (100 micrograms/ml), hydroxyurea (85 micrograms/ml), flurofamide (0.05 micrograms/ml), and EDTA (8 mM) inhibited enzyme activity by 50%. Cell lysates retained 50% of initial urease activity after 6 days and 40% activity after 18 days when stored at 4 degrees C in 20 mM sodium phosphate, pH 6.8. At -70 degrees C for 18 days, 1 mM EDTA or 15% glycerol preserved 40 or 34%, respectively, of initial activity. The urease of C. pylori appears to be biochemically unique from the enzymes of other common urease-producing species.

Campylobacter↗
PubMed1987

Uropathogenicity in rats and mice of Providencia stuartii from long-term catheterized patients.

Providencia stuartii, a frequent and persistent isolate from the urinary tract of chronically catheterized elderly patients, is multiply antibiotic resistant and may cause fatal bacteremia in those patients. We studied P. stuartii strains in rats and mice to determine differences in uropathogenicity. Strains studied varied in expression of factors which contribute to pathogenicity of other bacterial species. Urinary tract responses following challenge with P. stuartii strain HO (factors expressed) were similar to responses reported for uropathogenic E. coli strains both in bacterial persistence and histologic change. In animals similarly challenged with P. stuartii strain RO (factors not expressed), responses were similar to those reported for non-uropathogenic E. coli strains. Results indicate that: a) animal model studies may be useful in differentiating P. stuartii strains based on uropathogenicity, b) P. stuartii uropathogenicity may be related to identifiable factors associated with virulence in other species, and c) the CBA mouse model appears to be the most suitable for studies of P. stuartii uropathogenicity.

Animals↗
PubMed1987

Emergence of resistance to imipenem in Pseudomonas aeruginosa.

The emergence of resistance to imipenem by Pseudomonas aeruginosa was investigated with four pairs of isolates. Each pair represented pretherapy (susceptible) and posttherapy (resistant) specimens. In all cases, the imipenem-resistant isolates did not demonstrate changed susceptibilities to other beta-lactams. Agarose gel electrophoresis revealed no change in plasmid profiles between any pair of isolates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the Sarkosyl-insoluble membrane protein revealed the loss of an outer membrane protein of apparent molecular mass 48 to 49 kilodaltons in posttherapy strains when grown with imipenem selection (5 micrograms/ml). There was no significant difference in the binding of [14C]imipenem to the penicillin-binding proteins of the pre- and posttherapy strains. Trichloroacetic acid precipitation of membranes isolated after growth in the presence of [14C]imipenem revealed that significantly less drug was bound to Sarkosyl-soluble membrane protein in three of the four posttherapy strains than the membrane proteins of the respective pretherapy strains. beta-Lactamase activity against imipenem at 100 or 3 microM was not detected in any isolate either with or without induction. These data suggest that resistance to imipenem is associated with the loss of a 48- to 49-kilodalton outer membrane protein accompanied by, in three of four cases, decreased penetration of the antibiotic across the outer membrane.

Bacterial Outer Membrane Proteins↗
PubMed1987

Genetic and biochemical diversity of ureases of Proteus, Providencia, and Morganella species isolated from urinary tract infection.

Bacterial urease, particularly from Proteus mirabilis, has been implicated as a contributing factor in the formation of urinary and kidney stones, obstruction of urinary catheters, and pyelonephritis. Weekly urine specimens (n = 1,135) from 32 patients, residing at two chronic-care facilities, with urinary catheters in place for greater than or equal to 30 days yielded 5,088 phenotypically and serotypically diverse bacterial isolates at greater than or equal to 10(5) CFU/ml. A total of 86% of specimens contained at least one urease-positive species, and 46% of 3,939 gram-negative bacilli were urease positive. For investigation of genetic relatedness of urease determinants, whole-cell DNA from 50 urease-positive isolates each of Providencia stuartii, Providencia rettgeri, P. mirabilis, Proteus vulgaris, and Morganella morganii were hybridized with a urease gene probe derived from within the urease operon of Providencia stuartii BE2467. The percentage of strains hybridizing with the gene probe was 98 for Providencia stuartii, 100 for Providencia rettgeri, 70 for P. mirabilis, 2 for M. morganii, and 0 for P. vulgaris. Electrophoretic mobilities of ureases from representative isolates revealed nine different patterns among the five species. The urease gene probe hybridized with fragments of HindIII-digested chromosomal DNA from all isolates except M. morganii. Fragment sizes differed between species. Molecular sizes of the enzymes, determined by Sephacryl S-300 chromatography, were found to be 280 kilodaltons (kDa) (P. mirabilis), 323 to 337 kDa (Providencia stuartii, Providencia rettgeri, P. mirabilis, P. vulgaris), 620 kDa (providencia rettgeri), and greater than 700 kDa (M. morganii, Providencia rettgeri). Kms ranged from 0.7 mM urea for M. morganii to 60 mM urea for a P. mirabilis isolate. In general, P. mirabilis ureases demonstrated lower affinities for substrate but hydrolyzed urea at rates 6- to 25-fold faster than did enzymes from other species, which may explain the frequent association of this species with stone formation.

Aged↗
PubMed1987

Urease-positive bacteriuria and obstruction of long-term urinary catheters.

Long-term urethral catheterization (greater than or equal to 30 days), a management technique for urinary incontinence, results in polymicrobial bacteriuria. We frequently found urease-producing bacteria: of 1,135 weekly urine specimens from 32 long-term-catheterized patients, 86% had urease-positive bacterial species at greater than or equal to 10(5) CFU/ml. The most common species were Proteus mirabilis and Morganella morganii, each found in over half the specimens. P. mirabilis, but not other urease-positive species, was significantly associated with the 67 obstructions observed in 23 patients. M. morganii had a more complex association and in some way may protect the catheter from obstruction.

Aged↗
PubMed1987

Expression of type 1 fimbriae may be required for persistence of Escherichia coli in the catheterized urinary tract.

Long-term urinary catheterization results in polymicrobial bacteriuria and is complicated by fever, bacteremia, acute pyelonephritis, and death. Escherichia coli is a common urine isolate from catheterized patients and can persist for months. We hypothesized that fimbria-mediated adherence contributes to its persistence. For 1 year, urine specimens were collected from 51 patients greater than or equal to 65 years of age who were catheterized for greater than or equal to 30 days. E. coli was isolated at greater than or equal to 10(5) CFU/ml from 447 (36%) of 1,230 weekly urine specimens from 26 patients. Week 1 isolates from 52 definable episodes were tested for hemagglutination, hybridization with gene sequences from the pil and pap operons, in vitro adherence to catheter material, binding of 125I-labeled Tamm-Horsfall protein, hemolysin and colicin V production, and serum resistance. The proportions of isolates of short (1 week only), medium (2 to 11 weeks) and long (greater than or equal to 12 weeks) episodes of bacteriuria which expressed type 1 fimbriae as assayed by mannose-sensitive hemagglutination were 59, 65, and 92%, respectively. Isolates with the pil operon (the genome for type 1 fimbriae) from episodes lasting greater than 1 week expressed mannose-sensitive hemagglutination more frequently (P = 0.011) than pil-positive isolates from episodes of less than or equal to 1 week. Isolates from episodes of greater than 1 week also bound significantly more Tamm-Horsfall protein than isolates from episodes of less than or equal to 1 week (P = 0.044). Although nearly half of the isolates produced P fimbriae, an important virulence factor for the development of pyelonephritis, no correlation with persistence could be made. Overall, the E. coli isolates expressed traits similar to those of strains that caused cystitis. Type 1 fimbriae appear to be important for the persistence of E. coli in the long-term-catheterized urinary tract.

Aged↗
PubMed1987

Urease activity of Proteus penneri.

Ten strains of Proteus penneri isolated from geographically diverse laboratories were tested for urease activity. Cell lysates from urea-induced cells had a mean activity of 4.9 +/- 4.1 mumol of NH3 per min per mg of protein. On nondenaturing 6% polyacrylamide activity gels, the enzymes of P. penneri had very similar electrophoretic mobilities within species and within the Proteus genus but were distinct from the ureases of Providencia and Morganella species. On lower-percentage polyacrylamide, differences in mobilities of the ureases could be detected between the Proteus species. From representative strains, the P. penneri urease was found to be inducible by growth in urea and had an apparent molecular weight of 246,000 +/- 9,000, an isoelectric point of 5.1, and a Km for urea of 14 mM and was inhibitable by acetohydroxamic acid, hydroxyurea, and EDTA. In an in vitro model of struvite formation, a P. penneri strain produced abundant crystals on a glass rod submerged in synthetic urine in the absence but not presence of acetohydroxamic acid (500 micrograms/ml).

Chromatography, Gel↗
PubMed1986

Adherence to uroepithelial cells of Providencia stuartii isolated from the catheterized urinary tract.

The long-term catheterized urinary tract appears to offer a niche for Providencia stuartii, otherwise an unusual clinical isolate. P. stuartii, the most frequent and persistent isolate from the urine of 51 long-term catheterized patients, was recovered from 761 of 1230 (62%) weekly urine specimens. To test the hypothesis that prevalence of this species may be due to adherence properties of the organism, 20 selected strains from 14 patients at two nursing homes, representing six distinct serotypes and harbouring combinations of nine different plasmid species, were tested for adherence to uroepithelial cells (UEC). Optimal conditions were determined for differentiating strains on the basis of in vitro adherence to UEC. These strains, grown in nutrient broth, were incubated with UEC isolated from the urine of a healthy adult female (10(8) bacteria per 10(5) cells). Washed UEC, retained on 8 micron pore diameter filters, were transferred to slides, fixed and stained; bacteria were counted on each of 40 cells. Fourteen of the 20 strains were defined as adherent to UEC by comparison of mean adherent bacteria and percentage of uroepithelial cells with more than 10 bacteria. Adherence was compared to that of a P-fimbriated strain of Escherichia coli. It was not inhibited by 50 mM-mannose. We conclude that the majority of P. stuartii isolates are adherent to UEC in vitro and suggest that this may play a role in the persistence of this organism in the catheterized urinary tract.

Bacterial Adhesion↗
PubMed1986

Cloning of urease gene sequences from Providencia stuartii.

Providencia stuartii was the most prevalent isolate recovered from urine specimens taken weekly over a 1-year period from 51 nursing home patients with urinary catheters in place. Thirty percent of the isolates were urease positive. Urease, which is implicated in renal stone formation, was shown to be transmissible on an 82-kilobase conjugative plasmid in one isolate. Plasmid DNA isolated from this strain was digested with EcoRI, ligated into the EcoRI site of pBR322, and used to transform Escherichia coli HB101. Ampicillin-resistant clones were replica plated onto urea segregation agar, and a urease-positive clone, designated pMID101, was isolated. Recombinant and native urease from cell lysates had identical electrophoretic mobilities on nondenaturing polyacrylamide urease activity gels. The native enzyme was induced fourfold when cells were grown in the presence of 0.1% urea and had a km of 9.4 mM and a Vmax of 3.2 mumol of NH3 per min per mg of protein. Its molecular weight was estimated to be 375,000 +/- 35,000 by Sephacryl S-300 chromatography. The enzyme was cytoplasmic in P. stuartii, was inhibited in vitro by hydroxyurea, acetohydroxamic acid, and EDTA, and appears to have a complex subunit structure and a unique molecular size within genera of the Proteeae tribe.

Cell Compartmentation↗
PubMed1985

Separate resistances to arsenate and arsenite (antimonate) encoded by the arsenical resistance operon of R factor R773.

The arsenical resistance operon of R factor R773 was analyzed by subcloning and insertional inactivation. The operon was found to have two functional regions, the promoter-proximal region encoding resistance to arsenite and antimonate and the promoter-distal region encoding arsenate resistance. A unique 1.6-kilobase fragment was shown to be sufficient to encode arsenate resistance and produce arsenate extrusion from intact cells.

Arsenates↗
PubMed1985

Variable phenotypes of Providencia stuartii due to plasmid-encoded traits.

Weekly urine specimens from 51 long-term catheterized patients yielded 699 isolates of Providencia stuartii. Urease-positive strains represented 23.7% (166) of the isolates, sucrose-positive strains represented 24.5% (171), and lactose-utilizing strains represented 0.7% (5). Urease and sucrose traits were transferred by conjugation to Escherichia coli via an 82-kilobase plasmid; lactose fermentation was transferred by a 150-kilobase plasmid.

Bacteriuria↗
PubMed1984

Homology among arsenate resistance determinants of R factors in Escherichia coli.

Escherichia coli bearing R factors R773 or R46 or hybrid recombinant plasmids carrying the arsenic resistance determinants derived from these plasmids synthesized inducible polypeptides of similar apparent molecular weights when exposed to arsenite salts (R773 derivative, 64,000 and 16,000; R46 derivative, 62,000, 16,500, and 13,500). In addition, both plasmids encoded energy-dependent arsenate efflux systems and demonstrated DNA sequence homology by filter blot hybridization. Human isolates of arsenate- and arsenite-resistant enterobacteria were tested for homology with the arsenate operon of R773 by colony blot hybridization. Approximately one-third of the isolates hybridized strongly, and two-thirds showed little or no evidence of homology, suggesting the presence of two or more genetically distinct arsenate resistant determinants.

Arsenates↗
PubMed1984

Insertion and fate of the cell wall in Bacillus subtilis.

Cell wall assembly was studied in autolysin-deficient and -sufficient strains of Bacillus subtilis. Two independent probes, one for peptidoglycan and the other for surface-accessible teichoic acid, were employed to monitor cell surface changes during growth. Cell walls were specifically labeled with N-acetyl-D-[3H]glucosamine, and after growth, autoradiographs were prepared for both cell types. The locations of silver grains revealed that label was progressively lost from numerous sites on the cell cylinders, whereas label was retained on the cell poles, even after several generations. In the autolysin-deficient and chain-forming strain, it was found that the distance between densely labeled poles approximately doubled after each generation of growth. In the autolysin-sufficient strain, it was found that the numbers of labeled cell poles remained nearly constant for several generations, supporting the premise that completed septa and poles are largely conserved during growth. Fluorescein-conjugated concanavalin A was also used to determine the distribution of alpha-D-glucosylated teichoic acid on the surfaces of growing cells. Strains with temperature-sensitive phosphoglucomutase were used because in these mutants, glycosylation of cell wall teichoic acids can be controlled by temperature shifts. When the bacteria were grown at 45 degrees C, which stops the glucosylation of teichoic acid, the cells gradually lost their ability to bind concanavalin A on their cylindrical surfaces, but they retained concanavalin A-reactive sites on their poles. Discrete areas on the cylinder, defined by the binding of fluorescent concanavalin A, were absent when the synthesis of glucosylated teichoic acid was inhibited during growth for several generations at the nonpermissive temperature. When the mutant was shifted from a nonpermissive to a permissive temperature, all areas of the cylinder became able to bind the labeled concanavalin A after about one-half generation. Old cell poles were able to bind the lectin after nearly one generation at the permissive temperature, showing that new wall synthesis does occur in the cell poles, although it occurs slowly. These data, based on both qualitative and quantitative experiments, support a model for cell wall assembly in B. subtilis, in which cylinders elongate by inside-to-outside growth, with degradation of the stress-bearing old wall in wild-type organisms. Loss of wall material, by turnover, from many sites on the cylinder may be necessary for intercalation of new wall and normal length extension. Poles tend to retain their wall components during division and are turned over much more slowly.

Acetylglucosamine↗
PubMed1983

Cloning and expression of R-factor mediated arsenate resistance in Escherichia coli.

The resistance transfer factor R773 confers inducible arsenate, arsenite and antimony resistance on Escherichia coli. The genes for these resistances were cloned into the EcoRI site of plasmid pBR322 to produce a 33 kilobase plasmid. pUM1. Bacterial strains transformed with pUM1 synthesized a polypeptide of the apparent molecular weight 64,000 daltons when induced with arsenite. This polypeptide could be visualized on sodium dodecyl sulfate polyacrylamide gels stained with Coomassie blue. It was observed both in the membrane and cytosol fractions but not among the periplasmic proteins present in osmotic shock fluid. Minicells isolated from strain JR410(pUM1) incorporated [35S]methionine into an inducible 64,000 dalton polypeptide, as demonstrated on autoradiographs of electrophoresed [35S]-labeled minicell lysates, confirming that this polypeptide is a plasmid gene product. A 4.3 kilobase HindIII fragment of pUM1 was subcloned into the HindIII site of pBR322, producing recombinant plasmid pUM3. This plasmid conferred constitutive resistance ot arsenite and arsenate. Extensive synthesis of two polypeptides of 64,000 and 16,000 daltons was observed both in Coomassie stained gels of whole cells and autoradiographs of gels of [35S]methionine-labeled minicells. Synthesis of both polypeptides was constitutive.

Arsenates↗
PubMed1982

Energetics of plasmid-mediated arsenate resistance in Escherichia coli.

Plasmid R773, which codes for resistances to arsenate, arsenite, and antimony, was introduced into Escherichia coli strain AN120, a mutant deficient in the H+-translocating ATPase of oxidative phosphorylation. Cultures depleted of endogenous energy reserves were loaded with 74AsO3-4, and arsenate efflux was measured after dilution into medium containing various energy sources and inhibitors. Rapid extrusion of arsenate occurred when glucose was added. Arsenate was extruded both against and down a concentration gradient. In this strain glucose allows formation of both ATP via substrate-level phosphorylation and an electrochemical proton gradient (or protonmotive force) via oxidation of the products of glycolysis. When oxidation was inhibited by cyanide, glucose metabolism still produced arsenate efflux. Energy sources such as succinate, which supplies a protonmotive force but not ATP, did not result in efflux. Measurement of intracellular ATP concentration under each set of conditions demonstrated a direct correlation between the rate of efflux and ATP levels. Osmotically shocked cells lost the ability to extrude arsenate; however, no arsenate-binding activity was detected in osmotic shock fluid from induced cells. These results suggest that the arsenate efflux system is coupled to cellular ATP rather than an electrochemical proton gradient, possibly by an arsenate-translocating ATPase.

Arsenates↗