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1.
Front Cell Infect Microbiol ; 13: 1123228, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37780846

RESUMEN

Background: Over the last few decades, a growing body of evidence has suggested a role for various infectious agents in Alzheimer's disease (AD) pathogenesis. Despite diverse pathogens (virus, bacteria, fungi) being detected in AD subjects' brains, research has focused on individual pathogens and only a few studies investigated the hypothesis of a bacterial brain microbiome. We profiled the bacterial communities present in non-demented controls and AD subjects' brains. Results: We obtained postmortem samples from the brains of 32 individual subjects, comprising 16 AD and 16 control age-matched subjects with a total of 130 samples from the frontal and temporal lobes and the entorhinal cortex. We used full-length 16S rRNA gene amplification with Pacific Biosciences sequencing technology to identify bacteria. We detected bacteria in the brains of both cohorts with the principal bacteria comprising Cutibacterium acnes (formerly Propionibacterium acnes) and two species each of Acinetobacter and Comamonas genera. We used a hierarchical Bayesian method to detect differences in relative abundance among AD and control groups. Because of large abundance variances, we also employed a new analysis approach based on the Latent Dirichlet Allocation algorithm, used in computational linguistics. This allowed us to identify five sample classes, each revealing a different microbiota. Assuming that samples represented infections that began at different times, we ordered these classes in time, finding that the last class exclusively explained the existence or non-existence of AD. Conclusions: The AD-related pathogenicity of the brain microbiome seems to be based on a complex polymicrobial dynamic. The time ordering revealed a rise and fall of the abundance of C. acnes with pathogenicity occurring for an off-peak abundance level in association with at least one other bacterium from a set of genera that included Methylobacterium, Bacillus, Caulobacter, Delftia, and Variovorax. C. acnes may also be involved with outcompeting the Comamonas species, which were strongly associated with non-demented brain microbiota, whose early destruction could be the first stage of disease. Our results are also consistent with a leaky blood-brain barrier or lymphatic network that allows bacteria, viruses, fungi, or other pathogens to enter the brain.


Asunto(s)
Acné Vulgar , Enfermedad de Alzheimer , Microbiota , Humanos , Enfermedad de Alzheimer/microbiología , ARN Ribosómico 16S/genética , Teorema de Bayes , Bacterias/genética , Propionibacterium acnes , Encéfalo
2.
ACS Appl Bio Mater ; 6(1): 238-245, 2023 01 16.
Artículo en Inglés | MEDLINE | ID: mdl-36595712

RESUMEN

Since the onset of the SARS-CoV-2 pandemic, the world has witnessed over 617 million confirmed cases and more than 6.54 million confirmed deaths, but the actual totals are likely much higher. The virus has mutated at a significantly faster rate than initially projected, and positive cases continue to surge with the emergence of ever more transmissible variants. According to the CDC, and at the time of this manuscript submission, more than 77% of all current US cases are a result of the B.5 (omicron). The continued emergence of highly transmissible variants makes clear the need for more effective methods of mitigating disease spread. Herein, we have developed an antimicrobial fabric capable of destroying a myriad of microbes including betacoronaviruses. We have demonstrated the capability of this highly porous and nontoxic metal organic framework (MOF), γ-CD-MOF-1, to serve as a host for varied-length benzalkonium chlorides (BACs; active ingredient in Lysol). Molecular docking simulations predicted a binding affinity of up to -4.12 kcal·mol-1, which is comparable to that of other reported guest molecules for this MOF. Similar Raman spectra and powder X-ray diffraction patterns between the unloaded and loaded MOFs, accompanied by a decrease in the Brunauer-Emmett-Teller surface area from 616.20 and 155.55 m2 g-1 respectively, corroborate the suggested potential for pore occupation with BAC. The MOF was grown on polypropylene fabric, exposed to a BAC-loading bath, washed to remove excess BAC from the external surface, and evaluated for its microbicidal activity against various bacterial and viral classes. Significant antimicrobial character was observed against Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, bacteriophage, and betacoronavirus. This study shows that a common mask material (polypropylene) can be coated with BAC-loaded γ-CD-MOF-1 while maintaining the guest molecule's antimicrobial effects.


Asunto(s)
Antiinfecciosos , COVID-19 , Estructuras Metalorgánicas , Humanos , Estructuras Metalorgánicas/farmacología , Estructuras Metalorgánicas/química , Simulación del Acoplamiento Molecular , Tensoactivos , Polipropilenos , SARS-CoV-2
3.
BJU Int ; 129(1): 104-112, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34143561

RESUMEN

OBJECTIVE: To undertake the first comprehensive evaluation of the urinary microbiota associated with Hunner lesion (HL) interstitial cystitis/bladder pain syndrome (IC/BPS). Despite no previous identification of a distinct IC/BPS microbial urotype, HL IC/BPS, an inflammatory subtype of IC/BPS, was hypothesized most likely to be associated with a specific bacterial species or microbial pattern. PARTICIPANTS AND METHODS: The bacterial microbiota of midstream urine specimens from HL IC/BPS and age- and gender-matched IC/BPS patients without HL (non-HL IC/BPS) were examined using the pan-bacterial domain clinical-level molecular diagnostic Pacific Biosciences full-length 16S gene sequencing protocol, informatics pipeline and database. We characterized the differential presence, abundances, and diversity of species, as well as gender-specific differences between and among HL and non-HL IC/BPS patients. RESULTS: A total of 59 patients with IC/BPS were enrolled (29 HL, 30 non-HL; 43 women, 16 men) from a single centre and the microbiota in midstream urine specimens was available for comparison. The species abundance differentiation between the HL and non-HL groups (12 species) was not significantly different after Bonferroni adjustments for multiple comparisons. Similarly, the nine differentiating species noted between female HL and non-HL patients were not significantly different after similar statistical correction. However, four species abundances (out of the 10 species differences identified prior to correction) remained significantly different between male HL and non-HL subjects: Negativicoccus succinivorans, Porphyromonas somerae, Mobiluncus curtisii and Corynebacterium renale. Shannon diversity metrics showed significantly higher diversity among HL male patients than HL female patients (P = 0.045), but no significant diversity differences between HL and non-HL patients overall. CONCLUSIONS: We were not able to identify a unique pathogenic urinary microbiota that differentiates all HL from all non-HL IC/BPS. It is likely that the male-specific differences resulted from colonization/contamination remote from the bladder. We were not able to show that bacteria play an important role in patients with HL IC/BPS.


Asunto(s)
Bacterias/aislamiento & purificación , Cistitis Intersticial/microbiología , ADN Bacteriano/análisis , Microbiota , Orina/microbiología , Adulto , Anciano , Anciano de 80 o más Años , Corynebacterium/aislamiento & purificación , Cistitis Intersticial/orina , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mobiluncus/aislamiento & purificación , Porphyromonas/aislamiento & purificación , Factores Sexuales , Veillonellaceae/aislamiento & purificación
4.
Genet Test Mol Biomarkers ; 25(8): 551-562, 2021 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-34406842

RESUMEN

Background and Aims: Outbreaks of severe and chronic tick-borne diseases (TBDs) are on the rise. This is through the transmission of infectious disease agents to humans during tick feeding. The transmission rate and extent of microbial exchange, however, vary based on the tick microbiome composition. While select microbes are determined to be members of the normal tick microbiome and others are clearly recognized mammalian and/or avian pathogens, the status of many other members of the tick microbiota with respect to human and alternate host pathogenesis remains unclear. Moreover, the species-level 16S microbiome of prominent TBD vectors, including Ixodes pacificus, have not been extensively studied. To elucidate the I. pacificus microbiome composition, we performed a pan-domain species-specific characterization of the bacterial microbiome on adult I. pacificus ticks collected from two regional parks within Western California. Our methods provide for characterizing nuances within cohort microbiomes and their relationships to geo-locale of origin, surrounding fauna, and prevalences of known and suspected pathogens in relation to current TBD epidemiological zones. Methods: Ninety-two adult I. pacificus bacterial microbiomes were characterized using a high-fidelity, pan-domain, species-specific, full-length 16S rRNA amplification method using circular consensus sequencing performed on the Pacific Biosciences Sequel platform. Data analyses were performed with the MCSMRT data analysis package and database. Results: The species-specific I. pacificus microbiome composition illustrates a complex assortment of microflora, including over 900 eubacterial species with high taxonomic diversity, which was revealed to vary by sex and geo-locale, though the use of full-length 16S gene sequencing. The TBD-associated pathogens, such as Borrelia burgdorferi, Anaplasma phagocytophilum, and Rickettsia monacensis, were identified along with a host of bacteria previously unassociated with ticks. Conclusion: Species-level taxonomic classification of the I. pacificus microbiome revealed that full-length bacterial 16S gene sequencing is required for the granularity to elucidate the microbial diversity within and among ticks based on geo-locale.


Asunto(s)
Ixodes/genética , Ixodes/microbiología , Microbiota/genética , Animales , California , Ixodes/metabolismo , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ARN/métodos , Enfermedades por Picaduras de Garrapatas/genética
5.
Front Microbiol ; 12: 646303, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34122361

RESUMEN

Recent advances in 3D printing have led to a rise in the use of 3D printed materials in prosthetics and external medical devices. These devices, while inexpensive, have not been adequately studied for their ability to resist biofouling and biofilm buildup. Bacterial biofilms are a major cause of biofouling in the medical field and, therefore, hospital-acquired, and medical device infections. These surface-attached bacteria are highly recalcitrant to conventional antimicrobial agents and result in chronic infections. During the COVID-19 pandemic, the U.S. Food and Drug Administration and medical officials have considered 3D printed medical devices as alternatives to conventional devices, due to manufacturing shortages. This abundant use of 3D printed devices in the medical fields warrants studies to assess the ability of different microorganisms to attach and colonize to such surfaces. In this study, we describe methods to determine bacterial biofouling and biofilm formation on 3D printed materials. We explored the biofilm-forming ability of multiple opportunistic pathogens commonly found on the human body including Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus to colonize eight commonly used polylactic acid (PLA) polymers. Biofilm quantification, surface topography, digital optical microscopy, and 3D projections were employed to better understand the bacterial attachment to 3D printed surfaces. We found that biofilm formation depends on surface structure, hydrophobicity, and that there was a wide range of antimicrobial properties among the tested polymers. We compared our tested materials with commercially available antimicrobial PLA polymers.

6.
Microorganisms ; 8(9)2020 Aug 28.
Artículo en Inglés | MEDLINE | ID: mdl-32872142

RESUMEN

Biofilm infections have no approved effective medical treatments and can only be disrupted via physical means. This means that any biofilm infection that is not addressable surgically can never be eliminated and can only be managed as a chronic disease. Therefore, there is an urgent need for the development of new classes of drugs that can target the metabolic mechanisms within biofilms which render them recalcitrant to traditional antibiotics. Persister cells within the biofilm structure may play a large role in the enhanced antibiotic recalcitrance of bacteria biofilms. Biofilm persister cells can be resistant to up to 1000 times the minimal inhibitory concentrations of many antibiotics, as compared to their planktonic envirovars; they are thought to be the prokaryotic equivalent of metazoan stem cells. Their metabolic resistance has been demonstrated to be an active process induced by the stringent response that is triggered by the ribosomally-associated enzyme RelA in response to amino acid starvation. This 84-kD pyrophosphokinase produces the "magic spot" alarmones, collectively called (p)ppGpp. These alarmones act by directly regulating transcription by binding to RNA polymerase. These transcriptional changes lead to a major shift in cellular function to both upregulate oxidative stress-combating enzymes and down regulate major cellular functions associated with growth and replication. These changes in gene expression produce the quiescent persister cells. In this work, we describe a hybrid in silico laboratory pipeline for identifying and validating small-molecule inhibitors of RelA for use in the combinatorial treatment of bacterial biofilms as re-potentiators of classical antibiotics.

8.
World J Urol ; 38(2): 433-446, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31028455

RESUMEN

PURPOSE: To correlate the presence of fungi with symptom flares, pain and urinary severity in a prospective, longitudinal study of women with IC/BPS enrolled in the MAPP Research Network. METHODS: Flare status, pelvic pain, urinary severity, and midstream urine were collected at baseline, 6 and 12 months from female IC/BPS participants with at least one flare and age-matched participants with no reported flares. Multilocus PCR coupled with electrospray ionization/mass spectrometry was used for identification of fungal species and genus. Associations between "mycobiome" (species/genus presence, relative abundance, Shannon's/Chao1 diversity indices) and current flare status, pain, urinary severity were evaluated using generalized linear mixed models, permutational multivariate analysis of variance, Wilcoxon's rank-sum test. RESULTS: The most specific analysis detected 13 fungal species from 8 genera in 504 urine samples from 202 females. A more sensitive analysis detected 43 genera. No overall differences were observed in fungal species/genus composition or diversity by flare status or pain severity. Longitudinal analyses suggested greater fungal diversity (Chao1 Mean Ratio 3.8, 95% CI 1.3-11.2, p = 0.02) and a significantly greater likelihood of detecting any fungal species (OR = 5.26, 95% CI 1.1-25.8, p = 0.04) in high vs low urinary severity participants. Individual taxa analysis showed a trend toward increased presence and relative abundance of Candida (OR = 6.63, 95% CI 0.8-58.5, p = 0.088) and Malassezia (only identified in 'high' urinary severity phenotype) for high vs low urinary symptoms. CONCLUSION: This analysis suggests the possibility that greater urinary symptom severity is associated with the urinary mycobiome urine in some females with IC/BPS.


Asunto(s)
Cistitis Intersticial/orina , ADN de Hongos/análisis , Hongos/genética , Sistema Urinario/microbiología , Adulto , Cistitis Intersticial/microbiología , Femenino , Estudios de Seguimiento , Humanos , Fenotipo , Estudios Prospectivos , Factores de Tiempo
9.
BMC Genomics ; 20(1): 767, 2019 Oct 22.
Artículo en Inglés | MEDLINE | ID: mdl-31640553

RESUMEN

BACKGROUND: Escherichia coli C forms more robust biofilms than other laboratory strains. Biofilm formation and cell aggregation under a high shear force depend on temperature and salt concentrations. It is the last of five E. coli strains (C, K12, B, W, Crooks) designated as safe for laboratory purposes whose genome has not been sequenced. RESULTS: Here we present the complete genomic sequence of this strain in which we utilized both long-read PacBio-based sequencing and high resolution optical mapping to confirm a large inversion in comparison to the other laboratory strains. Notably, DNA sequence comparison revealed the absence of several genes thought to be involved in biofilm formation, including antigen 43, waaSBOJYZUL for lipopolysaccharide (LPS) synthesis, and cpsB for curli synthesis. The first main difference we identified that likely affects biofilm formation is the presence of an IS3-like insertion sequence in front of the carbon storage regulator csrA gene. This insertion is located 86 bp upstream of the csrA start codon inside the - 35 region of P4 promoter and blocks the transcription from the sigma32 and sigma70 promoters P1-P3 located further upstream. The second is the presence of an IS5/IS1182 in front of the csgD gene. And finally, E. coli C encodes an additional sigma70 subunit driven by the same IS3-like insertion sequence. Promoter analyses using GFP gene fusions provided insights into understanding this regulatory pathway in E. coli. CONCLUSIONS: Biofilms are crucial for bacterial survival, adaptation, and dissemination in natural, industrial, and medical environments. Most laboratory strains of E. coli grown for decades in vitro have evolved and lost their ability to form biofilm, while environmental isolates that can cause infections and diseases are not safe to work with. Here, we show that the historic laboratory strain of E. coli C produces a robust biofilm and can be used as a model organism for multicellular bacterial research. Furthermore, we ascertained the full genomic sequence of this classic strain, which provides for a base level of characterization and makes it useful for many biofilm-based applications.


Asunto(s)
Biopelículas/crecimiento & desarrollo , Escherichia coli/genética , Genoma Bacteriano/genética , Adhesión Bacteriana/genética , Mapeo Cromosómico , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Genes Reguladores/genética , Regiones Promotoras Genéticas , Estrés Salino/genética , Inversión de Secuencia , Temperatura , Factores de Transcripción/genética
10.
J Clin Med ; 8(3)2019 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-30917614

RESUMEN

We surveyed urine microbiota of females diagnosed with interstitial cystitis/bladder pain syndrome (IC/BPS) and matched control participants enrolled in the National Institutes of Health (NIH) Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network using the culture-independent methodology. Midstream urine specimens were analyzed with the Plex-ID molecular diagnostic platform that utilizes polymerase chain reaction⁻electrospray ionization⁻time-of-flight⁻mass spectrometry (PCR-ESI-TOF MS) to provide a comprehensive identification of bacterial and select fungal species. IC/BPS and control participants were evaluated for differences (presence, diversity, and abundance) in species and genus. Urine specimens obtained from 181 female IC/BPS and 182 female control participants detected a total of 92 species (41 genera). Mean (SD) species count was 2.49 (1.48) and 2.30 (1.28) among IC/BPS and control participants, respectively. Overall species composition did not significantly differ between IC/BPS and control participants at any level (p = 0.726 species level, p = 0.222 genus level). IC/BPS participants urine trended to an overabundance of Lactobacillus gasseri (p = 0.09) detected but had a lower prevalence of Corynebacterium compared with control participants (p = 0.002). The relative abundance data analysis mirrored the prevalence data differences with no significant differences in most species or genus abundance other than Lactobacillus gasseri and Corynebacterium (p = 0.08 and p = 0.001, respectively). No cause and/or effect conclusion can be drawn from this observation, but it suggests that a more comprehensive evaluation (vaginal, bowel, catheterized bladder and/or tissue-based specimens) of the lower urinary tract microbiota in IC/BPS patients is warranted.

11.
PLoS One ; 14(1): e0209688, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30620734

RESUMEN

Streptococcus pneumoniae displays increased resistance to antibiotic therapy following biofilm formation. A genome-wide search revealed that SP 0320 and SP 0675 (respectively annotated as 5-keto-D-gluconate-5-reductase and glucose dehydrogenase) contain the highest degree of homology to CsgA of Myxococcus xanthus, a signaling factor that promotes cell aggregation and biofilm formation. Single and double SP 0320 and SP 0675 knockout mutants were created in strain BS72; however, no differences were observed in the biofilm-forming phenotypes of mutants compared to the wild type strain. Using the chinchilla model of otitis media and invasive disease, all three mutants exhibited greatly increased virulence compared to the wild type strain (increased pus formation, tympanic membrane rupture, mortality rates). The SP 0320 gene is located in an operon with SP 0317, SP 0318 and SP 0319, which we bioinformatically annotated as being part of the Entner-Doudoroff pathway. Deletion of SP 0317 also resulted in increased mortality in chinchillas; however, mutations in SP 0318 and SP 0319 did not alter the virulence of bacteria compared to the wild type strain. Complementing the SP 0317, SP 0320 and SP 0675 mutant strains reversed the virulence phenotype. We prepared recombinant SP 0317, SP 0318, SP 0320 and SP 0675 proteins and confirmed their functions. These data reveal that disruption of genes involved in the degradation of ketogluconate, the Entner-Doudoroff pathway, and glucose dehydrogenase significantly increase the virulence of bacteria in vivo; two hypothetical models involving virulence triggered by reduced in carbon-flux through the glycolytic pathways are presented.


Asunto(s)
Infecciones Neumocócicas/genética , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas , Metabolismo de los Hidratos de Carbono , Chinchilla/microbiología , Glucosa/metabolismo , Glucosa 1-Deshidrogenasa/genética , Glucosa 1-Deshidrogenasa/metabolismo , Glucólisis , Otitis Media/microbiología , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Fenotipo , Infecciones Neumocócicas/microbiología , Eliminación de Secuencia , Virulencia
12.
PLoS One ; 13(12): e0209554, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30589862

RESUMEN

In E. coli, under high pH/high salt conditions, a major Na+/H+ antiporter (NhaA) is activated to maintain an internal pH level. Its expression is induced by a specific regulator NhaR, which is also responsible for osmC and pgaA regulation. Here I report that the NhaR regulator affects the carbon storage regulatory Csr system. I found that the expression of all major components of the Csr system-CsrA regulator, CsrB and CsrC small RNAs, and the CsrB and CsrC stability were indirectly affected by nhaR mutation under stress conditions. Using a combination of experimental and in silico analyses, I concluded that the mechanism of regulation included direct and indirect activation of a two-component system (TCS) response regulator-UvrY. NhaR regulation involved interactions with the regulators H-NS and SdiA and was affected by a naturally occurring spontaneous IS5 insertion in the promoter region. A regulatory circuit was proposed and discussed.


Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas de Escherichia coli/fisiología , Escherichia coli/fisiología , Concentración de Iones de Hidrógeno , Proteínas de Unión al ARN/fisiología , Proteínas Represoras/fisiología , Estrés Salino , Factores de Transcripción/fisiología , Secuencia de Bases , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Regulación Bacteriana de la Expresión Génica , Genes Reporteros , Modelos Biológicos , Mutación , Motivos de Nucleótidos , Regiones Promotoras Genéticas , Unión Proteica , Estabilidad del ARN , Temperatura
13.
Genome Biol ; 19(1): 123, 2018 08 24.
Artículo en Inglés | MEDLINE | ID: mdl-30143034

RESUMEN

BACKGROUND: Lung cancer is the leading cancer diagnosis worldwide and the number one cause of cancer deaths. Exposure to cigarette smoke, the primary risk factor in lung cancer, reduces epithelial barrier integrity and increases susceptibility to infections. Herein, we hypothesize that somatic mutations together with cigarette smoke generate a dysbiotic microbiota that is associated with lung carcinogenesis. Using lung tissue from 33 controls and 143 cancer cases, we conduct 16S ribosomal RNA (rRNA) bacterial gene sequencing, with RNA-sequencing data from lung cancer cases in The Cancer Genome Atlas serving as the validation cohort. RESULTS: Overall, we demonstrate a lower alpha diversity in normal lung as compared to non-tumor adjacent or tumor tissue. In squamous cell carcinoma specifically, a separate group of taxa are identified, in which Acidovorax is enriched in smokers. Acidovorax temporans is identified within tumor sections by fluorescent in situ hybridization and confirmed by two separate 16S rRNA strategies. Further, these taxa, including Acidovorax, exhibit higher abundance among the subset of squamous cell carcinoma cases with TP53 mutations, an association not seen in adenocarcinomas. CONCLUSIONS: The results of this comprehensive study show both microbiome-gene and microbiome-exposure interactions in squamous cell carcinoma lung cancer tissue. Specifically, tumors harboring TP53 mutations, which can impair epithelial function, have a unique bacterial consortium that is higher in relative abundance in smoking-associated tumors of this type. Given the significant need for clinical diagnostic tools in lung cancer, this study may provide novel biomarkers for early detection.


Asunto(s)
Neoplasias Pulmonares/genética , Neoplasias Pulmonares/microbiología , Microbiota/genética , Proteína p53 Supresora de Tumor/genética , Adulto , Anciano , Biodiversidad , Comamonadaceae/clasificación , Comamonadaceae/fisiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación/genética , Neoplasias de Células Escamosas/genética , Neoplasias de Células Escamosas/microbiología , Proteobacteria/metabolismo , Reproducibilidad de los Resultados , Fumadores , Proteína p53 Supresora de Tumor/metabolismo
14.
J Bacteriol ; 196(1): 129-39, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24142257

RESUMEN

Dental caries induced by Streptococcus mutans is one of the most prevalent chronic infectious diseases worldwide. The pathogenicity of S. mutans relies on the bacterium's ability to colonize tooth surfaces and survive a strongly acidic environment. We performed an ISS1 transposon mutagenesis to screen for acid-sensitive mutants of S. mutans and identified an SMU.746-SMU.747 gene cluster that is needed for aciduricity. SMU.746 and SMU.747 appear to be organized in an operon and encode a putative membrane-associated permease. SMU.746- and SMU.747-deficient mutants showed a reduced ability to grow in acidified medium. However, the short-term or long-term acid survival capacity and F1F0 ATPase activity remained unaffected in the mutants. Furthermore, deletion of both genes did not change cell membrane permeability and the oxidative and heat stress responses. Growth was severely affected even with slight acidification of the defined medium (pH 6.5). The ability of the mutant strain to acidify the defined medium during growth in the presence of glucose and sucrose was significantly reduced, although the glycolysis rate was only slightly affected. Surprisingly, deletion of the SMU.746-SMU.747 genes triggered increased biofilm formation in low-pH medium. The observed effects were more striking in a chemically defined medium. We speculate that the SMU.746-SMU.747 complex is responsible for amino acid transport, and we discuss its possible role in colonization and survival in the oral environment.


Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Ácidos Carboxílicos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Streptococcus mutans/fisiología , Proteínas Bacterianas/genética , Permeabilidad de la Membrana Celular/efectos de los fármacos , Medios de Cultivo/química , Elementos Transponibles de ADN , Eliminación de Gen , Glucosa/metabolismo , Concentración de Iones de Hidrógeno , Proteínas de Transporte de Membrana/genética , Viabilidad Microbiana/efectos de los fármacos , Familia de Multigenes , Mutagénesis Insercional , Operón , Streptococcus mutans/efectos de los fármacos , Streptococcus mutans/genética , Streptococcus mutans/metabolismo , Sacarosa/metabolismo
15.
Plasmid ; 70(1): 110-9, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23558148

RESUMEN

In spite of the contribution of plasmids to the spread of antibiotic resistance in human pathogens, little is known about the transferability of various drug resistance plasmids in bacterial biofilms. The goal of this study was to compare the efficiency of transfer of 19 multidrug resistance plasmids into Escherichia coli recipient biofilms and determine the effects of biofilm age, biofilm-donor exposure time, and donor-to-biofilm attachment on this process. An E. coli recipient biofilm was exposed separately to 19 E. coli donors, each with a different plasmid, and transconjugants were determined by plate counting. With few exceptions, plasmids that transferred well in a liquid environment also showed the highest transferability in biofilms. The difference in transfer frequency between the most and least transferable plasmid was almost a million-fold. The 'invasibility' of the biofilm by plasmids, or the proportion of biofilm cells that acquired plasmids within a few hours, depended not only on the type of plasmid, but also on the time of biofilm exposure to the donor and on the ability of the plasmid donor to attach to the biofilm, yet not on biofilm age. The efficiency of donor strain attachment to the biofilm was not affected by the presence of plasmids. The most invasive plasmid was pHH2-227, which based on genome sequence analysis is a hybrid between IncU-like and IncW plasmids. The wide range in transferability in an E. coli biofilm among plasmids needs to be taken into account in our fight against the spread of drug resistance.


Asunto(s)
Biopelículas/crecimiento & desarrollo , Conjugación Genética , ADN Bacteriano/genética , Farmacorresistencia Bacteriana/genética , Escherichia coli/genética , Plásmidos/genética , Antibacterianos/farmacología , Replicación del ADN , ADN Bacteriano/metabolismo , Farmacorresistencia Bacteriana/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Integrones , Pruebas de Sensibilidad Microbiana , Plásmidos/metabolismo , Factores de Tiempo
16.
Appl Environ Microbiol ; 77(15): 5079-88, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21642400

RESUMEN

Although biofilms represent a common bacterial lifestyle in clinically and environmentally important habitats, there is scant information on the extent of gene transfer in these spatially structured populations. The objective of this study was to gain insight into factors that affect transfer of the promiscuous multidrug resistance plasmid pB10 in Escherichia coli biofilms. Biofilms were grown in different experimental settings, and plasmid transfer was monitored using laser scanning confocal microscopy and plate counting. In closed flow cells, plasmid transfer in surface-attached submerged biofilms was negligible. In contrast, a high plasmid transfer efficiency was observed in a biofilm floating at the air-liquid interface in an open flow cell with low flow rates. A vertical flow cell and a batch culture biofilm reactor were then used to detect plasmid transfer at different depths away from the air-liquid interface. Extensive plasmid transfer occurred only in a narrow zone near that interface. The much lower transfer frequencies in the lower zones coincided with rapidly decreasing oxygen concentrations. However, when an E. coli csrA mutant was used as the recipient, a thick biofilm was obtained at all depths, and plasmid transfer occurred at similar frequencies throughout. These results and data from separate aerobic and anaerobic matings suggest that oxygen can affect IncP-1 plasmid transfer efficiency, not only directly but also indirectly, through influencing population densities and therefore colocalization of donors and recipients. In conclusion, the air-liquid interface can be a hot spot for plasmid-mediated gene transfer due to high densities of juxtaposed donor and recipient cells.


Asunto(s)
Biopelículas , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/genética , Transferencia de Gen Horizontal/genética , Plásmidos/genética , Plásmidos/metabolismo , Recuento de Células , Conjugación Genética/genética , Medios de Cultivo , Escherichia coli/fisiología , Microscopía Confocal , Mutación , Oxígeno/metabolismo
17.
Appl Environ Microbiol ; 76(13): 4553-6, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-20453134

RESUMEN

We designed a new genetic tool to detect plasmid transfer under anaerobic and aerobic conditions. The system is based on the T7 RNA polymerase gene and a T7 promoter-driven oxygen-independent green fluorescent protein, evoglow, alone or in combination with red fluorescent protein DsRed. Constructs are available as plasmids and mini-mariner transposons.


Asunto(s)
Conjugación Genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/genética , Vectores Genéticos , Plásmidos/genética , Aerobiosis , Anaerobiosis , Bacteriófago T7 , Elementos Transponibles de ADN , ARN Polimerasas Dirigidas por ADN , Escherichia coli/virología , Técnicas Genéticas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Regiones Promotoras Genéticas , Proteínas Virales , Proteína Fluorescente Roja
18.
Plasmid ; 63(2): 98-107, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20018208

RESUMEN

Plasmids of the incompatibility group IncP-1 are important agents of horizontal gene transfer and contribute to the spread of antibiotic resistance and xenobiotic degradation within bacterial communities. Even though some prototype plasmids have been studied in much detail, the diversity of this plasmid group was still greatly underestimated until recently, as only two of the five currently known divergent sub-groups had been described. To further improve our insight into the diversity and evolutionary history of this family of broad-host-range plasmids, we compared the complete nucleotide sequence of a new IncP-1delta plasmid pAKD4 to the genomes of other IncP-1 plasmids. Plasmid pAKD4 was previously isolated by exogenous plasmid isolation from an agricultural soil in Norway. Its 56,803bp nucleotide sequence shows high similarity in gene sequence and gene order to both plasmids pEST4011 and pIJB1, the only other IncP-1delta plasmids sequenced so far. While all three plasmids have a typical IncP-1 backbone comprising replication, transfer, and stable inheritance/control genes, the low sequence similarity in some regions and presence/absence of some backbone genes compared to other IncP-1 plasmids cluster them in a divergent sub-group. Therefore this study validates the presence of a real IncP-1delta clade with multiple plasmids. Moreover, since both pEST4011 and pIJB1 are missing a portion of their transfer genes, pAKD4 represents the first completely sequenced self-transferable plasmid with a complete IncP-1delta backbone. We therefore propose it to be the prototype IncP-1delta plasmid.


Asunto(s)
Genómica , Plásmidos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Replicación del ADN/efectos de los fármacos , Elementos Transponibles de ADN/genética , Evolución Molecular , Genes Bacterianos , Mercurio/toxicidad , Sistemas de Lectura Abierta/genética , Filogenia
19.
J Theor Biol ; 262(4): 711-9, 2010 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-19835890

RESUMEN

Plasmids are important vehicles for horizontal gene transfer and rapid adaptation in bacteria, including the spread of antibiotic resistance genes. Conjugative transfer of a plasmid from a plasmid-bearing to a plasmid-free bacterial cell requires contact and attachment of the cells followed by plasmid DNA transfer prior to detachment. We introduce a system of differential equations for plasmid transfer in well-mixed populations that accounts for attachment, DNA transfer, and detachment dynamics. These equations offer advantages over classical mass-action models that combine these three processes into a single "bulk" conjugation rate. By decomposing the process of plasmid transfer into its constituent parts, this new model provides a framework that facilitates meaningful comparisons of plasmid transfer rates in surface and liquid environments. The model also allows one to account for experimental and environmental effects such as mixing intensity. To test the adequacy of the model and further explore the effects of mixing on plasmid transfer, we performed batch culture experiments using three different plasmids and a range of different mixing intensities. The results show that plasmid transfer is optimized at low to moderate shaking speeds and that vigorous shaking negatively affects plasmid transfer. Using reasonable assumptions on attachment and detachment rates, the mathematical model predicts the same behavior.


Asunto(s)
Bacterias/genética , Plásmidos/metabolismo , Antibacterianos/farmacología , Bacterias/metabolismo , Biopelículas , Conjugación Genética , ADN/metabolismo , Fimbrias Bacterianas , Transferencia de Gen Horizontal , Bacterias Gramnegativas/metabolismo , Cinética , Modelos Biológicos , Modelos Teóricos
20.
Antonie Van Leeuwenhoek ; 96(2): 193-204, 2009 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-19259779

RESUMEN

The self-transmissible, broad-host-range (BHR) plasmid pMOL98 was previously isolated from polluted soil using a triparental plasmid capture approach and shown to possess a replicon similar to that of the BHR plasmids pSB102 and pIPO2. Here, complete sequence analysis and comparative genomics reveal that the 55.5 kb nucleotide sequence of pMOL98 shows extensive sequence similarity and synteny with the BHR plasmid family that now includes pIPO2, pSB102, pTER331, and pMRAD02. They share a plasmid backbone comprising replication, partitioning and conjugative transfer functions. Comparison of the variable accessory regions of these plasmids shows that the majority of natural transposons, as well as the mini-transposon used to mark the plasmids, are inserted in the parA locus. The transposon unique to pMOL98 appears to have inserted from the chromosome of the recipient strain used in the plasmid capture procedure. This demonstrates the necessity for careful screening of plasmids and host chromosomes to avoid mis-interpretation of plasmid genome content. The presence of very similar BHR plasmids with different accessory genes in geographically distinct locations suggests an important role in horizontal gene exchange and bacterial adaptation for this recently defined plasmid group, which we propose to name "PromA".


Asunto(s)
Cupriavidus/genética , Elementos Transponibles de ADN , Transferencia de Gen Horizontal , Plásmidos/genética , Secuencia de Bases , Biología Computacional , Conjugación Genética , Cupriavidus/metabolismo , Replicación del ADN , ADN Bacteriano/análisis , ADN Bacteriano/genética , Genómica , Datos de Secuencia Molecular , Plásmidos/clasificación , Análisis de Secuencia de ADN
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