RESUMEN
To comprehensively assess microbial diversity and abundance via molecular-analysis-based methods, procedures for sample collection, processing, and analysis were evaluated in depth. A model microbial community (MMC) of known composition, representative of a typical low-biomass surface sample, was used to examine the effects of variables in sampling matrices, target cell density/molecule concentration, and cryogenic storage on the overall efficacy of the sampling regimen. The MMC used in this study comprised 11 distinct species of bacterial, archaeal, and fungal lineages associated with either spacecraft or clean-room surfaces. A known cellular density of MMC was deposited onto stainless steel coupons, and after drying, a variety of sampling devices were used to recover cells and biomolecules. The biomolecules and cells/spores recovered from each collection device were assessed by cultivable and microscopic enumeration, and quantitative and species-specific PCR assays. rRNA gene-based quantitative PCR analysis showed that cotton swabs were superior to nylon-flocked swabs for sampling of small surface areas, and for larger surfaces, biological sampling kits significantly outperformed polyester wipes. Species-specific PCR revealed differential recovery of certain species dependent upon the sampling device employed. The results of this study empower current and future molecular-analysis-based microbial sampling and processing methodologies.
Asunto(s)
Archaea/aislamiento & purificación , Bacterias/aislamiento & purificación , Microbiología Ambiental , Hongos/aislamiento & purificación , Técnicas Microbiológicas/métodos , Archaea/genética , Archaea/crecimiento & desarrollo , Bacterias/genética , Bacterias/crecimiento & desarrollo , Hongos/genética , Hongos/crecimiento & desarrollo , Microscopía/métodos , Reacción en Cadena de la Polimerasa/métodosRESUMEN
BACKGROUND: The choice of optimal treatment strategies for T4b colon cancers has still been discussed, particularly the initiation of neoadjuvant therapy or surgery. We conducted this study to evaluate the safety and feasibility of laparoscopic multivisceral resection for T4b colon cancers. METHODS: We used the retrospective design to include all 43 patients with T4b colon cancer at a university hospital in Vietnam from March 2017 to March 2019. All patients were followed 30 days after the surgery, and information about the day of the first flatus, length of hospital stay, iatrogenic complications, postoperative morbidity, mortality, and adjuvant chemotherapy was collected. RESULTS: The mean operating time was 187 minutes (ranging from 80 to 310), the mean blood loss was 64.3 ml (5-200), and the conversion rate was 2.3%. The mean number of lymph nodes harvested was 15.5 (SD = 8.06), and 33 patients (76.7%) had at least 12 lymph nodes harvested. A total of 21 patients (48.8%) had lymph node metastases with a mean number of lymph node metastases of 1.89 (SD = 3.4). The radial resection margin was R0 in all 43 patients (100%). The median time until the first flatus and hospital stay were 3 days (2-5) and 7.1 (6-11) days, respectively. There was no mortality at 30 days postoperatively, and one patient had iatrogenic complication (2.3%). CONCLUSION: Laparoscopic radical colectomy was feasible and safe for patients with T4b colon cancer except those requiring major and complicated reconstruction.
Asunto(s)
Colectomía/métodos , Neoplasias del Colon/cirugía , Laparoscopía/métodos , Adulto , Anciano , Pérdida de Sangre Quirúrgica , Colectomía/efectos adversos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/mortalidad , Neoplasias del Colon/patología , Estudios de Factibilidad , Femenino , Humanos , Laparoscopía/efectos adversos , Tiempo de Internación , Escisión del Ganglio Linfático , Metástasis Linfática/patología , Masculino , Persona de Mediana Edad , Tempo Operativo , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Universal and species-specific quantitative polymerase chain reaction-based methods were employed to compare the effectiveness of four distinct materials used to collect biological samples from metal surfaces. Known cell densities of a model microbial community (MMC) were deposited onto metal surfaces and subsequently collected with cotton and nylon-flocked swabs for small surface areas and biological sampling kits (BiSKits) and polyester wipes for large surface areas. Ribosomal RNA gene-based quantitative PCR (qPCR) analyses revealed that cotton swabs were superior to nylon-flocked swabs for recovering nucleic acids (i.e., DNA) from small surface areas. Similarly, BiSKits outperformed polyester wipes for sampling large surface areas. Species-specific qPCR results show a differential recovery of rRNA genes of the various MMC constituents, seemingly dependent on the type of sampling device employed. Both cotton swabs and BiSKits recovered the rDNA of all nine of the MMC constituent microbes assayed, whereas nylon-flocked swabs and polyester wipes recovered the rDNA of only six and four of these MMC strains, respectively. The findings of this study demonstrate the importance and proficiency of molecular techniques in gauging the effectiveness and efficiency of various modes of biological sample collection from metal surfaces.
Asunto(s)
Bacterias/aislamiento & purificación , Contaminación de Equipos , Metales , Nave Espacial/instrumentación , Bacterias/clasificación , Bacterias/genética , ADN Bacteriano/análisis , ADN Ribosómico/análisis , ADN Ribosómico/aislamiento & purificación , Modelos Biológicos , Filogenia , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genéticaRESUMEN
The microbiology of the spacecraft assembly process is of paramount importance to planetary exploration, as the biological contamination that can result from remote-enabled spacecraft carries the potential to impact both life-detection experiments and extraterrestrial evolution. Accordingly, insights into the mechanisms and range of extremotolerance of Acinetobacter radioresistens 50v1, a Gram-negative bacterium isolated from the surface of the preflight Mars Odyssey orbiter, were gained by using a combination of microbiological, enzymatic, and proteomic methods. In summary, A. radioresistens 50v1 displayed a remarkable range of survival against hydrogen peroxide and the sequential exposures of desiccation, vapor and plasma phase hydrogen peroxide, and ultraviolet irradiation. The survival is among the highest reported for non-spore-forming and Gram-negative bacteria and is based upon contributions from the enzyme-based degradation of H(2)O(2) (catalase and alkyl hydroperoxide reductase), energy management (ATP synthase and alcohol dehydrogenase), and modulation of the membrane composition. Together, the biochemical and survival features of A. radioresistens 50v1 support a potential persistence on Mars (given an unintended or planned surface landing of the Mars Odyssey orbiter), which in turn may compromise the scientific integrity of future life-detection missions.
Asunto(s)
Acinetobacter/aislamiento & purificación , Marte , Nave Espacial , Contaminación de Equipos , Exobiología , Medio Ambiente Extraterrestre , Peróxido de Hidrógeno , Esporas Bacterianas/metabolismo , Esporas Bacterianas/efectos de la radiaciónRESUMEN
Rock varnish from Arizona's Whipple Mountains harbors a microbial community containing about 10(8) microorganisms g(-1) of varnish. Analyses of varnish phospholipid fatty acids and rRNA gene libraries reveal a community comprised of mostly Proteobacteria but also including Actinobacteria, eukaryota, and a few members of the Archaea. Rock varnish represents a significant niche for microbial colonization.
Asunto(s)
Ecosistema , Sedimentos Geológicos/microbiología , Actinobacteria/clasificación , Actinobacteria/genética , Actinobacteria/aislamiento & purificación , Archaea/clasificación , Archaea/genética , Archaea/aislamiento & purificación , Biodiversidad , California , Células Eucariotas , Datos de Secuencia Molecular , Filogenia , Proteobacteria/clasificación , Proteobacteria/genética , Proteobacteria/aislamiento & purificación , ARN de Archaea/genética , ARN de Archaea/aislamiento & purificación , ARN Bacteriano/genética , ARN Bacteriano/aislamiento & purificación , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/aislamiento & purificaciónRESUMEN
Rapid microbial monitoring technologies are invaluable in assessing contamination of spacecraft and associated environments. Universal and widespread elements of microbial structure and chemistry are logical targets for assessing microbial burden. Several biomarkers such as ATP, LPS, and DNA (ribosomal or spore-specific), were targeted to quantify either total bioburden or specific types of microbial contamination. The findings of these assays were compared with conventional, culture-dependent methods. This review evaluates the applicability and efficacy of some of these methods in monitoring the microbial burden of spacecraft and associated environments. Samples were collected from the surfaces of spacecraft, from surfaces of assembly facilities, and from drinking water reservoirs aboard the International Space Station (ISS). Culture-dependent techniques found species of Bacillus to be dominant on these surfaces. In contrast, rapid, culture-independent techniques revealed the presence of many Gram-positive and Gram-negative microorganisms, as well as actinomycetes and fungi. These included both cultivable and noncultivable microbes, findings further confirmed by DNA-based microbial detection techniques. Although the ISS drinking water was devoid of cultivable microbes, molecular-based techniques retrieved DNA sequences of numerous opportunistic pathogens. Each of the methods tested in this study has its advantages, and by coupling two or more of these techniques even more reliable information as to microbial burden is rapidly obtained.
Asunto(s)
Ambiente Controlado , Microbiología Ambiental , Monitoreo del Ambiente/métodos , Medio Ambiente Extraterrestre , Nave Espacial , Adenosina Trifosfato/análisis , Bacterias/genética , Bioensayo/métodos , Recuento de Colonia Microbiana , ADN/genética , ADN/aislamiento & purificación , Filogenia , ARN Ribosómico/genética , Análisis de Secuencia de ADNRESUMEN
Biology is believed to play a large role in the cycling of iron and manganese in many freshwater environments, but specific microbial groups indigenous to these systems have not been well characterized. To investigate the populations of Bacteria and Archaea associated with metal-rich sediments from Green Bay, WI, we extracted nucleic acids and analysed the phylogenetic relationships of cloned 16S rRNA genes. Because nucleic acids have not been routinely extracted from metal-rich samples, we investigated the bias inherent in DNA extraction and gene amplification from pure MnO2 using defined populations of whole cells or naked DNA. From the sediments, we screened for manganese-oxidizing bacteria using indicator media and found three isolates that were capable of manganese oxidation. In the phylogenetic analysis of bacterial 16S rRNA gene clones, we found two groups related to known metal-oxidizing genera, Leptothrix of the beta-Proteobacteria and Hyphomicrobium of the alpha-Proteobacteria, and a Fe(III)-reducing group related to the Magnetospirillum genus of the alpha-Proteobacteria. Groups related to the metal-reducing delta-Proteobacteria constituted 22% of the gene clones. In addition, gene sequences from one group of methanogens and a group of Crenarchaeota, identified in the archaeal gene clone library, were related to those found previously in Lake Michigan sediments.
Asunto(s)
Archaea/aislamiento & purificación , Bacterias/aislamiento & purificación , Agua Dulce/microbiología , Sedimentos Geológicos/microbiología , Hierro/metabolismo , Manganeso/metabolismo , Alphaproteobacteria/clasificación , Alphaproteobacteria/aislamiento & purificación , Alphaproteobacteria/metabolismo , Archaea/clasificación , Archaea/metabolismo , Bacterias/clasificación , Bacterias/metabolismo , Betaproteobacteria/clasificación , Betaproteobacteria/aislamiento & purificación , Betaproteobacteria/metabolismo , Crenarchaeota/clasificación , Crenarchaeota/aislamiento & purificación , Crenarchaeota/metabolismo , Compuestos Férricos/metabolismo , Bacterias Aerobias Gramnegativas/clasificación , Bacterias Aerobias Gramnegativas/aislamiento & purificación , Bacterias Aerobias Gramnegativas/metabolismo , Hyphomicrobium/clasificación , Hyphomicrobium/aislamiento & purificación , Hyphomicrobium/metabolismo , Compuestos de Manganeso/metabolismo , Datos de Secuencia Molecular , Oxidación-Reducción , Óxidos/metabolismo , Filogenia , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , WisconsinRESUMEN
Although there is significant interest in the potential interactions of microbes with gas hydrate, no direct physical association between them has been demonstrated. We examined several intact samples of naturally occurring gas hydrate from the Gulf of Mexico for evidence of microbes. All samples were collected from anaerobic hemipelagic mud within the gas hydrate stability zone, at water depths in the ca. 540- to 2,000-m range. The delta(13)C of hydrate-bound methane varied from -45.1 per thousand Peedee belemnite (PDB) to -74.7 per thousand PDB, reflecting different gas origins. Stable isotope composition data indicated microbial consumption of methane or propane in some of the samples. Evidence of the presence of microbes was initially determined by 4,6-diamidino 2-phenylindole dihydrochloride (DAPI) total direct counts of hydrate-associated sediments (mean = 1.5 x 10(9) cells g(-1)) and gas hydrate (mean = 1.0 x 10(6) cells ml(-1)). Small-subunit rRNA phylogenetic characterization was performed to assess the composition of the microbial community in one gas hydrate sample (AT425) that had no detectable associated sediment and showed evidence of microbial methane consumption. Bacteria were moderately diverse within AT425 and were dominated by gene sequences related to several groups of Proteobacteria, as well as Actinobacteria and low-G + C Firmicutes. In contrast, there was low diversity of Archaea, nearly all of which were related to methanogenic Archaea, with the majority specifically related to Methanosaeta spp. The results of this study suggest that there is a direct association between microbes and gas hydrate, a finding that may have significance for hydrocarbon flux into the Gulf of Mexico and for life in extreme environments.