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1.
Sci Rep ; 9(1): 19732, 2019 12 24.
Artículo en Inglés | MEDLINE | ID: mdl-31874981

RESUMEN

Human microbiomes are predicted to assemble in a reproducible and ordered manner yet there is limited knowledge on the development of the complex bacterial communities that constitute the oral microbiome. The oral microbiome plays major roles in many oral diseases including early childhood caries (ECC), which afflicts up to 70% of children in some countries. Saliva contains oral bacteria that are indicative of the whole oral microbiome and may have the ability to reflect the dysbiosis in supragingival plaque communities that initiates the clinical manifestations of ECC. The aim of this study was to determine the assembly of the oral microbiome during the first four years of life and compare it with the clinical development of ECC. The oral microbiomes of 134 children enrolled in a birth cohort study were determined at six ages between two months and four years-of-age and their mother's oral microbiome was determined at a single time point. We identified and quantified 356 operational taxonomic units (OTUs) of bacteria in saliva by sequencing the V4 region of the bacterial 16S RNA genes. Bacterial alpha diversity increased from a mean of 31 OTUs in the saliva of infants at 1.9 months-of-age to 84 OTUs at 39 months-of-age. The oral microbiome showed a distinct shift in composition as the children matured. The microbiome data were compared with the clinical development of ECC in the cohort at 39, 48, and 60 months-of-age as determined by ICDAS-II assessment. Streptococcus mutans was the most discriminatory oral bacterial species between health and current disease, with an increased abundance in disease. Overall our study demonstrates an ordered temporal development of the oral microbiome, describes a limited core oral microbiome and indicates that saliva testing of infants may help predict ECC risk.


Asunto(s)
Caries Dental/microbiología , Microbiota , Boca/microbiología , Saliva/microbiología , Streptococcus mutans , Preescolar , Caries Dental/genética , Femenino , Humanos , Lactante , Estudios Longitudinales , Masculino , Streptococcus mutans/clasificación , Streptococcus mutans/genética , Streptococcus mutans/crecimiento & desarrollo
2.
FEMS Immunol Med Microbiol ; 31(1): 73-81, 2001 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-11476985

RESUMEN

The gene organization in the lipopolysaccharide biosynthetic (rfb) locus was analyzed in seven Leptospira interrogans serovars within serogroup Icterohemorrhagiae, seven non-Icterohemorrhagiae serovars and one Leptospira borgpetersenii serovar. Two groups of loci were delineated based on DNA hybridization and sequence analysis. Group 1 contained the two Hardjo subtypes, Hardjoprajitno and Hardjobovis. Group 2 (containing Copenhageni, Pomona, Naam, Mwogolo, Smithi, Lai, Canicola, Autumnalis, Pyrogenes, Australis and Icterohemorrhagiae) differed from Group 1 in its organization upstream of orf11, where five ORFs (32, 33, 34, 35, 37) were identified that were not contained in the Group 1 loci. These ORFs encoded a putative epimerase (orf32), a glycosyltransferase (orf33), two integral membrane proteins (orfs 34 and 35), and a galactosyltransferase (orf37). Serovars Australis, Pomona and Autumnalis did not contain orf37. Serovar Bataviae was excluded from the grouping because of its unique genetic organization upstream of orf13. In the Group 2 loci, comparison of the genetic layout at the 5' end revealed differences which included mutations disrupting reading frames in either or both orf34 and orf35 and apparent allelic differences between orf33 homologs that may be sufficient to account for the genetic basis of serovar identity.


Asunto(s)
Genes Bacterianos , Leptospira/genética , Lipopolisacáridos/biosíntesis , Alelos , Proteínas Bacterianas/biosíntesis , Galactosiltransferasas/genética , Glicosiltransferasas/genética , Hexosiltransferasas/genética , Leptospira/enzimología , Leptospira interrogans/enzimología , Leptospira interrogans/genética , Datos de Secuencia Molecular , Antígenos O/biosíntesis , Sistemas de Lectura Abierta , Polimorfismo de Longitud del Fragmento de Restricción , Racemasas y Epimerasas/genética , Análisis de Secuencia
3.
J Mol Microbiol Biotechnol ; 2(4): 375-80, 2000 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-11075908

RESUMEN

Lipopolysaccharide is the major surface antigen of Leptospira. Variation in LPS structure is the basis for the more than 200 serovars that have been identified. Despite the importance of this antigen in immunity and diagnostics, there is relatively little known about the genetics and chemistry of leptospiral LPS, as compared to some members of the Enterobacteriaceae. The nucleotide sequence of the locus encoding enzymes for the biosynthesis of the O-antigen component of leptospiral LPS (rfb locus) has been determined for three serovars namely, L. interrogans serovar Pomona, L. interrogans serovar Hardjo subtype Hardjoprajitno and L. borgpetersenii serovar Hardjo subtype Hardjobovis. In the absence of data relating to the chemical structure or genetic tools to construct isogenic mutants in Leptospira, similarity analysis has been used to provide insight into the mechanisms by which the leptospiral O-antigen is assembled by comparison with characterized systems from other bacteria. In addition, comparison of the gene layout in each of the serovars provides an indication of the genetic basis for serovar diversity.


Asunto(s)
Leptospira/metabolismo , Lipopolisacáridos/biosíntesis , Enterobacteriaceae/inmunología , Enterobacteriaceae/metabolismo , Genes Bacterianos , Leptospira/genética , Leptospira/inmunología , Antígenos O/biosíntesis
4.
J Infect Dis ; 182(3): 941-4, 2000 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-10950794

RESUMEN

An investigation was done of the evidence for transmission of human immunodeficiency virus (HIV) from an HIV-positive man to several male and female sex contacts. Phylogenetic analysis of sequences from the gag and env genes showed a close relationship between the predominant virus strains from the source and 2 contacts. However, the likelihood that a female contact was infected by the source could not be determined, despite contact tracing indicating that this may have occurred. One male, shown by contact tracing and molecular evidence to have been infected by the source, subsequently transmitted HIV to his female sex partner. HIV sequence from a plasma sample used as a control in the phylogenetic analysis contained env and gag sequences that were closely related to those from the source. An epidemiologic link between these 2 individuals was subsequently confirmed by contact tracing.


Asunto(s)
Crimen , Infecciones por VIH/transmisión , VIH-1/genética , Adulto , Trazado de Contacto , Femenino , Productos del Gen env/genética , Productos del Gen gag/genética , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular
5.
Infect Immun ; 68(7): 3793-8, 2000 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-10858186

RESUMEN

Lipopolysaccharide (LPS) is a key antigen in immunity to leptospirosis. Its biosynthesis requires enzymes for the biosynthesis and polymerization of nucleotide sugars and the transport through and attachment to the bacterial membrane. The genes encoding these functions are commonly clustered into loci; for Leptospira borgpetersenii serovar Hardjo subtype Hardjobovis, this locus, named rfb, spans 36.7 kb and contains 31 open reading frames, of which 28 have been assigned putative functions on the basis of sequence similarity. Characterization of the function of these genes is hindered by the fact that it is not possible to construct isogenic mutant strains in Leptospira. We used two approaches to circumvent this problem. The first was to clone the entire locus into a heterologous host system and determine if a "recombinant" LPS or polysaccharide was synthesized in the new host. The second approach used putative functions to identify mutants in other bacterial species whose mutations might be complemented by genes on the leptospiral rfb locus. This approach was used to investigate the function of three genes in the leptospiral rfb locus and demonstrated function for orfH10, which complemented a wbpM strain of Pseudomonas aeruginosa, and orfH13, which complemented an rfbW strain of Vibrio cholerae. However, despite the similarity of OrfH11 to WecC, a wecC strain of E. coli was not complemented by orfH11. The predicted protein encoded by orfH8 is similar to GalE from a number of organisms. A Salmonella enterica serovar Typhimurium strain producing no GalE was used as a background in which orfH8 produced detectable GalE enzyme activity.


Asunto(s)
Proteínas Bacterianas/genética , Genes Bacterianos , Leptospira/genética , Animales , Proteínas Bacterianas/metabolismo , Carbohidrato Epimerasas/genética , Carbohidrato Epimerasas/metabolismo , Clonación Molecular , Escherichia coli/genética , Humanos , Leptospira/clasificación , Leptospira/metabolismo , Antígenos O/biosíntesis , Sistemas de Lectura Abierta , UDPglucosa 4-Epimerasa/genética , UDPglucosa 4-Epimerasa/metabolismo
6.
FEMS Microbiol Lett ; 177(2): 319-26, 1999 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-10474199

RESUMEN

Although Leptospira borgpetersenii subtype Hardjobovis and L. interrogans subtype Hardjoprajitno belong to different species, they are serologically indistinguishable and are therefore classified as serovar Hardjo. Since LPS is the major antigen involved in serological classification, this implies that the LPS of these subtypes is identical. Comparison of the LPS biosynthetic loci (rfb) of the subtypes revealed remarkable similarity, with 32 and 31 origins of replication (orfs) in the Hardjoprajitno and Hardjobovis rfb loci, respectively. The order and orientation of these orfs were identical with the exception of an additional orf in Hardjoprajitno between orfs 4 and 5 and intergenic sequences differing between the subtypes. The Hardjoprajitno rfb locus has been divided into four intercalated regions based on sequence similarity to other leptospiral rfb loci. orfJ1-orfJ14 as well as orfJ21-orfJ22 are more similar to regions of the rfb locus of L. borgpetersenii subtype Hardjobovis. orfJ15-orfJ20 as well as orfJ23-orfJ31 are almost identical to the corresponding orfs in L. interrogans serovar Copenhageni. We propose that the progenitor Hardjoprajitno strain, containing an rfb locus which closely resembled the Copenhageni locus, acquired orfs 1-14 and orfs 21-22 from subtype Hardjobovis resulting in two serologically indistinguishable subtypes of serovar Hardjo which in turn constituted the main bovine-adapted leptospiral serovar.


Asunto(s)
Proteínas Bacterianas/genética , Genes Bacterianos , Leptospira interrogans/clasificación , Leptospira interrogans/genética , Lipopolisacáridos/biosíntesis , Secuencia de Bases , Mapeo Cromosómico , Secuencia Conservada , Evolución Molecular , Datos de Secuencia Molecular , Familia de Multigenes , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Serotipificación
7.
Microb Pathog ; 27(2): 105-17, 1999 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-10458921

RESUMEN

Leptospiral LPS plays a critical role in immunity to leptospirosis and forms the basis for serological classification of Leptospira. However, neither the structure of leptospiral LPS nor the genetics of its biosynthesis have been elucidated. A probe derived from the rhamnose biosynthetic genes of L. interrogans serovar Copenhageni was used to identify the rfb locus of L. borgpetersenii serovar Hardjobovis. Chromosome walking and sequence analysis revealed an rfb locus spanning 36.7 kb, which consists of 31 ORFs transcribed in the same direction. Clusters of genes were identified which encode proteins related to enzymes involved in the biosynthesis of activated sugars including rhamnose. Additional ORFs in the locus encode glycosyltransferases for the assembly of the O-antigen subunit and integral membrane proteins for the transport of O-antigen subunits through the membrane and assembly into LPS.


Asunto(s)
Proteínas Bacterianas/genética , Leptospira/genética , Antígenos O/biosíntesis , Secuencia de Aminoácidos , Paseo de Cromosoma , ADN Bacteriano/análisis , ADN Bacteriano/genética , Glicosiltransferasas/genética , Leptospira/inmunología , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Ramnosa/biosíntesis , Ramnosa/genética , Alineación de Secuencia , Análisis de Secuencia de ADN
8.
J Invertebr Pathol ; 73(1): 59-73, 1999 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-9878291

RESUMEN

Two major clades, designated Groups I and II, of nucleopolyhedroviruses (NPVs) from lepidopteran hosts have been previously identified. To reveal more detailed relationships, a series of DNA polymerase nucleotide sequences from the taxa MbMNPV, SeMNPV, HzSNPV, HearNPV, SpltNPV, BusuNPV, and OranNPV have been determined using a polymerase chain reaction (PCR)-based approach. This technique enabled gene sequence determination using microliter samples of NPV-infected insect cadavers. Polyhedrin genes from HearNPV, OranNPV, SeMNPV, and SpltNPV were also isolated and sequenced using a similar approach. These sequences, together with other database entries, were aligned for positional homology of peptide sequences. Phylogenetic analysis of DNA polymerase molecular sequence alignments supports LdMNPV as a taxon of Group II and three Group II subclades, designated A, B, and C. Comparison of DNA polymerase trees with those estimated from occlusion protein molecular sequences enabled identification of three subclades of Group II. These are Subgroup II-A [MbMNPV, LeseNPV, MacoNPV, PaflNPV, SeMNPV, SpltNPV (India isolate), SfMNPV]; Subgroup II-B [SpliNPV, SpltNPV (Japan isolate), SpltNPV (Queensland isolate), and possibly HzSNPV, HearNPV, and ManeNPV], and Subgroup II-C [OpSNPV, OranNPV (S-type), BusuNPV (S-type), and possibly EcobNPV (S-type)]. Notably, all Subgroup II-A taxa are from noctuid hosts. Correlations of virus and host evolution within Group II taxa are discussed. The methods and data developed in this study will allow rapid sequencing of NPV DNA polymerase genes.


Asunto(s)
ADN Polimerasa Dirigida por ADN/genética , Nucleopoliedrovirus/clasificación , Proteínas Virales/genética , Secuencia de Aminoácidos , Animales , Cartilla de ADN , ADN Polimerasa Dirigida por ADN/clasificación , Amplificación de Genes , Genes Virales , Insectos/virología , Datos de Secuencia Molecular , Nucleopoliedrovirus/genética , Proteínas de la Matriz de Cuerpos de Oclusión , Péptidos/química , Filogenia , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Proteínas Virales/clasificación , Proteínas Estructurales Virales
9.
Epidemiol Infect ; 121(2): 473-5, 1998 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-9825801

RESUMEN

A set of 723 diagnostic sera from human patients, submitted for the microscopic agglutination test (MAT) for antibodies to a group of 6 leptospiral serovars, was also tested by MAT for antibodies to the recently-discovered Leptospira fainei serovar hurstbridge. MAT titres of > or = 128 to serovar hurstbridge were detected in 13.4% of these sera, and titres of > or = 512 in 7.2%. In contrast, none of 62 sera obtained from a control population of laboratory staff gave titres of > or = 128. The difference between the number of titres of > or = 128 given by the two groups of sera was highly significant (P < 0.01). The titres observed may have been due to cross-reactions with other leptospiral serovars, but this could not be demonstrated. An alternative explanation is that serovar hurstbridge is present in the human population.


Asunto(s)
Anticuerpos Antibacterianos/inmunología , Leptospira/inmunología , Leptospirosis/transmisión , Pruebas de Aglutinación , Australia , Humanos , Leptospirosis/epidemiología , Pruebas Serológicas
10.
Int J Syst Bacteriol ; 48 Pt 3: 851-8, 1998 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-9734039

RESUMEN

Pathogenic leptospires can be causative agents of reproductive problems in pigs. Cultures of uteri and kidneys from two pigs herds in New South Wales and Victoria (Australia) yielded five strains identified as Leptospira on morphological and cultural grounds. Phenotypic characteristics (growth at 13 and 30 degrees C, growth in the presence of 8-azaguanine) were intermediate between those of pathogenic and saprophytic leptospires. No cross-agglutination was observed with reference antisera representing the 24 pathogenic serogroups and the main saprophytic ones. Antiserum against one of the strains did not agglutinate reference stains representative of any serogroup. This provided evidence of a new serovar, designated hurstbridge. Genomic characterization of the five strains was achieved using five molecular approaches. Mapped restriction site polymorphisms in the rrs (16S rRNA) gene were not related to those of any reference strains. Arbitrarily primed PCR fingerprints suggested clonality of the five strains. The strains all showed an identical and unique PFGE profile. PCR, using primers specific for the rrs gene of pathologic leptospires, amplified corresponding sequences from the strains. DNA-DNA hybridization (and reciprocal experiments) using the S1 nucleas/TCA method was performed between one of the strains and the reference strains of Leptospira species. The homology ranged from 0 to 36% (the latter being was Leptospira inadai) thus satisfying the criterion of a new species, Leptospira fainei (type strain BUT 6T). Phylogenetic analysis of 16S rRNA sequence showed that L. fainei and L. inadai formed a clade separate from the previously recognized 'saprophyte' and 'pathogen' clades.


Asunto(s)
Leptospira/aislamiento & purificación , Porcinos/microbiología , Animales , Secuencia de Bases , ADN Bacteriano/análisis , Electroforesis en Gel de Campo Pulsado , Leptospira/clasificación , Leptospira/genética , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Fenotipo , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética , Serotipificación
11.
J Bacteriol ; 179(4): 1262-7, 1997 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-9023210

RESUMEN

Immunity to leptospirosis is principally humorally mediated and involves opsonization of leptospires for phagocytosis by macrophages and neutrophils. The only protective antigen identified to date is the leptospiral lipopolysaccharide (LPS), which biochemically resembles typical gram-negative LPS but has greatly reduced endotoxic activity. Little is known about the structure of leptospiral LPS. A 2.1-kb EcoRI fragment from the chromosome of serovar Copenhageni was cloned in pUC18 in Escherichia coli, after which flanking regions were cloned from a genomic library constructed in bacteriophage lambda GEM12. Sequence analysis identified four open reading frames which showed similarity to the rfbC, rfbD, rfbB, and rfbA genes, transcribed in that order, which encode the four enzymes involved in the biosynthesis of dTDP-rhamnose for the assembly of LPS in Salmonella enterica, E. coli, and Shigella flexneri. An additional open reading frame downstream of the rfbCDBA locus showed similarity with the rhamnosyltransferase genes of Shigella and Yersinia enterocolitica but not Salmonella. Comparison of deduced amino acid sequences showed up to 85% similarity of the leptospiral proteins with those of other gram-negative bacteria. Polyacrylamide gel electrophoresis of recombinant clones identified the putative RfbCDBA proteins, while reverse transcriptase-mediated PCR analysis indicated that the rfbCDBA gene cluster was expressed in Leptospira. Moreover, it could restore normal LPS phenotype to a defined rfbB::Tn5 mutant of S. flexneri which was deficient in all four genes, thereby confirming the functional identification of a part of the leptospiral rfb locus.


Asunto(s)
Genes Bacterianos , Leptospira interrogans/genética , Lipopolisacáridos/biosíntesis , Azúcares de Nucleósido Difosfato/biosíntesis , Nucleótidos de Timina/biosíntesis , Transportadoras de Casetes de Unión a ATP/genética , Proteínas Bacterianas/genética , Carbohidrato Epimerasas/genética , Clonación Molecular , Escherichia coli/genética , Prueba de Complementación Genética , Hexosiltransferasas/genética , Hidroliasas/genética , Leptospira interrogans/metabolismo , Manosa-6-Fosfato Isomerasa/genética , Datos de Secuencia Molecular , Azúcares de Nucleósido Difosfato/metabolismo , Nucleotidiltransferasas/genética , ARN Bacteriano/genética , ARN Mensajero/genética , Shigella flexneri/genética , Nucleótidos de Timina/metabolismo
12.
Arch Virol ; 113(1-2): 17-34, 1990.
Artículo en Inglés | MEDLINE | ID: mdl-2167057

RESUMEN

A clone library of 11 of 15 BamHI fragments representing 81% of the 140 kilobase DNA genome of the prototype bovine encephalitis herpesvirus strain N569 (BEHV.N569) was constructed. The clones were used to verify the BamHI, BstEII, EcoRI, and HindIII genomic maps for BEHV.N569 published by Engels et al. [Virus Res 6: 57-73 (1986)] for the same virus although some amendments/variations to the BamHI map were found in that 3 previously unidentified restriction sites were identified. Restriction site maps for BglII and KpnI were also derived for BEHV.N569. Southern blot analysis using 32P-labelled BEHV DNA as probe indicated that bovine herpesvirus 1 (BHV1), buffalo herpesvirus 1 (BuHV1) and caprine herpesvirus 1 (CaHV1) were similar and that the similarity occurred throughout the entire length of the genomes; CaHV1 was more distantly related to the other 3 viruses. Because of the similarities BEHV.N569 and BHV1. Cooper cloned DNA fragments were used to construct BamHI, BglII, BstEII, EcoRI, KpnI, and HindIII restriction site maps for the genome of BuHV1 and BamHI, BglII, and KpnI maps for the genome of BHV1.V155, a genital strain.


Asunto(s)
Biblioteca de Genes , Genes Virales , Herpesviridae/genética , Herpesvirus Bovino 1/genética , Animales , Southern Blotting , Búfalos/microbiología , Bovinos , Línea Celular , Clonación Molecular , Enzimas de Restricción del ADN , ADN Viral , Cabras/microbiología , Herpesviridae/clasificación , Herpesvirus Bovino 1/clasificación , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico
14.
Avian Pathol ; 17(3): 559-70, 1988.
Artículo en Inglés | MEDLINE | ID: mdl-18766716

RESUMEN

DNA from 10 Mycoplasma gallisepticum strains and one strain each of M. synoviae and M. gallinarum were studied by restriction endo-nuclease DNA analysis using endonucleases Eco RI, HindIII, BglII, BamHI, KpnI, and XhoI. Digestion patterns of DNA in agarose gels allowed easy differentiation of M. gallisepticum strains from different sources, while patterns obtained from one strain at the 6th and 100th in vitro passage levels were identical. The F strain and a field derivative obtained from a poultry farm where F strain vaccine had been previously used had almost identical patterns. This technique should be useful for comparing and differentiating M. gallisepticum strains in epidemiological and other studies. Strain differences were also noted by DNA-DNA hybridisation using a probe containing mycoplasma ribosomal RNA genes.

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