Culture-independent Methods Research Articles

Diversity of the protease-producing bacteria and their extracellular protease in the coastal mudflat of Jiaozhou Bay, China: in response to clam naturally growing and aquaculture.

The booming mudflat aquaculture poses an accumulation of organic matter and a certain environmental threat. Protease-producing bacteria are key players in regulating the nitrogen content in ecosystems. However, knowledge of the diversity of protease-producing bacteria in coastal mudflats is limited. This study investigated the bacterial diversity in the coastal mudflat, especially protease-producing bacteria and their extracellular proteases, by using culture-independent methods and culture-dependent methods. The clam aquaculture area exhibited a higher concentration of carbon, nitrogen, and phosphorus when compared with the non-clam area, and a lower richness and diversity of bacterial community when compared with the clam naturally growing area. The major classes in the coastal mud samples were Bacteroidia, Gammaproteobacteria, and Alphaproteobacteria. The Bacillus-like bacterial community was the dominant cultivated protease-producing group, accounting for 52.94% in the non-clam area, 30.77% in the clam naturally growing area, and 50% in the clam aquaculture area, respectively. Additionally, serine protease and metalloprotease were the principal extracellular protease of the isolated coastal bacteria. These findings shed light on the understanding of the microbes involved in organic nitrogen degradation in coastal mudflats and lays a foundation for the development of novel protease-producing bacterial agents for coastal mudflat purification.

Immunomagnetic separation coupled with flow cytometry for the analysis of Legionella pneumophila in aerosols

Legionella pneumophila are pathogenic bacteria that can be found in high concentrations in artificial water systems like evaporative cooling towers, which have been the source of frequent outbreaks in recent years. Since inhaled L. pneumophila can lead to Legionnaires’ disease, the development of suitable sampling and rapid analysis strategies for these bacteria in aerosols is therefore of great relevance. In this work, different concentrations of viable L. pneumophila Sg 1 were nebulized and sampled by the cyclone sampler Coriolis® µ under defined conditions in a bioaerosol chamber. To quantify intact Legionella cells, the collected bioaerosols were subsequently analyzed by immunomagnetic separation coupled with flow cytometry (IMS-FCM) on the platform rqmicro.COUNT. For analytical comparison, measurements with qPCR and cultivation were performed. Limits of detection (LOD) of 2.9 × 103 intact cells m−3 for IMS-FCM and 7.8 × 102 intact cells m−3 for qPCR indicating a comparable sensitivity as in culture (LOD = 1.5 × 103 culturable cells m−3). Over a working range of 103 − 106 cells mL−1, the analysis of nebulized and collected aerosol samples with IMS-FCM and qPCR provides higher recovery rates and more consistent results than by cultivation. Overall, IMS-FCM is a suitable culture-independent method for quantification of L. pneumophila in bioaerosols and is promising for field application due to its simplicity in sample preparation.Graphical abstract

Insight into Andrographis paniculata associated bacterial endomicrobiome and assessment of culturable bacterial endophytes for enhancement of industrially important andrographolide content

Andrographis paniculata is a common medicinal plant used worldwide for its preventive and curative properties. However, no information exists about the A. paniculata associated bacterial endophytes and their potential relationship with the plant-bioactive substances. In this study, we investigated the bacterial endophytic diversity of A. paniculata root and shoot tissues using culture-based and culture-independent high-throughput sequencing methods. The culture-independent analyses unveiled the presence of 465 operational taxonomic units (OTU’s) and 232 genera, respectively in the samples. Further, downstream categorization of OTU's revealed the dominance of Proteobacteria, Firmicutes, Actinobacteria and Bacteriodetes phylum members, with Bacillus being the most prevalent endophytic genera in the root, while Pseudomonas dominated the shoot tissues. A total of 39 bacterial endophytes from the roots, stems, leaves, and seeds of A. paniculata were isolated by a culture-dependent investigation whose subsequent examination using 16S rRNA gene sequencing confirmed a similar pattern of the dominance of the Bacillus and Pseudomonas genera. When all the isolates were further screened for their plant growth promotion activity, Micrococcus luteus (ASd6) significantly enhanced the biomass and andrographolide content by 68.01% and 44.16%, respectively. Taken together, the information captured through metagenomics and culture-dependent approach reveals some promising bacterial endophytes which could be employed in field for growth promotion and in planta secondary metabolite enhancement studies in near future.

Using Genomics to Decipher the Enigmatic Properties and Survival Adaptation of Candidate Phyla Radiation.

Microbial ecology is a critical field for understanding the composition, diversity, and functions of microorganisms in various environmental and health-related processes. The discovery of Candidate Phyla Radiation (CPR) through culture-independent methods has introduced a new division of microbes characterized by a symbiotic/parasitic lifestyle, small cell size, and small genome. Despite being poorly understood, CPRs have garnered significant attention in recent years due to their widespread detection in a variety of environmental and clinical samples. These microorganisms have been found to exhibit a high degree of genetic diversity compared to other microbes. Several studies have shed light on their potential importance in global biogeochemical cycles and their impact on various human activities. In this review, we provide a systematic overview of the discovery of CPRs. We then focus on describing how the genomic characteristics of CPRs have helped them interact with and adapt to other microbes in different ecological niches. Future works should focus on discovering the metabolic capacities of CPRs and, if possible, isolating them to obtain a better understanding of these microorganisms.

The Skin Microbiome: Current Techniques, Challenges, and Future Directions.

Skin acts as a barrier that promotes the colonization of bacteria, fungi, archaea, and viruses whose membership and function may differ depending on the various specialized niches or micro-environments of the skin. The group of microorganisms inhabiting the skin, also known as the skin microbiome, offers protection against pathogens while actively interacting with the host's immune system. Some members of the skin microbiome can also act as opportunistic pathogens. The skin microbiome is influenced by factors such as skin site, birth mode, genetics, environment, skin products, and skin conditions. The association(s) of the skin microbiome with health and disease has (have) been identified and characterized via culture-dependent and culture-independent methods. Culture-independent methods (such as high-throughput sequencing), in particular, have expanded our understanding of the skin microbiome's role in maintaining health or promoting disease. However, the intrinsic challenges associated with the low microbial biomass and high host content of skin microbiome samples have hindered advancements in the field. In addition, the limitations of current collection and extraction methods and biases derived from sample preparation and analysis have significantly influenced the results and conclusions of many skin microbiome studies. Therefore, the present review discusses the technical challenges associated with the collection and processing of skin microbiome samples, the advantages and disadvantages of current sequencing approaches, and potential future areas of focus for the field.

IN SILICO COMPARATIVE METAGENOMICS ANALYSIS OF HEAVY METALS’ AFFECTED MICROBIAL COMMUNITIES

Plethora of microorganisms is present on Earth and play crucial role in geochemical cycles. A major proportion of microorganisms is uncultured and therefore culture independent methods are most suited to study the microbial diversity comprehensively. This has led to microbial molecular ecology fields including metagenomics. In this study, metagenomics data of microbial communities of different sites contaminated with different heavy metals such as chromium, arsenic, lead, selenium, copper and cadmium were compared in silico to determine the microorganisms which are common in different samples and the microorganisms that are exclusive to certain samples only. For this purpose, 16S rRNA gene sequences of heavy metal contaminated sites was downloaded from NCBI Short Read Archive (SRA). The sequences were analyzed in Mothur tool through Galaxy server. The sequences were classified using Ribosomal Database Project (RDP) reference dataset and Operational Taxonomic Units (OTUs) were generated. Heatmaps, Venn diagrams, phylogenetic tree, and NMDS plots were generation. Diversity indices such as Shannon, Simpson and Chao, as well as relative abundance was determined. It was found that Proteobacteria, Actinobacteria, Acidobacteria, Fermicutes, Bacteriodetes, and Bacilli were the most abundant bacteria and were present in all heavy metal contaminated samples. Proteobacteria, and Beta-Proteobacteria, were present in all samples but were most abundant in cadmium affected samples. Xanthomonadales were most abundant in lead contaminated samples. Firmicutes were most abundant in chromium affected samples. Bacteriodetes were present in all arsenic contaminated samples but were not detected in any other samples. Rhizobiales were present in all arsenic and lead contaminated samples. Analysis of molecular variance (AMOVA) test was performed and it was found that the microbial communities of all the samples contaminated with different samples were found to be statistically different (p-value ?0.001). The bacteria which are present in all samples might have resistant against all these heavy metals and thus should be explored further for possible applications of bioremediation.

Exploring yeast diversity of dry-salted naturally black olives from Greek retail outlets with culture dependent and independent molecular methods

In the present study, the physicochemical (pH, water activity, moisture content, salt concentration) classical plate counts (total viable counts, yeasts, lactic acid bacteria, Staphylococcus aureus, Pseudomonas spp., Enterobacteriaceae) and amplicon sequencing of naturally black dry-salted olives obtained from different retail outlets of the Greek market were investigated. According to the results, the values of the physicochemical characteristics presented great variability among the samples. Specifically, pH and water activity (aw) values ranged between 4.0 and 5.0, as well as between 0.58 and 0.91, respectively. Moisture content varied between 17.3 and 56.7 % (g Η2Ο/100 g of olive pulp), whereas salt concentration ranged from 5.26 to 9.15 % (g NaCl/100 g of olive pulp). No lactic acid bacteria, S. aureus, Pseudomonas spp. and Enterobacteriaceae were detected. The mycobiota consisted of yeasts that were further characterized and identified by culture-dependent (rep-PCR, ITS-PCR, and RFLP) and amplicon target sequencing (ATS). Pichia membranifaciens, Candida sorbosivorans, Citeromyces nyonsensis, Candida etchelsii, Wickerhamomyces subpelliculosus, Candida apicola, Wickerhamomyces anomalus, Torulaspora delbrueckii and Candida versatilis were the dominant species according to ITS sequencing (culture-dependent), while ATS revealed the dominance of C. etchelsii, Pichia triangularis, P. membranifaciens, and C. versatilis among samples. The results of this study demonstrated considerable variability in quality attributes among the different commercial samples of dry-salted olives, reflecting a lack of standardization in the processing of this commercial style. However, the majority of the samples were characterized by satisfactory microbiological and hygienic quality and complied with the requirements of the trade standard for table olives of the International Olive Council (IOC) for this processing style in terms of salt concentration. In addition, the diversity of yeast species was elucidated for the first time in commercially available products, increasing our knowledge on the microbial ecology of this traditional food. Further investigation into the technological and multifunctional traits of the dominant yeast species may result in better control during dry-salting and enhance the quality and shelf-life of the final product.

Decoding the microbial universe with metagenomics: a brief insight.

A major part of any biological system on earth involves microorganisms, of which the majority are yet to be cultured. The conventional methods of culturing microbes have given fruitful outcomes yet have limitations. The curiosity for better understanding has led to the development of culture-independent molecular methods that help push aside the roadblocks of earlier methods. Metagenomics unifies the scientific community in search of a better understanding of the functioning of the ecosystem and its component organisms. This approach has opened a new paradigm in advanced research. It has brought to light the vast diversity and novelty among microbial communities and their genomes. This review focuses on the development of this field over time, the techniques and analysis of data generated through sequencing platforms, and its prominent interpretation and representation.

Yeast diversity during the spontaneous fermentation of wine with only the microbiota on grapes cultivated in Japan

Making wine via spontaneous fermentation without sulfur dioxide and commercial yeast (spontaneous winemaking) is increasing in recent year, but there is scant research regarding microbial communities present in Japan during spontaneous winemaking using culture-independent molecular methods. We analyzed fungal communities and populations during laboratory-scale spontaneous winemaking using sterilized labware to avoid winery-resident microbes. In the spontaneous fermentation of four grape varieties (Pinot Noir, Riesling, Koshu, and Koshusanjaku) grown in the same Japanese vineyard, our analysis of yeast and other fungal species by next-generation sequencing based on the ITS1 region demonstrated that Saccharomyces cerevisiae was eventually dominant in seven of 12 fermentation batches (three replications for each grape variety), whereas non-Saccharomyces species (e.g., Schizosaccharomyces japonicus, Lachancea dasiensis, and Hanseniaspora valbyensis) became dominant in four batches at the end of fermentation. In another batch, lactic acid bacteria (LAB) became dominant and the fermentation remained incomplete. Diverse microbes were involved in the spontaneous fermentation (particularly in Koshusanjaku), indicating that residual sugar remained and lactic and acetic acid largely increased. Compared to the control wine made with SO2 and commercial yeast, the concentration of lactic acid was 47-fold higher in the must dominated by L.dasiensis, and the concentrations of acetic acid and lactic acid were 10-fold and 20-fold higher in the must dominated by LAB, respectively. Even when indigenous S.cerevisiae became dominant, the finished wines obtained high sensory-analysis scores for complexity but low scores for varietal typicality, indicating the risk of fermentation with unselected wild yeast on the grapes grown in Japan.

Colistin Resistance Genes in Broiler Chickens in Tunisia.

Colistin is a polymyxin antibiotic that has been used in veterinary medicine for decades, as a treatment for enterobacterial digestive infections as well as a prophylactic treatment and growth promoter in livestock animals, leading to the emergence and spread of colistin-resistant Gram-negative bacteria and to a great public health concern, considering that colistin is one of the last-resort antibiotics against multidrug-resistant deadly infections in clinical practice. Previous studies performed on livestock animals in Tunisia using culture-dependent methods highlighted the presence of colistin-resistant Gram-negative bacteria. In the present survey, DNA extracted from cloacal swabs from 195 broiler chickens from six farms in Tunisia was tested via molecular methods for the ten mobilized colistin resistance (mcr) genes known so far. Of the 195 animals tested, 81 (41.5%) were mcr-1 positive. All the farms tested were positive, with a prevalence ranging from 13% to 93%. These results confirm the spread of colistin resistance in livestock animals in Tunisia and suggest that the investigation of antibiotic resistance genes by culture-independent methods could be a useful means of conducting epidemiological studies on the spread of antimicrobial resistance.

Endophytic Fungal Community of Stellera chamaejasme L. and Its Possible Role in Improving Host Plants' Ecological Flexibility in Degraded Grasslands.

Stellera chamaejasme L. is a widely distributed poisonous plant in Chinese degraded grasslands. To investigate the role of endophytic fungi (EF) in S. chamaejasme's quick spread in grasslands, the endophytic fungal community of S. chamaejasme was studied through culture-dependent and culture-independent methods, and the plant-growth-promoting (PGP) traits of some culturable isolates were tested. Further, the growth-promoting effects of 8 isolates which showed better PGP traits were evaluated by pot experiments. The results showed that a total of 546 culturable EF were isolated from 1114 plant tissue segments, and the colonization rate (CR) of EF in roots (33.27%) was significantly higher than that in shoots (22.39%). Consistent with this, the number of specific types of EF was greater in roots (8 genera) than in shoots (1 genus). The same phenomenon was found in culture-independent study. There were 95 specific genera found in roots, while only 18 specific genera were found in shoots. In addition, the dominant EF were different between the two study methods. Cladosporium (18.13%) and Penicillium (15.93%) were the dominant EF in culture-dependent study, while Apiotrichum (13.21%) and Athelopsis (5.62%) were the dominant EF in culture-independent study. PGP trait tests indicated that 91.30% of the tested isolates (69) showed phosphorus solubilization, IAA production, or siderophores production activity. The benefit of 8 isolates on host plants' growth was further studied by pot experiments, and the results indicated that all of the isolates can improve host plants' growth. Among them, STL3G74 (Aspergillus niger) showed the best growth-promotion effect; it can increase the plant's shoot and root dry biomass by 68.44% and 74.50%, respectively, when compared with the controls. Our findings revealed that S. chamaejasme has a wide range of fungal endophytic assemblages, and most of them possess PGP activities, which may play a key role in its quick spread in degraded grasslands.

Actinobacteria from Arctic and Atlantic deep-sea sediments-Biodiversity and bioactive potential.

The deep-sea covers over 70% of the Earth's surface and harbors predominantly uncharacterized bacterial communities. Actinobacteria are the major prokaryotic source of bioactive natural products that find their way into drug discovery programs, and the deep-sea is a promising source of biotechnologically relevant actinobacteria. Previous studies on actinobacteria in deep-sea sediments were either regionally restricted or did not combine a community characterization with the analysis of their bioactive potential. Here we characterized the actinobacterial communities of upper layers of deep-sea sediments from the Arctic and the Atlantic (Azores and Madeira) ocean basins, employing 16S rRNA metabarcoding, and studied the biosynthetic potential of cultivable actinobacteria retrieved from those samples. Metabarcoding analysis showed that the actinobacterial composition varied between the sampled regions, with higher abundance in the Arctic samples but higher diversity in the Atlantic ones. Twenty actinobacterial genera were detected using metabarcoding, as a culture-independent method, while culture-dependent methods only allowed the identification of nine genera. Isolation of actinobacteria resulted on the retrieval of 44 isolates, mainly associated with Brachybacterium, Microbacterium, and Brevibacterium genera. Some of these isolates were only identified on a specific sampled region. Chemical extracts of the actinobacterial isolates were subsequently screened for their antimicrobial, anticancer and anti-inflammatory activities. Extracts from two Streptomyces strains demonstrated activity against Candida albicans. Additionally, eight extracts (obtained from Brachybacterium, Brevibacterium, Microbacterium, Rhodococcus, and Streptomyces isolates) showed significant activity against at least one of the tested cancer cell lines (HepG2 and T-47D). Furthermore, 15 actinobacterial extracts showed anti-inflammatory potential in the RAW 264.4 cell model assay, with no concomitant cytotoxic response. Dereplication and molecular networking analysis of the bioactive actinobacterial extracts showed the presence of some metabolites associated with known natural products, but one of the analyzed clusters did not show any match with the natural products described as responsible for these bioactivities. Overall, we were able to recover taxonomically diverse actinobacteria with different bioactivities from the studied deep-sea samples. The conjugation of culture-dependent and -independent methods allows a better understanding of the actinobacterial diversity of deep-sea environments, which is important for the optimization of approaches to obtain novel chemically-rich isolates.

Shaping the subway microbiome through probiotic-based sanitation during the COVID-19 emergency: a pre–post case–control study

BackgroundThe COVID-19 pandemic has highlighted the extent to which the public transportation environment, such as in subways, may be important for the transmission of potential pathogenic microbes among humans, with the possibility of rapidly impacting large numbers of people. For these reasons, sanitation procedures, including massive use of chemical disinfection, were mandatorily introduced during the emergency and remain in place. However, most chemical disinfectants have temporary action and a high environmental impact, potentially enhancing antimicrobial resistance (AMR) of the treated microbes. By contrast, a biological and eco-sustainable probiotic-based sanitation (PBS) procedure was recently shown to stably shape the microbiome of treated environments, providing effective and long-term control of pathogens and AMR spread in addition to activity against SARS-CoV-2, the causative agent of COVID-19. Our study aims to assess the applicability and impact of PBS compared with chemical disinfectants based on their effects on the surface microbiome of a subway environment.ResultsThe train microbiome was characterized by both culture-based and culture-independent molecular methods, including 16S rRNA NGS and real-time qPCR microarray, for profiling the train bacteriome and its resistome and to identify and quantify specific human pathogens. SARS-CoV-2 presence was also assessed in parallel using digital droplet PCR. The results showed a clear and significant decrease in bacterial and fungal pathogens (p < 0.001) as well as of SARS-CoV-2 presence (p < 0.01), in the PBS-treated train compared with the chemically disinfected control train. In addition, NGS profiling evidenced diverse clusters in the population of air vs. surface while demonstrating the specific action of PBS against pathogens rather than the entire train bacteriome.ConclusionsThe data presented here provide the first direct assessment of the impact of different sanitation procedures on the subway microbiome, allowing a better understanding of its composition and dynamics and showing that a biological sanitation approach may be highly effective in counteracting pathogens and AMR spread in our increasingly urbanized and interconnected environment.2f-kwnMvWGEoeLQ9ACdzrQVideo

Bacterial communities of Aedes aegypti mosquitoes differ between crop and midgut tissues.

Microbiota studies of Aedes aegypti and other mosquitoes generally focus on the bacterial communities found in adult female midguts. However, other compartments of the digestive tract maintain communities of bacteria which remain almost entirely unstudied. For example, the Dipteran crop is a food storage organ, but few studies have looked at the microbiome of crops in mosquitoes, and only a single previous study has investigated the crop in Ae. aegypti. In this study, we used both culture-dependent and culture-independent methods to compare the bacterial communities in midguts and crops of laboratory reared Ae. aegypti. Both methods revealed a trend towards higher abundance, but also higher variability, of bacteria in the midgut than the crop. When present, bacteria from the genus Elizabethkingia (family Weeksellaceae) dominated midgut bacterial communities. In crops, we found a higher diversity of bacteria, and these communities were generally dominated by acetic acid bacteria (family Acetobacteriaceae) from the genera Tanticharoenia and Asaia. These three taxa drove significant community structure differences between the tissues. We used FAPROTAX to predict the metabolic functions of these communities and found that crop bacterial communities were significantly more likely to contain bacteria capable of methanol oxidation and methylotrophy. Both the presence of acetic acid bacteria (which commonly catabolize sugar to produce acetic acid) and the functional profile that includes methanol oxidation (which is correlated with bacteria found with natural sources like nectar) may relate to the presence of sugar, which is stored in the mosquito crop. A better understanding of what bacteria are present in the digestive tract of mosquitoes and how these communities assemble will inform how the microbiota impacts mosquito physiology and the full spectrum of functions provided by the microbiota. It may also facilitate better methods of engineering the mosquito microbiome for vector control or prevention of disease transmission.

Microbial Community Associated with Ground waters Discharge in Transylvania (Romania) and Balaton Highland (Hungary)

In the present study, water samples from 8 sites known as: Taploca, Nagy-borvíz, Piricske (located in Romania) and Szent Jakab, Kiskút, Kossuth Lajos, Polányi kút and Berzsenyi (located in Balaton highland in Hungary) and characterized with low nutrient content, were studied using cultivation independent methods. Diversity indices and cell counts were determined to assess the species richness in relation to the cell counts within the samples. Next generation sequencing was used to reveal the existing microbial community, and taxon specific PCR was used to detect the presence of some species with hygienic. 18 bacterial phyla above a ratio of 2% were identified in addition to 13 archaeal phyla using amplicon sequencing.

A narrative review of the potential role of microaspiration and a dysregulated aerodigestive microbiome in lung disease

: When initiated the human microbiome project did not include the lungs and airways in its sampling sites, indicating an under appreciation of the role of the human lung microbiome in health and disease. This paradigm has recently changed through the use of culture independent methods to characterise the human lung microbiome. The original thinking, that the normal lung was essentially sterile, had previously been challenged by findings of microaspiration in normal volunteers and in patients with decreased levels of consciousness. The sterile lung was also questioned by findings of clinically occult infection markers in lung allograft recipients. What is arguably a “rediscovery” of the importance of the human lung microbiome may still underappreciate physiological and patho-physiological inter-relationships between organ systems, studied in separate research disciplines. In particular, microaspiration may be an important, direct mechanism through which the lung microbiome is modulated. As well as aspiration related to gastro-oesophageal reflux and microaspiration the authors feel that the importance of dysphagia in chronic lung disease, will be increasingly recognised in frailty related microbiome exchange between the oropharynx into the lung. This review therefore discusses interconnections in the human microbiome, with a focus on the potential for aerodigestive pathophysiology and microaspiration. Potential connections with human lung disease are discussed and contextualised within a developing literature. This review therefore highlights much needed new targets for translational intervention in lung pathophysiology and underlies the importance of a mixed disciplinary team approach for the future.

A clinical review of the potential role of microaspiration and a dysregulated aerodigestive microbiome in lung disease

: When initiated the human microbiome project did not include the lungs and airways in its sampling sites, indicating an under appreciation of the role of the human lung microbiome in health and disease. This paradigm has recently changed through the use of culture independent methods to characterise the human lung microbiome. The original thinking, that the normal lung was essentially sterile, had previously been challenged by findings of microaspiration in normal volunteers and in patients with decreased levels of consciousness. The sterile lung was also questioned by findings of clinically occult infection markers in lung allograft recipients. What is arguably a “rediscovery” of the importance of the human lung microbiome may still underappreciate physiological and patho-physiological inter-relationships between organ systems, studied in separate research disciplines. In particular, microaspiration may be an important, direct mechanism through which the lung microbiome is modulated. As well as aspiration related to gastro-oesophageal reflux and microaspiration the authors feel that the importance of dysphagia in chronic lung disease, will be increasingly recognised in frailty related microbiome exchange between the oropharynx into the lung. This review therefore discusses interconnections in the human microbiome, with a focus on the potential for aerodigestive pathophysiology and microaspiration. Potential connections with human lung disease are discussed and contextualised within a developing literature. This review therefore highlights much needed new targets for translational intervention in lung pathophysiology and underlies the importance of a mixed disciplinary team approach for the future.

Sediment prokaryotic microbial community and potential biogeochemical cycle from saline lakes shaped by habitat

The microbial diversity and ecological function in different saline lakes was reduced or disappeared as the influence of climate change and human activities even before they were known. However, reports about prokaryotic microbial of saline lakes from Xinjiang are very limited especially in large-scale investigations. In this study, a total of 6 saline lakes represented three different habitats, including hypersaline lake (HSL), arid saline lake (ASL), and light saltwater lake (LSL) were involved. The distribution pattern and potential functions of prokaryotes were investigated by using the cultivation-independent method of amplicon sequencing. The results showed that Proteobacteria was the predominant community and was widely distributed in all kinds of saline lakes, Desulfobacterota was the representative community in hypersaline lakes, Firmicutes and Acidobacteriota were mainly distributed in arid saline lake samples, and Chloroflexi was more abundant in light saltwater lakes. Specifically, the archaeal community was mainly distributed in the HSL and ASL samples, whereas it was very rare in the LSL lakes. The functional group showed that fermentation was the main metabolic process of microbes in all saline lakes and covered 8 phyla, including Actinobacteriota, Bacteroidota, Desulfobacterota, Firmicutes, Halanaerobiaeota, Proteobacteria, Spirochaetota, and Verrucomicrobiota. Among the 15 functional phyla, Proteobacteria was a distinctly important community in saline lakes, as it exhibited wide functions in the biogeochemical cycle. According to the correlation of environmental factors, SO42-, Na+, CO32-, and TN were significantly affected in the microbial community from saline lakes in this study. Overall, our study provided more detailed information about microbial community composition and distribution from three different habitats of saline lakes, especially the potential functions of carbon, nitrogen, and sulfur cycles, which provided new insight for understanding the complex microbiota adapt to the extreme environment and new perspectives on evaluating microbial contributions to degraded saline lakes under environmental change.

Risk of Exposure to Coccidioides spp. in the Temblor Special Recreation Management Area (SRMA), Kern County, CA

The Temblor Mountain Special Recreation Area (SRMA) on the western flank of the San Joaquin Valley, CA, is located in the endemic area of Coccidioides, a fungal pathogen responsible for the increasing incidence of coccidioidomycosis (Valley fever). Recreationists in the SRMA, such as off-highway vehicle (OHV) drivers and mountain bikers who disturb the soils, are at risk of being exposed to airborne arthroconidia (asexual spores) of the pathogen. To reduce the risk of pathogen exposure for visitors, the Bureau of Land Management (BLM) plans to limit recreational activities to areas with a reduced pathogen presence. They envision an official OHV park in the future, by also restricting access to areas with ongoing restoration efforts and by limiting soil erosion in sensitive areas. To investigate which soils in the Temblor SRMA are most likely to support the growth of Coccidioides spp., soil samples were collected over a 3-year period from dominant soil types in a northern and a southern sampling area and analyzed for the pathogen using a culture-independent PCR-based method. In addition, soil pH and electrical conductivity were determined. The results of this study revealed slight genetic variance in the Coccidioides sequences obtained from the soils of the Temblor SRMA. An analysis of variance (ANOVA) could not confirm differences in soil pH and electrical conductivity (EC) between the different soil types investigated and between sites where the pathogen was detected compared to sites where it could not be found. However, the year of sampling appeared to have an influence on observed soil pH and EC, and the presence of the pathogen. Of all dominant soil types investigated, those belonging to the Littlesignal-Cochora association were the least likely to contain the pathogen, whereas soils of the Beam-Panoza-Hillbrick association appeared more supportive. In addition to pointing out OHV areas with lower pathogen exposure risk in the Temblor SRMA, recommendations were made to educate visitors and BLM workers about the risk of contracting Valley fever.

Listeria monocytogenes loss of cultivability on carrot is associated with the formation of mesosome-like structures

Raw carrot is known to have antimicrobial activity against Listeria monocytogenes, but the mechanism of action has not been fully elucidated. In this study, we examined carrot antilisterial activity against several strains of Listeria species (including L. grayi, L. innocua, L. seeligeri, and L. welshimeri) and L. monocytogenes. A representative strain of L. monocytogenes was subsequently used for further characterizing carrot antilisterial activity. Exposure to fresh-cut carrot for 15 min resulted in a similar loss of cultivability, ranging from 2.5 to 4.7 log units, across all Listeria strains evaluated. L. monocytogenes recovered from the fresh-cut surface of different raw carrots was 1.6 to 4.1 log lower than levels obtained from paired boiled carrot samples with abolished antilisterial activity. L. monocytogenes levels recovered from fresh-cut carrot were 2.8 to 3.1 log lower when enumerated by culture-dependent methods than by the culture-independent method of PMAxx-qPCR, a qPCR assay that is performed using DNA pre-treated to selectively sequester DNA from cells with injured membranes. These results suggested that L. monocytogenes loss of cultivability on fresh-cut carrot was not associated with a loss of L. monocytogenes cell membrane integrity and putative cell viability. Transmission electron microscopy imaging revealed that L. monocytogenes rapidly formed mesosome-like structures upon exposure to carrot fresh-cut surface but not upon exposure to boiled carrot surface, suggesting there may be an association between the formation of these mesosome-like structures and a loss of cultivability in L. monocytogenes. However, further research is necessary to conclude the causality of this association.