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T L Stull

Publications and source records attributed to T L Stull.

At least 55 records · Page 3Linked to original sources
PubMed1990

In-vitro activities of trospectomycin, cefpodoxime, and second-generation cephalosporins against Haemophilus influenzae type b.

The in-vitro activities of trospectomycin, cefpodoxime, cefamandole, cefonicid, and cefuroxime against beta-lactamase-negative and -positive invasive clinical isolates of Haemophilus influenzae type b were determined by the agar dilution method. Trospectomycin and cefpodoxime inhibited 90% of the strains at concentrations of 5 and 0.06 mg/l, respectively, and no differences between the susceptibilities of the beta-lactamase-negative and -positive strains were noted. The activity of cefpodoxime was minimally affected by increased inoculum size, but significant inoculum effects were noted with cefamandole, cefonicid, and cefuroxime with beta-lactamase positive strains.

Anti-Bacterial Agents↗
PubMed1990

Problems with current recommendations for susceptibility testing of Haemophilus influenzae.

We compared results of MIC and disk susceptibility tests on Haemophilus test medium (HTM) and those on comparative media. Ampicillin MICs were determined with seven ampicillin-resistant, non-beta-lactamase-producing (AmprNBLP) isolates by using HTM and supplemented brain heart infusion (sBHI) agar. Ampicillin and amoxicillin-clavulanate disk tests with 16 AmprNBLP strains, 18 ampicillin-susceptible (Amps) isolates, and 17 ampicillin-resistant, beta-lactamase-producing (AmprBLP) strains were performed by using five media: laboratory-prepared HTM (PHTM), commercial HTM (CHTM), sBHI, enriched chocolate agar, and Mueller-Hinton chocolate agar. We observed that five of seven and three of seven AmprNBLP strains were misclassified as susceptible with PHTM (MIC, less than 2 micrograms/ml) with inocula of 10(3) and 10(5) CFU, respectively, but were resistant with sBHI (MIC, greater than or equal to 2 micrograms/ml). Whereas Mueller-Hinton chocolate agar and enriched chocolate agar plates supported the growth of all 51 strains by the disk tests, 37% (19 of 51) and 8% (4 of 51) of strains did not grow on PHTM and CHTM, respectively. Lack of growth on PHTM was observed for all three phenotypes; 7 of 18 Amps, 4 of 17 AmprBLP, and 8 of 16 AmprNBLP strains did not grow. The four strains that did not grow on CHTM were all AmprNBLP isolates. Zone sizes were significantly larger on PHTM than on the other media. Of the strains that were evaluable by the new National Committee for Clinical Laboratory Standards guidelines with either PHTM or CHTM, all Amps strains were classified as susceptible. Among the AmprBLP strains, CHTM correctly identified all as resistant, whereas PHTM detected two isolates to be intermediate. Among the AmprNBLP strains, CHTM and PHTM misclassified four (33%) and five (62%) isolates, respectively, as susceptible; an additional isolate was identified as intermediate on both media. We conclude that there is strain-dependent growth on HTM, that adoption of this medium for routine Haemophilus susceptibility testing is problematic due to this growth variability, and that detection of AmprNBLP isolates would be unreliable.

Ampicillin Resistance↗
PubMed1990

Haemocin, the bacteriocin produced by Haemophilus influenzae: species distribution and role in colonization.

Four hundred thirty-eight strains of Haemophilus influenzae were examined for production of and sensitivity to haemocin, a bacteriocin produced by some members of this species. Whereas 199 of 212 (94%) type b isolates produced haemocin, 131 of 134 (98%) nontypeable and 91 of 92 (99%) encapsulated non-type b isolates were sensitive to haemocin. Among strains previously genetically characterized by multilocus enzyme electrophoresis, haemocin production was detected in type b isolates belonging to 25 of 29 (86%) clonally distinct electrophoretic types. None of 60 clonally distinct nontypeable strains produced this substance, and all were sensitive to it in vitro. The genes encoding haemocin production were transformed independently of the genes necessary for capsule expression from a prototypic type b strain to a nontypeable strain. After intranasal inoculation of infant rats with an equal mixture of a non-haemocin-producing strain and its haemocin-producing transformant, organisms capable of haemocin production predominated in both nasopharyngeal and blood cultures. These data demonstrate that haemocin production is strongly associated with type b encapsulated members of this species and suggest a mechanism by which haemocin might play a role in host nasopharyngeal colonization by this pathogen.

Animals↗
PubMed1989

DNA polymorphisms among Escherichia coli isolated from bacteriuric women.

Restriction fragment length polymorphism (RFLP) patterns, plasmid profiles, and antimicrobial susceptibility patterns of paired sequential Escherichia coli isolates from antibiotic-treated and untreated elderly women were analyzed. Isolates from 26 of 27 subjects who were treated successfully with antibiotics but became reinfected differed by RFLP analysis, whereas 10 of 12 subjects who failed treatment and 11 of 14 untreated subjects had paired isolates with identical RFLP banding patterns. Only 40 of the 53 pairs of isolates could be analyzed by plasmid profiles; 36 of these 40 were concordant with RFLP analysis. Antimicrobial susceptibility patterns showed poor concordance with RFLP analysis. This study demonstrates the utility of RFLP analysis and indicates that isolation of E. coli from elderly women after sterilization of the urinary tract usually results from introduction of a new strain; elderly women who fail antibiotic therapy or receive no therapy may remain persistently infected with the same E. coli strain.

Aged↗
PubMed1989

In vitro activities and targets of three cephem antibiotics against Haemophilus influenzae.

The antimicrobial activities of cefixime, cefpodoxime, and ceftibuten were determined with 18 ampicillin-susceptible (Amps), 13 ampicillin-resistant beta-lactamase-producing (AmprBLP), and 7 ampicillin-resistant non-beta-lactamase-producing (AmprNBLP) strains of Haemophilus influenzae. An effect of inoculum density on apparent MIC, the bactericidal activity of these agents, and the targets of the three cephems were determined. The MICs of cefixime, cefpodoxime, and ceftibuten for 90% of the Amps and AmprBLP isolates were 0.04, 0.08, and 0.08 microgram/ml, respectively. In contrast, the MICs for 90% of the AmprNBLP strains were 0.96, 1.92, and 7.68 micrograms/ml. No significant inoculum effect was observed for any group of strains comparing inocula of 10(3) and 10(5) CFU, whereas only the AmprNBLP isolates showed a marked effect at an inoculum of 10(6) CFU. Although bactericidal levels were achieved for the Amps and AmprBLP strains, tolerance to cefixime and ceftibuten was observed. The bactericidal activity for the AmprNBLP strains was limited, with cefixime showing the highest activity of the three cephems. Penicillin-binding proteins 2, 4, and 5 revealed high affinity, with 50% inhibitory concentration levels below the MIC for all three cephems, suggesting that these are important targets of these agents in H. influenzae. We conclude that the cephems are highly active in vitro against Amps and AmprBLP strains of H. influenzae, but less so against AmprNBLP isolates.

Ampicillin↗
PubMed1988

A broad-spectrum probe for molecular epidemiology of bacteria: ribosomal RNA.

We investigated the use of ribosomal RNA (rRNA) as a probe for molecular epidemiology of bacterial pathogens. The chromosomal DNA of Escherichia coli, Pseudomonas cepacia, and nontypable Haemophilus influenzae was digested with EcoRI. Agarose gel electrophoresis, Southern blotting, and hybridization by 32P-labeled rRNA revealed eight to 13 bands. The P. cepacia and H. influenzae banding patterns, observed by using an E. coli rRNA probe, were identical to those produced with homologous rRNA probes. Polymorphism of several hybridization bands distinguished all E. coli isolates, nine of 10 H. influenzae isolates, and seven of eight P. cepacia isolates. Two to four bands were common to all P. cepacia and E. coli isolates. The banding patterns of H. influenzae isolates cultured from the trachea and blood of an infant and from the mother's cervix were identical. These data demonstrate that this method is a widely applicable system for determining the molecular epidemiology of genetically diverse gram-negative organisms.

DNA Restriction Enzymes↗
PubMed1988

Iron acquisition by Haemophilus influenzae.

The mechanisms for acquisition of iron by Haemophilus influenzae and their role in pathogenesis are not known. Heme and nonheme sources of iron were evaluated for their effect on growth of type b and nontypable strains of H. influenzae in an iron-restricted, defined medium. All 13 strains acquired iron from heme, hemoglobin, hemoglobin-haptoglobin, and heme-hemopexin. Among nonheme sources of protein-bound iron, growth of H. influenzae was enhanced by partially saturated human transferrin but not by lactoferrin or ferritin. Purified ferrienterochelin and ferridesferrioxamine failed to provide iron to H. influenzae, and the supernatants of H. influenzae E1a grown in iron-restricted medium failed to enhance iron-restricted growth of siderophore-dependent strains of Escherichia coli, Salmonella typhimurium, and Arthrobacter terregens. Marked alterations in the profile of outer membrane proteins of H. influenzae were observed when the level of free iron was varied between 1 microM and 1 mM. Catechols were not detected in the supernatants of strain E1a; however, iron-related hydroxamate production was detected by two biochemical assays. We conclude that the sources of iron for H. influenzae are diverse. The significance of hydroxamate production and iron-related outer membrane proteins to H. influenzae iron acquisition is not yet clear.

Bacterial Outer Membrane Proteins↗
PubMed1988

Human antibody response to outer membrane proteins and fimbriae of Haemophilus influenzae type b.

We investigated the prevalence of antibodies in childrens' sera directed against outer membrane proteins (OMP) and fimbriae of Haemophilus influenzae type b. Invasive isolates of H. influenzae type b were enriched for fimbriae production; OMP and fimbriae were resolved by SDS-PAGE. After blotting to nitrocellulose, the proteins were incubated with homologous patient sera or with sera from healthy children. IgG antibodies bound to OMP were detected by immunoperoxidase staining. Immunoblotting was also performed using purified, nondenatured fimbriae as antigen. Nine of the 10 patients studied had antibodies in the acute serum directed against one or more of the OMP. Neither the acute nor the convalescent serum of the remaining patient contained antibodies against OMP. Antibodies against a greater number of OMP were present in the convalescent serum, in comparison to the acute serum, in 4 of the 10 patients. Five of 10 patients had antibodies against the purified fimbriae of an unrelated invasive isolate in either the acute or the convalescent serum. Acute sera from patients more frequently contained antibodies directed against OMP 60K (p less than or equal to 0.01) and OMP 51K (p less than or equal to 0.003) compared with the sera of healthy controls. In contrast, the sera of healthy children more frequently contained antibodies directed against OMP 40K (p less than or equal to 0.04). Sera from both patients and controls contained antibodies against commensal Haemophilus. We conclude that although antibodies against OMP are commonly present in healthy children, antibodies against certain OMP may be markers for susceptibility or protection.

Antibodies, Bacterial↗
PubMed1987

Protein sources of heme for Haemophilus influenzae.

Although Haemophilus influenzae requires heme for growth, the source of heme during invasive infections is not known. We compared heme, lactoperoxidase, catalase, cytochrome c, myoglobin, and hemoglobin as sources of heme for growth in defined media. The minimum concentration of heme permitting unrestricted growth of strain E1a, an H. influenzae type b isolate from cerebrospinal fluid, was 0.02 micrograms/ml. Using molar equivalents of heme as lactoperoxidase, catalase, cytochrome c, myoglobin, and hemoglobin, we determined that myoglobin and hemoglobin permitted unrestricted growth at this concentration. To determine the ability of host defenses to sequester heme from H. influenzae, we used affinity chromatography to purify human haptoglobin and hemopexin, serum proteins which bind hemoglobin and heme. Plate assays revealed that 12 strains of H. influenzae acquired heme from hemoglobin, hemoglobin-haptoglobin, heme-hemopexin, and heme-albumin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins of strain E1a grown in heme-replete and heme-restricted conditions revealed a heme-repressible outer membrane protein with an apparent molecular mass of 38 kilodaltons. These results demonstrated that, unlike Escherichia coli, H. influenzae may acquire heme from hemoglobin-haptoglobin. H. influenzae also may acquire heme from hemopexin and albumin, which have not been previously investigated. The role of outer membrane proteins in the acquisition of heme is not yet clear.

Bacterial Outer Membrane Proteins↗
PubMed1986

Failure to detect ampicillin-resistant, non-beta-lactamase-producing Haemophilus influenzae by standard disk susceptibility testing.

We questioned whether the apparent rarity of ampicillin-resistant, non-beta-lactamase-producing (NBLP) Haemophilus influenzae was due to failure of detection. We identified ampicillin-resistant and ampicillin-susceptible H. influenzae by the agar dilution technique, using 10(5)- and 10(3)-CFU inocula. We compared the disk susceptibility of 18 ampicillin-resistant NBLP strains, 13 ampicillin-resistant, beta-lactamase-producing strains, and 10 ampicillin-susceptible strains by using standard 10- and 2-micrograms ampicillin disks on two different media. We also investigated the possibility that disks containing 10 micrograms of clavulanic acid and 2 micrograms of ampicillin could be used to distinguish between the two ampicillin-resistant populations. Using the disk containing 2 micrograms of ampicillin, we correctly differentiated all the ampicillin-resistant isolates from the ampicillin-susceptible isolates on both media (a zone diameter of less than or equal to 15 mm was considered resistant). In contrast, the 10-micrograms ampicillin disk failed to detect 44% (8 of 18) of the ampicillin-resistant NBLP strains (a zone diameter of less than or equal to 19 mm was considered resistant). The MIC of ampicillin with the 10(3)-CFU inoculum correlated better with zone diameters than with the 10(5)-CFU inoculum. A difference in zone diameters of greater than or equal to 8 mm between the disk containing 10 micrograms of clavulanic acid and 2 micrograms of ampicillin and the disk containing only 2 micrograms of ampicillin correctly identified all beta-lactamase-producing strains. We conclude that the 2-micrograms ampicillin disk tests more readily identify ampicillin resistance in H. influenzae than do the 10-micrograms ampicillin disk tests. Future investigation should determine whether this in vitro resistance correlates with clinical treatment failures.

Ampicillin↗
PubMed1985

A comparison of techniques for isolation of the outer membrane proteins of Haemophilus influenzae type b.

We compared several rapid techniques used for extraction of outer membrane proteins from gram-negative enteric bacteria to Haemophilus influenzae type b. After lysis of cells with a French press, the inner and outer membranes were separated by isopycnic centrifugation. Each membrane was identified by density, morphology, enzymatic activity, and susceptibility to solid-phase iodination of intact cells. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we identified 10 polypeptides which were enriched in the outer membrane band compared to the inner membrane band. Using these proteins, we compared the polypeptide pattern of outer membranes with that obtained by (1) selective solubilization with sodium dodecyl-beta-D-maltoside, octyl-beta-D-glucopyranoside, Triton X-100, sodium, or cholamidopropyl dimethylaminopropanesulfonate; (2) extraction with chaotropic agents and heat; and (3) differential centrifugation of vesicles shed during transition from log growth phase to stationary growth phase. There were definable differences between the polypeptide pattern of membranes obtained with each rapid technique compared to the polypeptide pattern of isolated outer membranes. The polypeptide pattern of lithium extracts and the Triton X-100 insoluble fractions of total membranes most closely approximated the polypeptide pattern of isopycnically isolated outer membranes. Depending on the outer membrane protein sought, one of these rapid techniques can be utilized when a rapid method of outer membrane protein isolation is required.

Bacterial Outer Membrane Proteins↗
PubMed1985

A permeability barrier as a mechanism of chloramphenicol resistance in Haemophilus influenzae.

Chloramphenicol resistance in Haemophilus influenzae occurs most frequently via plasmid-mediated chloramphenicol acetyltransferase production. We studied four strains with high-level chloramphenicol resistance (MIC greater than 20 micrograms/ml) which did not have detectable chloramphenicol acetyltransferase activity. The chloramphenicol resistance determinant was transformed into a chloramphenicol-susceptible laboratory H. influenzae strain from each of the four wild-type strains, enabling isogenic comparisons. By thin-layer chromatography and a bioassay, there was no evidence of non-chloramphenicol acetyltransferase modification of chloramphenicol. In vitro protein synthesis in the presence of chloramphenicol was equivalently inhibited in the chloramphenicol-resistant transformants and in the susceptible recipient. Chloramphenicol uptake by these strains during logarithmic growth was compared by high-pressure liquid chromatographic quantitation; at chloramphenicol concentrations of 5, 10, and 20 micrograms/ml the four transformants showed a decreased rate of uptake of chloramphenicol compared with the isogenic chloramphenicol-susceptible recipient. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of outer membrane proteins revealed a markedly diminished 40-kilodalton protein in the resistant transformants. We propose that the mechanism of chloramphenicol resistance in these strains is a relative permeability barrier due to the loss of an outer membrane protein.

Acetyltransferases↗
PubMed1984

Human serum bactericidal activity against Haemophilus influenzae type b.

We examined bactericidal and opsonizing activity of pooled adult 'immune' serum against Haemophilus influenzae type b with and without the addition of phagocytes. Four type b strains from cerebrospinal fluid (CSF) and three such strains from the nasopharynx (NP) of healthy children were examined. Duplicate reaction mixtures contained organisms in exponential (E) or stationary phase (S) of growth, serum, a complement source (human agammaglobulinaemic serum), and culture medium (bactericidal assay); separate assays contained the above components and polymorphonuclear leucocytes (opsonization system). A decrease in bacterial density of greater than or equal to 1 log10 unit was considered significant. All four S-CSF strains, three of four E-CSF strains and one of three S-NP strains were sensitive to the bactericidal activity of pooled serum. The other E-CSF strain, two S-NP strains and all three E-NP strains were resistant to the bactericidal activity of pooled serum. Two of three E-NP strains were opsonized by pooled serum; the other strains resistant to the bactericidal activity of pooled serum were also resistant to opsonization. Bactericidal and opsonizing activity of serum from an immunized adult was greater than or equal to that of pooled serum against each strain. Assuming normal adults are immune to invasive H. influenzae type b infection, an experimental test reflecting this immunity is the bactericidal activity against CSF isolates tested in stationary phase. We conclude that protection against invasive disease due to H. influenzae type b appears more complex than the presence of bactericidal and opsonizing activity in serum.

Adult↗
PubMed1984

Characterization of non-beta-lactamase-mediated ampicillin resistance in Haemophilus influenzae.

Ampicillin resistance in Haemophilus influenzae is most often due to the plasmid-mediated production of TEM beta-lactamase. We studied four strains with high-level ampicillin resistance (MIC of 32 micrograms/ml with an inoculum of 10(5) CFU on solid media) which did not produce detectable beta-lactamase activity with two different detection methods. Two of the four strains contained extrachromosomal DNA by agarose gel electrophoresis. Conjugation failed to transfer ampicillin resistance; in contrast, transformation yielded ampicillin-resistant transformants in three of the four strains. These transformants did not contain detectable extrachromosomal DNA. In addition, mobilization of the resistance determinant by transformation to, or conjugation with, recombination-deficient strains was unsuccessful. DNA-DNA hybridization experiments revealed no homology of the DNA of these strains with two R plasmids (one coding for ampicillin resistance, the other for chloramphenicol and tetracycline resistance). We conclude that the genetic basis of the non-beta-lactamase ampicillin resistance in these strains appears to be chromosomally mediated. We investigated the mechanism of resistance in these strains. Enzymatic modification of penicillin was not detected by autoradiography of a thin-layer chromatogram of cell sonic extracts of three ampicillin-resistant transformant strains incubated with [14C]penicillin. To assess changes in permeability of the cell envelope, a plasmid coding for beta-lactamase was conjugated into these strains, and the hydrolysis of penicillin by intact cells and cell sonic extracts was compared. Only one of three transformant strains had significantly diminished permeability. Outer membrane proteins of these strains analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed apparent differences in comparison with the isogenic ampicillin-susceptible recipient strain. Autofluorography of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Sarkosyl-solubilized crude membrane (the putative inner membranes) from these ampicillin-resistant transformant strains incubated with [3H]penicillin compared with the isogenic ampicillin-susceptible recipient strain revealed reduced binding to PBP 3 and 6, 3 and 4, or 4. In addition, affinity binding studies revealed decreased affinity of PBP 4 for ampicillin of all four transformants tested. We conclude that the major mechanism of resistance in these strains is altered penicillin-binding proteins; however, other mechanisms, including permeability, may also play a role.

Ampicillin↗