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1.
Int Urogynecol J ; 27(5): 723-33, 2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-26423260

RESUMEN

INTRODUCTION AND HYPOTHESIS: Many adult women have resident urinary bacteria (urinary microbiome/microbiota). In adult women affected by urinary urgency incontinence (UUI), the etiologic and/or therapeutic role of the urinary microbiome/microbiota remains unknown. We hypothesized that microbiome/microbiota characteristics would relate to clinically relevant treatment response to UUI medication per os. METHODS: Adult women initiating medication treatment orally for UUI and a comparator group of unaffected women were recruited in a tertiary care health-care system. All participants provided baseline clinical data and urine samples. Women with UUI were given 5 mg solifenacin, with potential dose escalation to 10 mg for inadequate UUI symptom control at 4 weeks. Additional data and urine samples were collected from women with UUI at 4 and 12 weeks. The samples were assessed using 16S ribosomal RNA (rRNA) gene sequencing and enhanced quantitative urine culturing. The primary outcome was treatment response as measured by the validated Patient Global Symptom Control (PGSC) questionnaire. Clinically relevant UUI symptom control was defined as a 4 or 5 score on the PGSC. RESULTS: Diversity and composition of the urinary microbiome/microbiota of women with and without UUI differed at baseline. Women with UUI had more bacteria and a more diverse microbiome/microbiota. The clinical response to solifenacin in UUI participants was related to baseline microbiome/microbiota, with responders more likely to have fewer bacteria and a less diverse community at baseline. Nonresponders had a more diverse community that often included bacteria not typically found in responders. CONCLUSIONS: Knowledge of an individual's urinary microbiome/microbiota may help refine UUI treatment. Complementary tools, DNA sequencing, and expanded urine culture provide information about bacteria that appear to be related to UUI incontinence status and treatment response in this population of adult women.


Asunto(s)
Bacteriuria/microbiología , Microbiota , Antagonistas Muscarínicos/uso terapéutico , ARN Ribosómico 16S/análisis , Succinato de Solifenacina/uso terapéutico , Incontinencia Urinaria de Urgencia/tratamiento farmacológico , Incontinencia Urinaria de Urgencia/microbiología , Sistema Urinario/microbiología , Actinomyces/aislamiento & purificación , Administración Oral , Adulto , Anciano , Estudios de Casos y Controles , Recuento de Colonia Microbiana , Corynebacterium/aislamiento & purificación , Femenino , Humanos , Lactobacillus/aislamiento & purificación , Persona de Mediana Edad , Antagonistas Muscarínicos/administración & dosificación , Estudios Prospectivos , Succinato de Solifenacina/administración & dosificación , Streptococcus/aislamiento & purificación , Resultado del Tratamiento
2.
Am J Obstet Gynecol ; 213(3): 347.e1-11, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26210757

RESUMEN

OBJECTIVE: The purpose of this study was to characterize the urinary microbiota in women who are planning treatment for urgency urinary incontinence and to describe clinical associations with urinary symptoms, urinary tract infection, and treatment outcomes. STUDY DESIGN: Catheterized urine samples were collected from multisite randomized trial participants who had no clinical evidence of urinary tract infection; 16S ribosomal RNA gene sequencing was used to dichotomize participants as either DNA sequence-positive or sequence-negative. Associations with demographics, urinary symptoms, urinary tract infection risk, and treatment outcomes were determined. In sequence-positive samples, microbiotas were characterized on the basis of their dominant microorganisms. RESULTS: More than one-half (51.1%; 93/182) of the participants' urine samples were sequence-positive. Sequence-positive participants were younger (55.8 vs 61.3 years old; P = .0007), had a higher body mass index (33.7 vs 30.1 kg/m(2); P = .0009), had a higher mean baseline daily urgency urinary incontinence episodes (5.7 vs 4.2 episodes; P < .0001), responded better to treatment (decrease in urgency urinary incontinence episodes, -4.4 vs -3.3; P = .0013), and were less likely to experience urinary tract infection (9% vs 27%; P = .0011). In sequence-positive samples, 8 major bacterial clusters were identified; 7 clusters were dominated not only by a single genus, most commonly Lactobacillus (45%) or Gardnerella (17%), but also by other taxa (25%). The remaining cluster had no dominant genus (13%). CONCLUSION: DNA sequencing confirmed urinary bacterial DNA in many women with urgency urinary incontinence who had no signs of infection. Sequence status was associated with baseline urgency urinary incontinence episodes, treatment response, and posttreatment urinary tract infection risk.


Asunto(s)
Bacteriuria/microbiología , Infecciones por Bacteroidaceae/microbiología , Infecciones por Bacterias Grampositivas/microbiología , Microbiota/genética , ARN Ribosómico 16S/análisis , Incontinencia Urinaria de Urgencia/microbiología , Sistema Urinario/microbiología , Inhibidores de la Liberación de Acetilcolina/uso terapéutico , Adulto , Factores de Edad , Anciano , Bacteriuria/epidemiología , Infecciones por Bacteroidaceae/epidemiología , Índice de Masa Corporal , Toxinas Botulínicas Tipo A/uso terapéutico , Antagonistas Colinérgicos/uso terapéutico , Femenino , Gardnerella/genética , Gardnerella/aislamiento & purificación , Infecciones por Bacterias Grampositivas/epidemiología , Humanos , Lactobacillus/genética , Lactobacillus/aislamiento & purificación , Persona de Mediana Edad , Obesidad/epidemiología , Prevotella/genética , Prevotella/aislamiento & purificación , Calidad de Vida , Resultado del Tratamiento , Incontinencia Urinaria de Urgencia/epidemiología , Incontinencia Urinaria de Urgencia/terapia , Infecciones Urinarias/epidemiología , Infecciones Urinarias/microbiología
3.
J Clin Microbiol ; 52(3): 871-6, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24371246

RESUMEN

Our previous study showed that bacterial genomes can be identified using 16S rRNA sequencing in urine specimens of both symptomatic and asymptomatic patients who are culture negative according to standard urine culture protocols. In the present study, we used a modified culture protocol that included plating larger volumes of urine, incubation under varied atmospheric conditions, and prolonged incubation times to demonstrate that many of the organisms identified in urine by 16S rRNA gene sequencing are, in fact, cultivable using an expanded quantitative urine culture (EQUC) protocol. Sixty-five urine specimens (from 41 patients with overactive bladder and 24 controls) were examined using both the standard and EQUC culture techniques. Fifty-two of the 65 urine samples (80%) grew bacterial species using EQUC, while the majority of these (48/52 [92%]) were reported as no growth at 10(3) CFU/ml by the clinical microbiology laboratory using the standard urine culture protocol. Thirty-five different genera and 85 different species were identified by EQUC. The most prevalent genera isolated were Lactobacillus (15%), followed by Corynebacterium (14.2%), Streptococcus (11.9%), Actinomyces (6.9%), and Staphylococcus (6.9%). Other genera commonly isolated include Aerococcus, Gardnerella, Bifidobacterium, and Actinobaculum. Our current study demonstrates that urine contains communities of living bacteria that comprise a resident female urine microbiota.


Asunto(s)
Bacterias/aislamiento & purificación , Técnicas Microbiológicas/métodos , Manejo de Especímenes/métodos , Vejiga Urinaria/microbiología , Orina/microbiología , Adulto , Bacterias/clasificación , Femenino , Humanos , Sensibilidad y Especificidad
4.
Infect Immun ; 81(5): 1399-410, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23429532

RESUMEN

Type II protein secretion (T2S) by Legionella pneumophila is required for intracellular infection of host cells, including macrophages and the amoebae Acanthamoeba castellanii and Hartmannella vermiformis. Previous proteomic analysis revealed that T2S by L. pneumophila 130b mediates the export of >25 proteins, including several that appeared to be novel. Following confirmation that they are unlike known proteins, T2S substrates NttA, NttB, and LegP were targeted for mutation. nttA mutants were impaired for intracellular multiplication in A. castellanii but not H. vermiformis or macrophages, suggesting that novel exoproteins which are specific to Legionella are especially important for infection. Because the importance of NttA was host cell dependent, we examined a panel of T2S substrate mutants that had not been tested before in more than one amoeba. As a result, RNase SrnA, acyltransferase PlaC, and metalloprotease ProA all proved to be required for optimal intracellular multiplication in H. vermiformis but not A. castellanii. Further examination of an lspF mutant lacking the T2S apparatus documented that T2S is also critical for infection of the amoeba Naegleria lovaniensis. Mutants lacking SrnA, PlaC, or ProA, but not those deficient for NttA, were defective in N. lovaniensis. Based upon analysis of a double mutant lacking PlaC and ProA, the role of ProA in H. vermiformis was connected to its ability to activate PlaC, whereas in N. lovaniensis, ProA appeared to have multiple functions. Together, these data document that the T2S system exports multiple effectors, including a novel one, which contribute in different ways to the broad host range of L. pneumophila.


Asunto(s)
Acanthamoeba castellanii/microbiología , Proteínas Bacterianas/metabolismo , Hartmannella/microbiología , Legionella pneumophila/metabolismo , Naegleria/microbiología , Southern Blotting , ADN Bacteriano/análisis , Humanos , Legionella pneumophila/genética , Legionella pneumophila/crecimiento & desarrollo , Macrófagos/microbiología , ARN Bacteriano/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN
5.
Int J Syst Evol Microbiol ; 62(Pt 12): 2946-2954, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22286905

RESUMEN

A Gram-negative, rod-shaped bacterium, designated H63(T), was isolated from aortic valve tissue of a patient with native valve endocarditis. 16S rRNA gene sequencing revealed that H63(T) belongs to the genus Legionella, with its closest neighbours being the type strains of Legionella brunensis (98.8% similarity), L. londiniensis (97.0%), L. jordanis (96.8%), L. erythra (96.2%), L. dresdenensis (96.0%) and L. rubrilucens, L. feeleii, L. pneumophila and L. birminghamensis (95.7%). DNA-DNA hybridization studies yielded values of <70% relatedness between strain H63(T) and its nearest neighbours in terms of 16S rRNA gene sequence similarity, indicating that the strain represents a novel species. Phylogenetic analysis of the 16S rRNA, macrophage infectivity potentiator (mip) and RNase P (rnpB) genes confirmed that H63(T) represents a distinct species, with L. brunensis being its closest sister taxon. Fatty acid composition and biochemical traits, such as the inability to ferment glucose and reduce nitrate, supported the affiliation of H63(T) to the genus Legionella. H63(T) was distinguishable from its neighbours based on it being positive for hippurate hydrolysis. H63(T) was further differentiated by its inability to grow on BCYE agar at 17 °C, its poor growth on low-iron medium and the absence of sliding motility. Also, H63(T) did not react with antisera generated from type strains of Legionella species. H63(T) replicated within macrophages. It also grew in mouse lungs, inducing histopathological evidence of pneumonia and dissemination to the spleen. Together, these results confirm that H63(T) represents a novel, pathogenic Legionella species, for which the name Legionella cardiaca sp. nov. is proposed. The type strain is H63(T) ( = ATCC BAA-2315(T)  = DSM 25049(T)  = JCM 17854(T)).


Asunto(s)
Válvula Aórtica/microbiología , Endocarditis/microbiología , Legionella/clasificación , Legionella/aislamiento & purificación , Filogenia , Animales , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Ácidos Grasos/análisis , Humanos , Legionella/genética , Ratones , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN
7.
PLoS One ; 9(10): e111375, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25354343

RESUMEN

Human urinary disorders are generally studied in rodent models due to limitations of functional in vitro culture models of primary human urothelial cells (HUCs). Current HUC culture models are often derived from immortalized cancer cell lines, which likely have functional characteristics differ from healthy human urothelium. Here, we described a simple explant culture technique to generate HUCs and assessed their in vitro functions. Using transmission electron microscopy, we assessed morphology and heterogeneity of the generated HUCs and characterized their intercellular membrane structural proteins relative to ex vivo urothelium tissue. We demonstrated that our cultured HUCs are free of fibroblasts. They are also heterogeneous, containing cells characteristic of both immature basal cells and mature superficial urothelial cells. The cultured HUCs expressed muscarinic receptors (MR1 and MR2), carnitine acetyltransferase (CarAT), immunoregulatory cytokines IL7, IL15, and IL23, as well as the chemokine CCL20. HUCs also expressed epithelial cell-specific molecules essential for forming intercellular structures that maintain the functional capacity to form the physiological barrier of the human bladder urothelium. A subset of HUCs, identified by the high expression of CD44, expressed the Toll-like receptor 4 (TLR4) along with its co-receptor CD14. We demonstrated that HUCs express, at the mRNA level, both forms of the IL22 receptor, the membrane-associated (IL22RA1) and the secreted soluble (IL22RA2) forms; in turn, IL22 inhibited expression of MR1 and induced expression of CarAT and two antimicrobial peptides (S100A9 and lipocalin-2). While the cellular sources of IL22 have yet to be identified, the HUC cytokine and chemokine profiles support the concept that IL22-producing cells are present in the human bladder mucosa tissue and that IL22 plays a regulatory role in HUC functions. Thus, the described explant technique is clearly capable of generating functional HUCs suitable for the study of human urinary tract disorders, including interactions between urothelium and IL22-producing cells.


Asunto(s)
Acetilcolina/metabolismo , Calgranulina B/metabolismo , Interleucinas/farmacología , Lipocalinas/metabolismo , Urotelio/metabolismo , Calgranulina B/genética , Células Cultivadas , Quimiocina CCL20/genética , Quimiocina CCL20/metabolismo , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Lipocalinas/genética , Receptores de Lipopolisacáridos/genética , Receptores de Lipopolisacáridos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Urotelio/efectos de los fármacos , Urotelio/ultraestructura , Interleucina-22
8.
mBio ; 5(4): e01283-14, 2014 Jul 08.
Artículo en Inglés | MEDLINE | ID: mdl-25006228

RESUMEN

Bacterial DNA and live bacteria have been detected in human urine in the absence of clinical infection, challenging the prevailing dogma that urine is normally sterile. Urgency urinary incontinence (UUI) is a poorly understood urinary condition characterized by symptoms that overlap urinary infection, including urinary urgency and increased frequency with urinary incontinence. The recent discovery of the urinary microbiome warrants investigation into whether bacteria contribute to UUI. In this study, we used 16S rRNA gene sequencing to classify bacterial DNA and expanded quantitative urine culture (EQUC) techniques to isolate live bacteria in urine collected by using a transurethral catheter from women with UUI and, in comparison, a cohort without UUI. For these cohorts, we demonstrated that the UUI and non-UUI urinary microbiomes differ by group based on both sequence and culture evidences. Compared to the non-UUI microbiome, sequencing experiments revealed that the UUI microbiome was composed of increased Gardnerella and decreased Lactobacillus. Nine genera (Actinobaculum, Actinomyces, Aerococcus, Arthrobacter, Corynebacterium, Gardnerella, Oligella, Staphylococcus, and Streptococcus) were more frequently cultured from the UUI cohort. Although Lactobacillus was isolated from both cohorts, distinctions existed at the species level, with Lactobacillus gasseri detected more frequently in the UUI cohort and Lactobacillus crispatus most frequently detected in controls. Combined, these data suggest that potentially important differences exist in the urinary microbiomes of women with and without UUI, which have strong implications in prevention, diagnosis, or treatment of UUI. Importance: New evidence indicates that the human urinary tract contains microbial communities; however, the role of these communities in urinary health remains to be elucidated. Urgency urinary incontinence (UUI) is a highly prevalent yet poorly understood urinary condition characterized by urgency, frequency, and urinary incontinence. Given the significant overlap of UUI symptoms with those of urinary tract infections, it is possible that UUI may have a microbial component. We compared the urinary microbiomes of women affected by UUI to those of a comparison group without UUI, using both high-throughput sequencing and extended culture techniques. We identified statistically significant differences in the frequency and abundance of bacteria present. These differences suggest a potential role for the urinary microbiome in female urinary health.


Asunto(s)
Microbiota/fisiología , ARN Ribosómico 16S/genética , Incontinencia Urinaria/microbiología , Sistema Urinario/microbiología , Actinomyces/genética , Actinomyces/aislamiento & purificación , Aerococcus/genética , Aerococcus/aislamiento & purificación , Anciano , Arthrobacter/genética , Arthrobacter/aislamiento & purificación , Corynebacterium/genética , Corynebacterium/aislamiento & purificación , Femenino , Gardnerella/genética , Gardnerella/aislamiento & purificación , Humanos , Lactobacillus/genética , Lactobacillus/aislamiento & purificación , Microbiota/genética , Persona de Mediana Edad , Staphylococcus/genética , Staphylococcus/aislamiento & purificación , Streptococcus/genética , Streptococcus/aislamiento & purificación
9.
FEMS Microbiol Lett ; 300(2): 256-64, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19817866

RESUMEN

Examination of cell-free culture supernatants revealed that Legionella pneumophila strains secrete an endoglucanase activity. Legionella pneumophila lspF mutants were deficient for this activity, indicating that the endoglucanase is secreted by the bacterium's type II protein secretion (T2S) system. Inactivation of celA, encoding a member of the family-5 of glycosyl hydrolases, abolished the endoglucanase activity in L. pneumophila culture supernatants. The cloned celA gene conferred activity upon recombinant Escherichia coli. Thus, CelA is the major secreted endoglucanase of L. pneumophila. Mutants inactivated for celA grew normally in protozoa and macrophage, indicating that CelA is not required for the intracellular phase of L. pneumophila. The CelA endoglucanase is one of at least 25 proteins secreted by the type II system of L. pneumophila and the 17th type of enzyme effector associated with this pathway. Only a subset of the other Legionella species tested expressed secreted endoglucanase activity, suggesting that the T2S output differs among the different legionellae. Overall, this study represents the first documentation of an endoglucanase (EC 3.2.1.4) being produced by a strain of Legionella.


Asunto(s)
Celulasa/metabolismo , Legionella pneumophila/enzimología , Acanthamoeba castellanii/microbiología , Animales , Células Cultivadas , Clonación Molecular , Recuento de Colonia Microbiana , Escherichia coli/genética , Escherichia coli/metabolismo , Técnicas de Inactivación de Genes , Hartmannella/microbiología , Macrófagos/microbiología , Ratones , Ratones Endogámicos A , Virulencia
10.
Appl Environ Microbiol ; 73(10): 3173-82, 2007 May.
Artículo en Inglés | MEDLINE | ID: mdl-17369329

RESUMEN

"Photobacterium mandapamensis" (proposed name) and Photobacterium leiognathi are closely related, phenotypically similar marine bacteria that form bioluminescent symbioses with marine animals. Despite their similarity, however, these bacteria can be distinguished phylogenetically by sequence divergence of their luminescence genes, luxCDAB(F)E, by the presence (P. mandapamensis) or the absence (P. leiognathi) of luxF and, as shown here, by the sequence divergence of genes involved in the synthesis of riboflavin, ribBHA. To gain insight into the possibility that P. mandapamensis and P. leiognathi are ecologically distinct, we used these phylogenetic criteria to determine the incidence of P. mandapamensis as a bioluminescent symbiont of marine animals. Five fish species, Acropoma japonicum (Perciformes, Acropomatidae), Photopectoralis panayensis and Photopectoralis bindus (Perciformes, Leiognathidae), Siphamia versicolor (Perciformes, Apogonidae), and Gadella jordani (Gadiformes, Moridae), were found to harbor P. mandapamensis in their light organs. Specimens of A. japonicus, P. panayensis, and P. bindus harbored P. mandapamensis and P. leiognathi together as cosymbionts of the same light organ. Regardless of cosymbiosis, P. mandapamensis was the predominant symbiont of A. japonicum, and it was the apparently exclusive symbiont of S. versicolor and G. jordani. In contrast, P. leiognathi was found to be the predominant symbiont of P. panayensis and P. bindus, and it appears to be the exclusive symbiont of other leiognathid fishes and a loliginid squid. A phylogenetic test for cospeciation revealed no evidence of codivergence between P. mandapamensis and its host fishes, indicating that coevolution apparently is not the basis for this bacterium's host preferences. These results, which are the first report of bacterial cosymbiosis in fish light organs and the first demonstration that P. leiognathi is not the exclusive light organ symbiont of leiognathid fishes, demonstrate that the host species ranges of P. mandapamensis and P. leiognathi are substantially distinct. The host range difference underscores possible differences in the environmental distributions and physiologies of these two bacterial species.


Asunto(s)
Biodiversidad , Ecosistema , Peces/microbiología , Photobacterium/fisiología , Simbiosis , Animales , Proteínas Bacterianas/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Luminiscencia , Datos de Secuencia Molecular , Photobacterium/clasificación , Photobacterium/aislamiento & purificación , Filogenia , Riboflavina/genética , Análisis de Secuencia de ADN , Homología de Secuencia
11.
Environ Microbiol ; 6(2): 145-58, 2004 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-14756879

RESUMEN

Photobacterium leiognathi forms a bioluminescent symbiosis with leiognathid fishes, colonizing the internal light organ of the fish and providing its host with light used in bioluminescence displays. Strains symbiotic with different species of the fish exhibit substantial phenotypic differences in symbiosis and in culture, including differences in 2-D PAGE protein patterns and profiles of indigenous plasmids. To determine if such differences might reflect a genetically based symbiont-strain/host-species specificity, we profiled the genomes of P. leiognathi strains from leiognathid fishes using PFGE. Individual strains from 10 species of leiognathid fishes exhibited substantial genomic polymorphism, with no obvious similarity among strains; these strains were nonetheless identified as P. leiognathi by 16S rDNA sequence analysis. Profiling of multiple strains from individual host specimens revealed an oligoclonal structure to the symbiont populations; typically one or two genomotypes dominated each population. However, analysis of multiple strains from multiple specimens of the same host species, to determine if the same strain types consistently colonize a host species, demonstrated substantial heterogeneity, with the same genomotype only rarely observed among the symbiont populations of different specimens of the same host species. Colonization of the leiognathid light organ to initiate the symbiosis therefore is likely to be oliogoclonal, and specificity of the P. leiognathi/leiognathid fish symbiosis apparently is maintained at the bacterial species level rather than at the level of individual, genomotypically defined strain types.


Asunto(s)
Peces/microbiología , Photobacterium/genética , Polimorfismo Genético , Simbiosis/fisiología , Animales , ADN Bacteriano/análisis , Genoma , Photobacterium/clasificación , Photobacterium/fisiología , Filogenia , Plásmidos/genética , Plásmidos/metabolismo , Proteoma
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