Culture-based Approach Research Articles

Diversity of bacteria in cloud water collected at a National Atmospheric Monitoring Station in Southern China

Bacteria in the atmosphere affect human health and atmospheric characteristics, however, few studies have investigated bacteria in high altitude areas in China, especially in clouds and fog. In this study, the bacterial community structures of three cloud water samples, collected at the Nanling national atmospheric monitoring station (112°53′56″E, 24°41′56″N) in China, were studied by combining culture-based approach and high-throughput sequencing technology. The results showed that the dominant bacteria in cloud water samples were Proteobacteria (71.36%), followed by Actinobacteria (21.72%) and bacteroidetes (6.43%), but bacterial species identified using a culture-based approach were <1% of the total bacteria. NO2, SO42−, and pH value were related to the community structures of bacterial, indicating anthropogenic emissions possibly enhanced the variety of bacterial communities in cloud water.

A multiscale study of fungal endophyte communities of the foliar endosphere of native rubber trees in Eastern Amazon

Hevea brasiliensis is a native hyperdiverse tree species in the Amazon basin with great economic importance since it produces the highest quality natural rubber. H. brasiliensis, in its natural habitat, may harbor fungal endophytes that help defend against phytopathogenic fungi. In this work, we investigated the fungal endophytic communities in two pristine areas in Eastern Amazon (Anavilhanas National Park – ANP and Caxiuanã National Forest – CNF) at different spatial scales: regional, local, individual (tree), and intra-individual (leaflet). Using a culture-based approach, 210 fungal endophytes were isolated from 240 sampling units and assigned to 46 distinct MOTUs based on sequencing of the nrITS DNA. The community compositions of the endophytomes are different at both regional and local scales, dominated by very few taxa and highly skewed toward rare taxa, with many endophytes infrequently isolated across hosts in sampled space. Colletotrichum sp. 1, a probably latent pathogen, was the most abundant endophytic putative species and was obtained from all individual host trees in both study areas. Although the second most abundant putative species differed between the two collection sites, Clonostachys sp. 1 and Trichoderma sp. 1, they are phylogenetically related (Hypocreales) mycoparasites. Thus, they probably exhibit the same ecological function in the foliar endosphere of rubber tree as antagonists of its fungal pathogens.

Implementing corporate social responsibility strategies in the hospitality and tourism firms: A culture-based approach

In accordance with the strategic importance of corporate social responsibility (CSR), multiple CSR studies have existed in the tourism and hospitality literature. Although appropriate CSR strategies and stakeholders’ interests and perceptions with regard to CSR may differ from one national culture to another, CSR studies incorporating cultural influences are still rare in the tourism and hospitality field. Particularly, given a high degree of internationalization and cultural diversity of stakeholder groups in each local market, CSR study on the basis of national culture is needed in the tourism and hospitality industry. In order to enrich CSR literature and provide practical implications, this study aims to propose a culture-based CSR implementation model for the hospitality and tourism firms, incorporating national culture.

Integrated Bacterial Identification and Antimicrobial Susceptibility Testing for Polymicrobial Infections Using Digital PCR and Digital High-Resolution Melt in a Microfluidic Array Platform.

In diagnosing bacterial infection, rapid bacterial identification (ID) and antimicrobial susceptibility testing (AST) are critical to clinicians in order to provide an effective treatment in a timely manner. The gold standard, culture-based approach provides both ID and antimicrobial susceptibility but requires several days of turnaround time. Especially in polymicrobial infections, where there are more than one organisms interacting collectively that can complicate the treatment. Here, we demonstrate a rapid bacterial diagnostic approach that is capable of bacterial ID/AST in heterogeneous samples within less than 4 hours by using digital PCR (dPCR) and digital high-resolution melt via microfluidic devices. By utilizing dPCR, we are able to quantify amount of nucleic acid, which correlates to phenotypic responses of {\bf individual pathogens in a mixed sample and also shorten the required time of antibiotic exposure. In addition, we employ a machine learning algorithm to automatically identify bacterial species based on melt profiles of 16S rRNA gene.

Coins and Cosmologies in Iron Age Western Britain

This paper offers a material culture-based approach to British Western Iron Age coins (i.e. those often attributed to the Dobunni). Through analysis of the materiality and imagery of these objects, the author explores the embodiment of later Iron Age cosmologies. In doing so, the cycles of day and night and of life and death are discussed. The ways in which these cosmologies could have been transposed onto the landscape through coin production and depositional contexts helps to demonstrate how Iron Age societies in Western Britain may have understood their world and confirmed their space within it.

Diurnal patterns of airborne algae in the Hawaiian Islands: a preliminary study

Although the literature on the diversity of airborne algal communities in various locations around the world is increasing, little is known about their temporal and spatial patterns. We compared airborne algal communities from Honolulu, Hawai‘i, USA, over three 24-h sampling periods to examine diurnal patterns in diversity and abundance. Using a culture-based approach, 192 algal colonies were characterized and identified as 31 operational taxonomic units. A combination of microscopy and Sanger sequencing (of the UPA marker) was used for characterizations. More airborne algal colonies were identified from nighttime collections (127 of 192 colonies) than daytime collections (65 of 192 colonies) (p < 0.0001). Similarly, 95% of the daytime collections were Cyanobacteria, and 87% of the nighttime collections were Chlorophyta, and the trends of more Cyanobacteria being collected during the day and more Chlorophyta at night were significant (p < 0.0001). Meteorological analyses for the sampling periods indicated that air masses sampled during the three trials consistently arrived in the Hawaiian Islands on a northeast trade wind pattern, but with different origins in the Pacific Ocean, and that low-to-trace levels of rain fell during the sampling periods. Land breeze and sea breeze effects, which are common temperature-driven phenomena on tropical islands, may have played a role in the diurnal pattern observed in the current study.

Microbiome analysis and bacterial isolation from Lejía Lake soil in Atacama Desert.

As a consequence of the severe climatic change affecting our entire world, many lakes in the Andes Cordillera are likely to disappear within a few decades. One of these lakes is Lejía Lake, located in the central Atacama Desert. The objectives of this study were: (1) to characterize the bacterial community from Lejía Lake shore soil (LLS) using 16S rRNA sequencing and (2) to test a culture-based approach using a soil extract medium (SEM) to recover soil bacteria. This extreme ecosystem was dominated by three phyla: Bacteroidetes, Proteobacteria, and Firmicutes with 29.2, 28.2 and 28.1% of the relative abundance, respectively. Using SEM, we recovered 7.4% of the operational taxonomic units from LLS, all of which belonged to the same three dominant phyla from LLS (6.9% of Bacteroidetes, 77.6% of Proteobacteria, and 15.3% of Firmicutes). In addition, we used SEM to recover isolates from LLS and supplemented the culture medium with increasing salt concentrations to isolate microbial representatives of salt tolerance (Halomonas spp.). The results of this study complement the list of microbial taxa diversity from the Atacama Desert and assess a pipeline to isolate selective bacteria that could represent useful elements for biotechnological approaches.

Celebrating centuries: Pink-pigmented bacteria from rosy patinas in the House of Bicentenary (Herculaneum, Italy)

Abstract The archaeological site of Herculaneum (Campania, Italy), which was buried as a result of the eruption of Mt. Vesuvius in the first Century C.E., was first excavated in the XVIII century. It has been included on the UNESCO World Heritage List since 1997, and in 2001 the Herculaneum Conservation Project (HCP) was started, which has carried out, among other activities, studies and conservation interventions across the site. Up until this time, little data has been available on the growth of biological agents that could cause biodeterioration of wall paintings and archaeological structures. Particularly, the presence of rosy discoloration is frequent on ancient monuments of the Vesuvian area, even if such phenomenon has so far been largely neglected. In this study, we describe, for the first time, the pink patina distribution and the microbial species isolated from the House of the Bicentenary in Herculaneum. By combining culture-based approaches with molecular and phylogenetic analyses we reliably isolated the pink-producing bacterial species and attributed them primarily to Arthrobacter agilis, and secondarily to Rhodococcus corynebacterioides, and Methylobacterium extorquens. Strains closely related to Dietzia maris and Gordonia rubripertincta were also isolated. With the exception of M. extorquens, a proteobacterium, all of the other isolates belong to the phylum Actinobacteria. All isolates produced carotenoid pigments, suggesting that they can participate in the development of such peculiar coloration. Our data indicate the presence of a large number of pink-pigmented bacterial species in the community, even if the presence of bacteria in viable but not-culturable state, such as Rubrobacter radiotolerans, is not excludible. The culture-based approach had the advantage of (i) obtaining bacterial isolates, (ii) showing their differential ability to produce pink discolorations, and (iii) testing conditions for in vitro growth. Moreover, field observations showed an association of pink patinas with dry conditions, saline efflorescence and moderate solar radiation. Some seasonal variations were also detected, with an increase in late spring and summer.

Distributions of ectomycorrhizal and foliar endophytic fungal communities associated with Pinus ponderosa along a spatially constrained elevation gradient.

Understanding distributions of plant-symbiotic fungi is important for projecting responses to environmental change. Many coniferous trees host ectomycorrhizal fungi (EM) in association with roots and foliar endophytic fungi (FE) in leaves. We examined how EM and FE associated with Pinus ponderosa each vary in abundance, diversity, and community structure over a spatially constrained elevation gradient that traverses four plant communities, 4°C in mean annual temperature, and 15 cm in mean annual precipitation. We sampled 63 individuals of Pinus ponderosa in 10 sites along a 635 m elevation gradient that encompassed a geographic distance of 9.8 km. We used standard methods to characterize each fungal group (amplified and sequenced EM from root tips; isolated and sequenced FE from leaves). Abundance and diversity of EM were similar across sites, but community composition and distributions of the most common EM differed with elevation (i.e., with climate, soil chemistry, and plant communities). Abundance and composition of FE did not differ with elevation, but diversity peaked in mid-to-high elevations. Our results suggest relatively tight linkages between EM and climate, soil chemistry, and plant communities. That FE appear less linked with these factors may speak to limitations of a culture-based approach, but more likely reflects the small spatial scale encompassed by our study. Future work should consider comparable methods for characterizing these functional groups, and additional transects to understand relationships of EM and FE to environmental factors that are likely to shift as a function of climate change.

Culture-free bacterial detection and identification from blood with rapid, phenotypic, antibiotic susceptibility testing

The current culture-based approach for the diagnosis of bloodstreams infection is incommensurate with timely treatment and curbing the prevalence of multi-drug resistant organisms (MDROs) due to its long time-to-result. Bloodstream infections typically involve extremely low (e.g., <10 colony-forming unit (CFU)/mL) bacterial concentrations that require a labor-intensive process and as much as 72 hours to yield a diagnosis. Here, we demonstrate a culture-free approach to achieve rapid diagnosis of bloodstream infections. An immuno-detection platform with intrinsic signal current amplification was developed for the ultrasensitive, rapid detection, identification (ID) and antibiotic susceptibility testing (AST) of infections. With its capability of monitoring short-term (1–2 hours) bacterial growth in blood, the platform is able to provide 84-minute simultaneous detection and ID in blood samples below the 10 CFU/mL level and 204-minute AST. The susceptible-intermediate-resistant AST capacity was demonstrated.

Handout Matematika berbantuan Etnomatematika Berbasis Budaya Lokal

This study aims to develop teaching materials in the form of handouts through a local culture-based ethnomathematics approach to the wake-up material. The research model used is the research of Borg and Gall development, but in this study only limited to seven steps of research and development, namely: 1) Potential and Problems; 2) Gathering information; 3) Product Design; 4) Design Validation; 5) Design Revisions; 6) Product Trial; and 7) Revision of the Ahir Phase. Data collection techniques used in this study, namely interviews, observation, and questionnaires (questionnaires). Data analysis techniques using descriptive qualitative analysis that describes the results of product development. Based on the result of data analysis, it is evaluated from the quality aspect of the prevalence of teaching materials to get the average score of 3.97 with the category of "Valid". While the learner's response to a limited trial earned an average grade of 3.67 with the "Practical" category, and the questionnaire response of learners on a large trial earned an average of 4.02 under the "Pull" category. It can be concluded that the developed teaching materials have valid, practical, and interesting criteria that can be used as teaching materials in teaching and learning activities.

Effect of Dairy Manure Storage Conditions on the Survival of E. coli O157:H7 and Listeria.

Dairy manure is regularly applied to crop fields as a solid or liquid to improve the soil nutrient status. However, pathogens may survive during manure storage and enter the environment during application. In this study, three storage practices were evaluated to understand the survival patterns of O157:H7 and spp. in dairy manure using a culture-based approach. To replicate common farm manure storage techniques, solid manure was stacked as piles with periodic turning or as static piles without turning, whereas liquid manure (feces, urine, and water) was stored as a slurry in small tanks to simulate lagoon conditions. The and levels in the manure samples were determined for 29 wk. Results showed that there was an initial reduction in bacteria levels in the first month; however, both and managed to survive in the solid manure piles for the full study period. In slurry samples, was not detected after 14 wk, but survived until the end of the experiment at relatively lower levels than in the solid manure piles. Ambient weather and pile size were identified as the main reasons for bacteria survival during the course of the experiment. The outcome of this study is important in terms of understanding pathogen survival in manure piles and slurries prior to their application to crop fields.

Determination of the Bacteriophage Host Range: Culture-Based Approach.

The bacteriophage host range is one of the most practically important characteristics of each bacterial virus. Here the classical plate-culture-based approach for bacteriophage host range determination is described. The important considerations related to interpretation of the data and limitations of the methods are discussed.

Strategies for preventing group B streptococcal infections in newborns: a nation-wide survey of Italian policies

BackgroundThere are no Italian data regarding the strategies for preventing neonatal group B streptococcal (GBS) infection. We conducted a national survey in order to explore obstetrical, neonatal and microbiological practices for the GBS prevention.MethodsThree distinct questionnaires were sent to obstetricians, neonatologists and microbiologists. Questionnaires included data on prenatal GBS screening, maternal risk factors, intrapartum antibiotic prophylaxis, microbiological information concerning specimen processing and GBS antimicrobial susceptibility.ResultsAll respondent obstetrical units used the culture-based screening approach to identify women who should receive intrapartum antibiotic prophylaxis, and more than half of the microbiological laboratories (58%) reported using specimen processing consistent with CDC guidelines. Most neonatal units (89 out of 107, 82%) reported using protocols for preventing GBS early-onset sepsis consistent with CDC guidelines.ConclusionsThe screening-based strategy is largely prevalent in Italy, and most protocols for preventing GBS early-onset sepsis are consistent with CDC guidelines. However, we found discrepancies in practices among centers that may reflect the lack of Italian guidelines issued by public health organizations.

Tissue Culture (Somatic Embryogenesis)-Induced Tnt1 Retrotransposon-Based Mutagenesis in Brachypodium distachyon.

Brachypodium distachyon is a model grass species for economically important cereal crops. Efforts are in progress to develop useful functional genomic resources in Brachypodium. A tobacco retrotransposon, Tnt1, has been used successfully in recent past to generate insertional mutagenesis in several dicot plant species. Tnt1 retrotransposon replicates, transposes, and inserts at multiple random genomic locations in the plant genome. Transposition occurs only during somatic embryogenesis but not during seed transmission. We developed Brachypodium transgenic plants that can express the Tnt1 element. Here, we describe an efficient tissue culture-based approach to generate Tnt1 insertional mutant population using transgenic Brachypodium line expressing the Tnt1 retrotransposon.

Diversity of the cultivable gut bacterial communities associated with the fruit flies Bactrocera dorsalis and Bactrocera cucurbitae (Diptera: Tephritidae)

Gut microbes play an important role in insect morphogenesis, nutrition, development of resistance against parasitoids and detoxification of toxic compounds. A culture-based approach is therefore an useful tool for the characterization of cultivable microbial communities associated with the insect gut. In the present study an attempt was made to decipher the gender specificity of gut bacterial communities of two major fruit fly species of India viz., Bactrocera dorsalis (Hendel) and Bactrocera cucurbitae (Conquillett) (Diptera: Tephritidae). Based on molecular identification, B. dorsalis females were found to predominantly harbor the bacterial species Enterobacter cloacae, Enterobacter asburiae and Citrobacter freundii, while B. dorsalis males were found to harbor Providencia rettgerii, Klebsiella oxytoca, Enterococcus faecalis and Pseudomonas aeruginosa The cultivable diversity from females of B. cucurbitae comprised mainly of Morganella morganii and Bacillus pumilis while B.cucurbitae males were predominantly colonized by aerobic endospore formers viz., Bacillus cereus, B. licheniformis and B. subtilis. The above findings have thrown light on a distinct pattern of gender specific gut bacterial colonization in fruit flies, which have to be factored in for the formulation of fruit fly management strategies.

Phylogenetic diversity and functional characterization of the Manila clam microbiota: a culture-based approach.

According to the hologenome theory, the microbiota contributes to the fitness of the holobiont having an important role in its adaptation, survival, development, health, and evolution. Environmental stress also affects the microbiota and its capability to assist the holobiont in coping with stress factors. Here, we analyzed the diversity of cultivable bacteria associated with Manila clam tissues (mantle, gills, hemolymph) in two non-contaminated sites (Portugal and France) and one metal-contaminated site (Portugal). A total of 240 isolates were obtained. Representative isolates (n=198) of the overall diversity were identified by 16S rDNA sequencing and subjected to functional characterization. Isolates affiliated with Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. Proteobacteria (mostly Pseudoalteromonadaceae and Vibrionaceae) were dominant in non-contaminated sites while Actinobacteria (mostly Microbacteriaceae) dominated in the metal-contaminated site. The main factor affecting the microbiota composition was contamination. No significant differences were observed between clam tissues and geographic regions. Several isolates tested positive for antibacterial activity, biofilm formation, protease, and siderophore production. The results show that the Manila clam harbors a diverse microbiota that may contribute to clam protection and overall fitness, as well as to its adaptation to stressful environments. In addition, the Manila clam microbiota is revealed as a promising source of novel probiotics with potential application in aquaculture.

Impact of three different fungicides on fungal epi- and endophytic communities of common bean (Phaseolus vulgaris) and broad bean (Vicia faba)

ABSTRACTIn this study, the impacts of three different fungicides to fungal phyllosphere communities on broad bean (Vicia faba, Fabaceae) and common bean (Phaseolus vulgaris, Fabaceae) were analyzed. The fungicides included copper, sulfur, and azoxystrobin. The plants were sowed, grown, and treated under conditions occurring in conventional and organic farming. A culture-based approach was used to identify changes in the phyllosphere fungal community after the treatment. Different effects on species richness and growth index of the epiphytic and endophytic communities for common bean and broad bean could be shown. Treatments with sulfur showed the weakest effect, followed by those based on copper and the systemic azoxystrobin, which showed the strongest effect especially on endophytic communities. The epiphytic fungal community took five weeks to recover after treatment with azoxystrobin. However, the effect of azoxystrobin on the endophytic community lasted more than five weeks. Finally, the data suggest that the surface structure of the host leaves have a huge impact on the mode of action that the fungicides exert.

The fungal composition of natural biofinishes on oil-treated wood

BackgroundBiofinished wood is considered to be a decorative and protective material for outdoor constructions, showing advantages compared to traditional treated wood in terms of sustainability and self-repair. Natural dark wood staining fungi are essential to biofinish formation on wood. Although all sorts of outdoor situated timber are subjected to fungal staining, the homogenous dark staining called biofinish has only been detected on specific vegetable oil-treated substrates. Revealing the fungal composition of various natural biofinishes on wood is a first step to understand and control biofinish formation for industrial application.ResultsA culture-based survey of fungi in natural biofinishes on oil-treated wood samples showed the common wood stain fungus Aureobasidium and the recently described genus Superstratomyces to be predominant constituents. A culture-independent approach, based on amplification of the internal transcribed spacer regions, cloning and Sanger sequencing, resulted in clone libraries of two types of biofinishes. Aureobasidium was present in both biofinish types, but was only predominant in biofinishes on pine sapwood treated with raw linseed oil. Most cloned sequences of the other biofinish type (pine sapwood treated with olive oil) could not be identified. In addition, a more in-depth overview of the fungal composition of biofinishes was obtained with Illumina amplicon sequencing that targeted the internal transcribed spacer region 1. All investigated samples, that varied in wood species, (oil) treatments and exposure times, contained Aureobasidium and this genus was predominant in the biofinishes on pine sapwood treated with raw linseed oil. Lapidomyces was the predominant genus in most of the other biofinishes and present in all other samples. Surprisingly, Superstratomyces, which was predominantly detected by the cultivation-based approach, could not be found with the Illumina sequencing approach, while Lapidomyces was not detected in the culture-based approach.ConclusionsOverall, the culture-based approach and two culture-independent methods that were used in this study revealed that natural biofinishes were composed of multiple fungal genera always containing the common wood staining mould Aureobasidium. Besides Aureobasidium, the use of other fungal genera for the production of biofinished wood has to be considered.

Oomycete Species Associated with Soybean Seedlings in North America-Part II: Diversity and Ecology in Relation to Environmental and Edaphic Factors.

Soybean (Glycine max (L.) Merr.) is produced across a vast swath of North America, with the greatest concentration in the Midwest. Root rot diseases and damping-off are a major concern for production, and the primary causal agents include oomycetes and fungi. In this study, we focused on examination of oomycete species distribution in this soybean production system and how environmental and soil (edaphic) factors correlate with oomycete community composition at early plant growth stages. Using a culture-based approach, 3,418 oomycete isolates were collected from 11 major soybean-producing states and most were identified to genus and species using the internal transcribed spacer region of the ribosomal DNA. Pythium was the predominant genus isolated and investigated in this study. An ecology approach was taken to understand the diversity and distribution of oomycete species across geographical locations of soybean production. Metadata associated with field sample locations were collected using geographical information systems. Operational taxonomic units (OTU) were used in this study to investigate diversity by location, with OTU being defined as isolate sequences with 97% identity to one another. The mean number of OTU ranged from 2.5 to 14 per field at the state level. Most OTU in this study, classified as Pythium clades, were present in each field in every state; however, major differences were observed in the relative abundance of each clade, which resulted in clustering of states in close proximity. Because there was similar community composition (presence or absence) but differences in OTU abundance by state, the ordination analysis did not show strong patterns of aggregation. Incorporation of 37 environmental and edaphic factors using vector-fitting and Mantel tests identified 15 factors that correlate with the community composition in this survey. Further investigation using redundancy analysis identified latitude, longitude, precipitation, and temperature as factors that contribute to the variability observed in community composition. Soil parameters such as clay content and electrical conductivity also affected distribution of oomycete species. The present study suggests that oomycete species composition across geographical locations of soybean production is affected by a combination of environmental and edaphic conditions. This knowledge provides the basis to understand the ecology and distribution of oomycete species, especially those able to cause diseases in soybean, providing cues to develop management strategies.