RESUMEN
BACKGROUND: Characterizing microbial communities via next-generation sequencing is subject to a number of pitfalls involving sample processing. The observed community composition can be a severe distortion of the quantities of bacteria actually present in the microbiome, hampering analysis and threatening the validity of conclusions from metagenomic studies. We introduce an experimental protocol using mock communities for quantifying and characterizing bias introduced in the sample processing pipeline. We used 80 bacterial mock communities comprised of prescribed proportions of cells from seven vaginally-relevant bacterial strains to assess the bias introduced in the sample processing pipeline. We created two additional sets of 80 mock communities by mixing prescribed quantities of DNA and PCR product to quantify the relative contribution to bias of (1) DNA extraction, (2) PCR amplification, and (3) sequencing and taxonomic classification for particular choices of protocols for each step. We developed models to predict the "true" composition of environmental samples based on the observed proportions, and applied them to a set of clinical vaginal samples from a single subject during four visits. RESULTS: We observed that using different DNA extraction kits can produce dramatically different results but bias is introduced regardless of the choice of kit. We observed error rates from bias of over 85% in some samples, while technical variation was very low at less than 5% for most bacteria. The effects of DNA extraction and PCR amplification for our protocols were much larger than those due to sequencing and classification. The processing steps affected different bacteria in different ways, resulting in amplified and suppressed observed proportions of a community. When predictive models were applied to clinical samples from a subject, the predicted microbiome profiles were better reflections of the physiology and diagnosis of the subject at the visits than the observed community compositions. CONCLUSIONS: Bias in 16S studies due to DNA extraction and PCR amplification will continue to require attention despite further advances in sequencing technology. Analysis of mock communities can help assess bias and facilitate the interpretation of results from environmental samples.
Asunto(s)
Artefactos , Bacterias/genética , ADN Bacteriano/genética , Genes de ARNr , ARN Ribosómico 16S/genética , Manejo de Especímenes/normas , Bacterias/clasificación , Bacterias/aislamiento & purificación , Sesgo , ADN Bacteriano/aislamiento & purificación , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Humanos , Metagenómica/instrumentación , Metagenómica/métodos , Metagenómica/normas , Consorcios Microbianos/genética , Microbiota/genética , Modelos Biológicos , Filogenia , Reacción en Cadena de la Polimerasa/normas , ARN Ribosómico 16S/aislamiento & purificación , Vagina/microbiologíaRESUMEN
Bacterial vaginosis is the leading vaginal disorder but the transition from health to this dysbiotic condition remains poorly characterized. Our goal was to quantify the ability of BV-associated anaerobes to adhere to epithelial cells in the presence of lactobacilli. Gardnerella vaginalis outcompeted Lactobacillus crispatus and Lactobacillus iners actually enhanced its adherence.
Asunto(s)
Bacterias Anaerobias/fisiología , Adhesión Bacteriana , Células Epiteliales/microbiología , Gardnerella vaginalis/fisiología , Lactobacillus/fisiología , Vaginosis Bacteriana/microbiología , Femenino , HumanosRESUMEN
BACKGROUND: Bacteria of the genus Sneathia are emerging as potential pathogens of the female reproductive tract. Species of Sneathia, which were formerly grouped with Leptotrichia, can be part of the normal microbiota of the genitourinary tracts of men and women, but they are also associated with a variety of clinical conditions including bacterial vaginosis, preeclampsia, preterm labor, spontaneous abortion, post-partum bacteremia and other invasive infections. Sneathia species also exhibit a significant correlation with sexually transmitted diseases and cervical cancer. Because Sneathia species are fastidious and rarely cultured successfully in vitro; and the genomes of members of the genus had until now not been characterized, very little is known about the physiology or the virulence of these organisms. RESULTS: Here, we describe a novel species, Sneathia amnii sp. nov, which closely resembles bacteria previously designated "Leptotrichia amnionii". As part of the Vaginal Human Microbiome Project at VCU, a vaginal isolate of S. amnii sp. nov. was identified, successfully cultured and bacteriologically cloned. The biochemical characteristics and virulence properties of the organism were examined in vitro, and the genome of the organism was sequenced, annotated and analyzed. The analysis revealed a reduced circular genome of ~1.34 Mbp, containing ~1,282 protein-coding genes. Metabolic reconstruction of the bacterium reflected its biochemical phenotype, and several genes potentially associated with pathogenicity were identified. CONCLUSIONS: Bacteria with complex growth requirements frequently remain poorly characterized and, as a consequence, their roles in health and disease are unclear. Elucidation of the physiology and identification of genes putatively involved in the metabolism and virulence of S. amnii may lead to a better understanding of the role of this potential pathogen in bacterial vaginosis, preterm birth, and other issues associated with vaginal and reproductive health.
Asunto(s)
Genoma Bacteriano , Leptotrichia/genética , Análisis de Secuencia de ADN , Antibacterianos/farmacología , Femenino , Humanos , Leptotrichia/clasificación , Leptotrichia/efectos de los fármacos , Metagenoma , Pruebas de Sensibilidad Microbiana , Fenotipo , Filogenia , ARN Ribosómico 16S/genética , Vagina/microbiología , Virulencia/genéticaRESUMEN
Phage-mediated transfer of microbial genetic elements plays a crucial role in bacterial life style and evolution. In this study, we identify the RinA family of phage-encoded proteins as activators required for transcription of the late operon in a large group of temperate staphylococcal phages. RinA binds to a tightly regulated promoter region, situated upstream of the terS gene, that controls expression of the morphogenetic and lysis modules of the phage, activating their transcription. As expected, rinA deletion eliminated formation of functional phage particles and significantly decreased the transfer of phage and pathogenicity island encoded virulence factors. A genetic analysis of the late promoter region showed that a fragment of 272 bp contains both the promoter and the region necessary for activation by RinA. In addition, we demonstrated that RinA is the only phage-encoded protein required for the activation of this promoter region. This region was shown to be divergent among different phages. Consequently, phages with divergent promoter regions carried allelic variants of the RinA protein, which specifically recognize its own promoter sequence. Finally, most Gram-postive bacteria carry bacteriophages encoding RinA homologue proteins. Characterization of several of these proteins demonstrated that control by RinA of the phage-mediated packaging and transfer of virulence factor is a conserved mechanism regulating horizontal gene transfer.
Asunto(s)
Regulación Viral de la Expresión Génica , Transferencia de Gen Horizontal , Fagos de Staphylococcus/genética , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas Virales/metabolismo , Factores de Virulencia/genética , Ensamble de Virus/genética , Islas Genómicas , Bacterias Grampositivas/patogenicidad , Bacterias Grampositivas/virología , Lisogenia/genética , Operón , Regiones Promotoras Genéticas , Eliminación de Secuencia , Transactivadores/genética , Factores de Transcripción/genética , Activación Transcripcional , Proteínas Virales/genética , Virión/metabolismoRESUMEN
BACKGROUND: Worldwide, bacterial vaginosis (BV) is the most common vaginal disorder. It is associated with risk for preterm birth and HIV infection. The etiology of the condition has been debated for nearly half a century and the lack of knowledge about its cause and progression has stymied efforts to improve therapy and prevention. Gardnerella vaginalis was originally identified as the causative agent, but subsequent findings that it is commonly isolated from seemingly healthy women cast doubt on this claim. Recent studies shedding light on the virulence properties of G. vaginalis, however, have drawn the species back into the spotlight. RESULTS: In this study, we sequenced the genomes of a strain of G. vaginalis from a healthy woman, and one from a woman with bacterial vaginosis. Comparative analysis of the genomes revealed significant divergence and in vitro studies indicated disparities in the virulence potential of the two strains. The commensal isolate exhibited reduced cytotoxicity and yet the cytolysin proteins encoded by the two strains were nearly identical, differing at a single amino acid, and were transcribed at similar levels. The BV-associated strain encoded a different variant of a biofilm associated protein gene and demonstrated greater adherence, aggregation, and biofilm formation. Using filters with different pore sizes, we found that direct contact between the bacteria and epithelial cells is required for cytotoxicity. CONCLUSIONS: The results indicated that contact is required for cytotoxicity and suggested that reduced cytotoxicity in the commensal isolate could be due to impaired adherence. This study outlines two distinct genotypic variants of G. vaginalis, one apparently commensal and one pathogenic, and presents evidence for disparate virulence potentials.
Asunto(s)
Cuello del Útero/citología , Células Epiteliales/microbiología , Gardnerella vaginalis/genética , Gardnerella vaginalis/patogenicidad , Genoma Bacteriano/genética , Genómica , Antibacterianos/farmacología , Adhesión Bacteriana/genética , Proteínas Bacterianas/genética , Biopelículas , Farmacorresistencia Bacteriana/genética , Células Epiteliales/citología , Femenino , Gardnerella vaginalis/efectos de los fármacos , Gardnerella vaginalis/metabolismo , Humanos , Redes y Vías Metabólicas/genética , Análisis de Secuencia de ADN , Especificidad de la Especie , Factores de Virulencia/genéticaRESUMEN
The opportunistic pathogen Pseudomonas aeruginosa causes chronic biofilm infections in cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by overproduction of the exopolysaccharide alginate. Here we show that AlgK, a protein essential for production of high molecular weight alginate, is an outer membrane lipoprotein that contributes to the correct localization of the porin AlgE. Our 2.5 A structure shows AlgK is composed of 9.5 tetratricopeptide-like repeats, and three putative sites of protein-protein interaction have been identified. Bioinformatics analysis suggests that BcsA, PgaA, and PelB, involved in the production and export of cellulose, poly-beta-1,6-N-Acetyl-D-glucosamine, and Pel exopolysaccharide, respectively, share the same topology as AlgK/E. Together, our data suggest that AlgK plays a role in the assembly of the alginate biosynthetic complex and represents the periplasmic component of a new type of outer membrane secretin that differs from canonical bacterial capsular polysaccharide secretion systems.