RESUMEN
Sonication technology has recently been touted to decrease composite viscosity during delivery and may allow better cavity preparation adaptation and minimize voids. The purpose of this investigation was to evaluate the difference between conventional, hand-placed, incremental application of a standard hybrid resin-based composite (RBC) and sonicated application of a bulk-fill RBC in box-type and cylindrical cavity preparations. Experimental restorations were fabricated using molds of box-type or cylindrical preparations. For bulk-filled specimens, a single compule of bulk-fill composite was dispensed with a sonic handpiece. The conventional hybrid material was placed in 3 increments (2 mm, 2 mm, and 1 mm). Microfocus x-ray computed tomography was used to analyze voids for percentage and total volume porosity as well as number of actual pores. An analysis of variance indicated that RBC restorations that were applied to cylindrical cavities using a sonicated bulk-filled application method exhibited significantly less porosity (1.42%; P < 0.001) than incrementally placed cylindrical restorations (2.87%); sonicated bulk-filled, cube-shaped restorations (3.12%); and incrementally placed cube-shaped restorations (5.16%). When the groups were subcategorized into the specific characteristics of shape (cube vs cylinder) and application method (bulk vs incremental), the cylindrical group, which included both bulk-filled and incrementally placed specimens, demonstrated significantly less porosity (2.00%; P < 0.001) than other groups. Restorations that were incrementally placed into cube-shaped cavities produced the largest amount of porosity.
Asunto(s)
Resinas Compuestas/uso terapéutico , Restauración Dental Permanente/métodos , Microtomografía por Rayos X , Resinas Compuestas/administración & dosificación , Preparación de la Cavidad Dental/métodos , Humanos , Porosidad , Microtomografía por Rayos X/métodosRESUMEN
We are investigating a nontypeable Haemophilus influenzae (NTHI) strain, R2866, isolated from a child with meningitis. R2866 is unusually resistant to killing by normal human serum. The serum 50% inhibitory concentration (IC50) for this strain is 18%, approaching that of encapsulated H. influenzae. R3392 is a derivative of R2866 that was found to have increased sensitivity to human serum (IC50, 1.5%). Analysis of tetrameric repeat regions within lipooligosaccharide (LOS) biosynthetic genes in both strains indicated that the glycosyltransferase gene lgtC was out of frame ("off") in most colonies of R3392 but in frame with its start codon ("on") in most colonies of the parent. We sought antigenic and biochemical evidence for modification of the LOS structure. In a whole-cell enzyme-linked immunosorbent assay, strain R3392 displayed reduced binding of the Galalpha1,4Gal-specific monoclonal antibody 4C4. Mass spectrometry analysis of LOS from strain R2866 indicated that the primary oligosaccharide glycoform contained four heptose and four hexose residues, while that of R3392 contained four heptose and three hexose residues. We conclude that the R2866 lgtC gene encodes a galactosyltransferase involved in synthesis of the 4C4 epitope, as in other strains, and that expression of lgtC is associated with the high-level serum resistance that has been observed for this strain. This is the first description of the genetic basis of high-level serum resistance in NTHI, as well as the first description of LOS composition in an NTHI strain for which the complete genome sequence has been determined.
Asunto(s)
Proteínas Bacterianas/fisiología , Actividad Bactericida de la Sangre , Galactosiltransferasas/fisiología , Infecciones por Haemophilus/enzimología , Haemophilus influenzae/enzimología , Haemophilus influenzae/inmunología , Hexosiltransferasas/fisiología , Animales , Animales Recién Nacidos , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Galactosiltransferasas/biosíntesis , Galactosiltransferasas/sangre , Infecciones por Haemophilus/sangre , Infecciones por Haemophilus/inmunología , Haemophilus influenzae/genética , Hexosiltransferasas/genética , Humanos , Inmunidad Innata , Concentración 50 Inhibidora , Lipopolisacáridos/sangre , RatasRESUMEN
To gain insight into the role of luxSHi in disease pathogenesis, we inactivated that gene in several non-typeable Haemophilus influenzae isolates with an antibiotic resistance cassette. Gene inactivation was confirmed by PCR and by Southern blot analysis in each strain. Culture filtrates from luxSHi mutants contained a decreased amount of autoinducer-2 (AI-2) activity in comparison to the wild-type isolates using the Vibrio harveyi BB170 bioassay. Culture filtrates from Escherichia coli strain DH5alpha expressing a cloned luxSHi contained 350-fold more AI-2 activity per cell than E. coli DH5alpha containing the vector alone. The growth rate in several liquid media, and the cell density after overnight growth were not significantly different between the parents and the luxSHi mutants. Two clinical H. influenzae and their luxSHi mutants produced an identical biofilm in a flow system. Invasion of human cells by the luxSHi mutants, in comparison to the wild-type parents was strain-dependent, and cell type-dependent, but the luxSHi mutants tended to be more invasive. The luxSHi mutant of an otitis media isolate, strain R3157 appeared more virulent in the chinchilla model of otitis media: there were more bacteria in the middle ear, a greater inflammatory response and more goblet cell hyperplasia 10 days after the inoculation. We conclude that the H. influenzae homologue of luxS modulates certain virulence traits.
Asunto(s)
Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , Infecciones por Haemophilus/microbiología , Haemophilus influenzae/patogenicidad , Animales , Liasas de Carbono-Azufre , Línea Celular Tumoral , Chinchilla , ADN Bacteriano/química , ADN Bacteriano/genética , Femenino , Haemophilus influenzae/genética , Haemophilus influenzae/crecimiento & desarrollo , Haemophilus influenzae/ultraestructura , Homoserina/análogos & derivados , Homoserina/genética , Homoserina/inmunología , Humanos , Lactonas/inmunología , Microscopía Electrónica de Rastreo , Mutagénesis Insercional , Otitis Media/microbiología , VirulenciaRESUMEN
Mutants in deoxyadenosine methyltransferase (dam) from many Gram-negative pathogens suggest multiple roles for Dam methylase: directing post-replicative DNA mismatch repair to the correct strand, guiding the temporal control of DNA replication and regulating the expression of multiple genes (including virulence factors) by differential promoter methylation. Dam methylase (HI0209) in strain Rd KW20 was inactivated in Haemophilus influenzae strains Rd KW20, Strain 12 and INT-1; restriction with Dam methylation-sensitive enzymes DpnI and DpnII confirmed the absence of Dam methylation, which was restored by complementation with a single copy of dam ectopically expressed in cis. Despite the lack of increased mutation frequency, the dam mutants had a 2-aminopurine-susceptible phenotype that could be suppressed by secondary mutations in mutS, suggesting a role for Dam in H. influenzae DNA mismatch repair. Invasion of human brain microvascular endothelial cells (HBMECs) and human respiratory epithelial cells (NCI-H292) by the dam mutants was significantly attenuated in all strains, suggesting the absence of a Dam-regulated event necessary for uptake or invasion of host cells. Intracellular replication was inhibited only in the Strain 12 dam mutant, whereas in the infant rat model of infection, the INT-1 dam mutant was less virulent. Dam activity appears to be necessary for both in vitro and in vivo virulence in a strain-dependent fashion and may function as a regulator of gene expression including virulence factors.
Asunto(s)
Haemophilus influenzae/enzimología , Haemophilus influenzae/patogenicidad , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/genética , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismo , 2-Aminopurina/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/fisiología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Línea Celular , Citoplasma/microbiología , Reparación del ADN , ADN Bacteriano/aislamiento & purificación , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/microbiología , Células Epiteliales/microbiología , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Prueba de Complementación Genética , Infecciones por Haemophilus/microbiología , Humanos , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN , Mutagénesis Insercional , Ratas , Virulencia/genéticaRESUMEN
BACKGROUND: Certain strains of an obligate parasite of the human upper respiratory tract, nontypeable Haemophilus influenzae (NTHi), can cause invasive diseases such as septicemia and meningitis, as well as chronic mucosal infections such as otitis media. To do this, the organism must invade and survive within both epithelial and endothelial cells. We have identified a facilitator of NTHi survival inside human cells, virulence-associated protein D (vapDHi, encoded by gene HI0450). Both vapDHi and a flanking gene, HI0451, exhibit the genetic and physical characteristics of a toxin/antitoxin (TA) locus, with VapDHi serving as the toxin moiety and HI0451 as the antitoxin. We propose the name VapXHi for the HI0451 antitoxin protein. Originally identified on plasmids, TA loci have been found on the chromosomes of a number of bacterial pathogens, and have been implicated in the control of translation during stressful conditions. Translation arrest would enhance survival within human cells and facilitate persistent or chronic mucosal infections. RESULTS: Isogenic mutants in vapDHi were attenuated for survival inside human respiratory epithelial cells (NCI-H292) and human brain microvascular endothelial cells (HBMEC), the in vitro models of mucosal infection and the blood-brain barrier, respectively. Transcomplementation with a vapDHi allele restored wild-type NTHi survival within both cell lines. A PCR survey of 59 H. influenzae strains isolated from various anatomical sites determined the presence of a vapDHiallele in 100% of strains. Two isoforms of the gene were identified in this population; one that was 91 residues in length, and another that was truncated to 45 amino acids due to an in-frame deletion. The truncated allele failed to transcomplement the NTHi vapDHi survival defect in HBMEC. Subunits of full-length VapDHi homodimerized, but subunits of the truncated protein did not. However, truncated protein subunits did interact with full-length subunits, and this interaction resulted in a dominant-negative phenotype. Although Escherichia coli does not contain a homologue of either vapDHi or vapXHi, overexpression of the VapDHi toxin in trans resulted in E. coli cell growth arrest. This arrest could be rescued by providing the VapXHi antitoxin on a compatible plasmid. CONCLUSION: We conclude that vapDHi and vapXHi may constitute a H. influenzae TA locus that functions to enhance NTHi survival within human epithelial and endothelial cells.
Asunto(s)
Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Haemophilus influenzae/genética , Glicoproteínas de Membrana/genética , Alelos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/fisiología , Toxinas Bacterianas/antagonistas & inhibidores , Barrera Hematoencefálica , Encéfalo/irrigación sanguínea , Células Cultivadas , Mapeo Cromosómico , Dimerización , Endotelio Vascular/citología , Endotelio Vascular/microbiología , Escherichia coli/genética , Prueba de Complementación Genética , Haemophilus influenzae/patogenicidad , Haemophilus influenzae/fisiología , Humanos , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/fisiología , Mutación , Proteínas Recombinantes/biosíntesis , Mucosa Respiratoria/citología , Mucosa Respiratoria/microbiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Virulencia/genéticaRESUMEN
A chemically defined medium that supports the growth of both encapsulated and nontypeable Haemophilus influenzae strains in broth to densities that are >/= 10(9) CFU/ml or on agar plates is described. The mean generation time of a panel of clinical isolates was comparable to that in rich, chemically undefined media (brain-heart infusion broth supplemented with heme and beta-NAD).