The threat of microplastics and microbial degradation potential; a current perspective.

Microplastics in marine environments come from various sources, and over the years, their buildup in marine environments suggests an inevitable need for the safe mitigation of plastic pollution. Microplastics are one of the chief and hazardous components of marine pollution, as they are transferred through the food chain to different trophic levels, affecting living organisms. They are also a source of transfer for pathogenic organisms. Upon transfer to humans, several toxic effects can occur. This review aims to assess the accumulation of microplastics in marine environments globally, the threat posed to humans, and the biodegradation potential of bacteria and fungi for future mitigation strategies. The versatility of bacteria and fungi in the biodegradation of different types of plastics has been discussed, with a focus on the microbial majority that has been cultivated in labs from the marine environment. We also propose that the exploration of yet-to-be-cultivated microbial majority can be a way forward for employing future strategies to mitigate microplastics.

Leveraging genomic information to predict environmental preferences of bacteria.

Genomic information is now available for a broad diversity of bacteria, including uncultivated taxa. However, we have corresponding knowledge on environmental preferences (i.e. bacterial growth responses across gradients in oxygen, pH, temperature, salinity, and other environmental conditions) for a relatively narrow swath of bacterial diversity. These limits to our understanding of bacterial ecologies constrain our ability to predict how assemblages will shift in response to global change factors, design effective probiotics, or guide cultivation efforts. We need innovative approaches that take advantage of expanding genome databases to accurately infer the environmental preferences of bacteria and validate the accuracy of these inferences. By doing so, we can broaden our quantitative understanding of the environmental preferences of the majority of bacterial taxa that remain uncharacterized. With this perspective, we highlight why it is important to infer environmental preferences from genomic information and discuss the range of potential strategies for doing so. In particular, we highlight concrete examples of how both cultivation-independent and cultivation-dependent approaches can be integrated with genomic data to develop predictive models. We also emphasize the limitations and pitfalls of these approaches and the specific knowledge gaps that need to be addressed to successfully expand our understanding of the environmental preferences of bacteria.

Phenylobacterium montanum sp. nov., an oligotrophic, slightly acidophilic mesophile isolated from sandy soil.

A bacterial strain, designated S6T, was isolated from the sandy soil on a rocky mountain in South China. Cells of S6T were Gram-stain-negative, aerobic, non-spore-forming, non-motile and non-prosthecae-producing. 16S rRNA gene sequence analysis revealed the highest similarities to 12 uncultured bacteria, followed by Phenylobacterium sp. B6.10-61 (97.14 %). The closest related validly published strains are Caulobacter henricii ATCC 15253T (96.15 %), Phenylobacterium conjunctum FWC 21T (96.08 %) and Caulobacter mirabilis FWC 38T (96.08 %). Phylogenetic analysis based on 16S rRNA gene, genome and proteome sequences demonstrated that S6T formed a separated lineage in the genus Phenylobacterium. Strain S6T contained Q-10 (97.5 %) as the major ubiquinone and C18 : 1 ω7c and C16 : 0 as the major fatty acids. The polar lipid profile consisted of phosphatidylglycerol, an unknown phosphoglycolipid and three unknown glycolipids. The assembled genome comprises a chromosome with a length of 5.5 Mb and a plasmid of 96 014 bp. The G+C content was 67.6 mol%. The morphological, physiological, chemotaxonomic and phylogenetic analyses clearly distinguished this strain from its closest phylogenetic neighbours. Thus it is proposed that strain S6T represents a novel species in the genus Phenylobacterium, for which the name Phenylobacterium montanum sp. nov. is proposed. The type strain is S6T (=NBRC 115419T=GCMCC 1.18594T).

Cultivating the uncultured: Harnessing the "sandwich agar plate" approach to isolate heme-dependent bacteria from marine sediment.

In the classical microbial isolation technique, the isolation process inevitably destroys all microbial interactions and thus makes it difficult to culture the many microorganisms that rely on these interactions for survival. In this study, we designed a simple coculture technique named the "sandwich agar plate method," which maintains microbial interactions throughout the isolation and pure culture processes. The total yield of uncultured species in sandwich agar plates based on eight helper strains was almost 10-fold that of the control group. Many uncultured species displayed commensal lifestyles. Further study found that heme was the growth-promoting factor of some marine commensal bacteria. Subsequent genomic analysis revealed that heme auxotrophies were common in various biotopes and prevalent in many uncultured microbial taxa. Moreover, our study supported that the survival strategies of heme auxotrophy in different habitats varied considerably. These findings highlight that cocultivation based on the "sandwich agar plate method" could be developed and used to isolate more uncultured bacteria.

Dual-Mechanism Peptide SR25 has Broad Antimicrobial Activity and Potential Application for Healing Bacteria-infected Diabetic Wounds.

The rise of antibiotic resistance poses a significant public health crisis, particularly due to limited antimicrobial options for the treatment of infections with Gram-negative pathogens. Here, an antimicrobial peptide (AMP) SR25 is characterized, which effectively kills both Gram-negative and Gram-positive bacteria through a unique dual-targeting mechanism without detectable resistance. Meanwhile, an SR25-functionalized hydrogel is developed for the efficient treatment of infected diabetic wounds. SR25 is obtained through genome mining from an uncultured bovine enteric actinomycete named Nonomuraea Jilinensis sp. nov. Investigations reveal that SR25 has two independent cellular targets, disrupting bacterial membrane integrity and restraining the activity of succinate:quinone oxidoreductase (SQR). In a diabetic mice wound infection model, the SR25-incorporated hydrogel exhibits high efficacy against mixed infections of Escherichia coli (E. coli) and methicillin-resistant Staphylococcus aureus (MRSA), accelerating wound healing. Overall, these findings demonstrate the therapeutic potential of SR25 and highlight the value of mining drugs with multiple mechanisms from uncultured animal commensals for combating challenging bacterial pathogens.

Metagenomics of two aquifers with thermal anomalies in Mallorca Island, and proposal of new uncultivated taxa named following the rules of SeqCode.

Groundwater offers an intriguing blend of distinctive physical and chemical conditions, constituting a challenge for microbial life. In Mallorca, the largest island of Balearic archipelago, harbours a variety of thermal anomalies (i.e., geothermal manifestation where surface aquifers exhibiting temperatures exceeding the regional average). The metagenomes of two aquifers in the centre and southern of the island showed Pseudomonadota to be the most represented phylum when using extracted 16S rRNA gene sequences. However, the microbial structures within and between aquifers were remarkably diverse but similar in their metabolic profiles as revealed by the metagenome-assembled genomes (MAGs) pointing to a prevalence of aerobic chemolithoautotrophic and heterotrophic metabolisms, especially in the Llucmajor aquifer. Also, some evidences of anaerobic lifestyles were detected, which would indicate that these environments either could suffer episodes of oxygen depletion or the anaerobes had been transported from deeper waters. We believe that the local environmental factors (temperature, external inputs or chemistry) seem to be more relevant than the connection and, eventually, transport of microbial cells within the aquifer in determining the highly divergent structures. Notably, most of the reconstructed genomes belonged to undescribed bacterial lineages and from them two high-quality MAGs could be classified as novel taxa named following the rules of the Code for Nomenclature of Prokaryotes Described from Sequence Data (SeqCode). Accordingly, we propose the new species and genus Costitxia debesea gen. nov., sp. nov., affiliated with the novel family Costitxiaceae fam. nov., order Costitxiales ord. nov. and class Costitxiia class. nov.; and the new new species and genus Lloretia debesea gen. nov. sp. nov. affiliated with the novel family Lloretiaceae fam. nov.

Application Progress of Culturomics in the Isolated Culture of Rhizobacteria: A Review.

Comprehending the structure and function of rhizobacteria components and their regulation are crucial for sustainable agricultural management. However, obtaining comprehensive species information for most bacteria in the natural environment, particularly rhizobacteria, presents a challenge using traditional culture methods. To obtain diverse and pure cultures of rhizobacteria, this study primarily reviews the evolution of rhizobacteria culturomics and associated culture methods. Furthermore, it explores new strategies for enhancing the application of culturomics, providing valuable insights into efficiently enriching and isolate target bacterial strains/groups from the environment. The findings will help improve rhizobacteria's culturability and enrich the functional bacterial library.

An antibiotic from an uncultured bacterium binds to an immutable target.

Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C55PP, lipid II, and lipid IIIWTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development.

Uncovering the Secrets of Slow-Growing Bacteria in Tropical Savanna Soil Through Isolation and Genomic Analysis.

One gram of soil holds ten billion bacteria of thousands of different species, but most remain unknown, and one of the serious issues is intrinsic to slow-growing bacteria. In this study, we aimed to isolate and characterize slow-growing bacteria from Brazilian Cerrado soil. Over a period of 4 weeks, we conducted an incubation process and selected a total of 92 isolates. These isolates, consisting mostly of slow-growing bacteria, have the ability to thrive in low-water conditions and possess features that promote plant growth. To identify the isolated bacteria, we performed 16S rRNA sequencing analysis and found that the slow-growing strains were genetically similar to known bacterial species but also belonged to a novel group of species. The new strains identified were Caballeronia sp., Neobacillus sp., Bradyrhizobium sp., and high GC Gram-positive species. Furthermore, we conducted growth experiments using various culture media and temperature conditions. These experiments revealed an extended lag phase for five strains, indicating their slow growth characteristics. Genomic analysis of these five slow-growing bacteria showed their potential to participate in biogeochemical cycles, metabolize various carbohydrates, encode proteins with a role in promoting plant growth and have biosynthetic potential for secondary metabolites. Taken together, our findings reveal the untapped potential of slow-growing bacteria in tropical savanna soils.

Anaerobic degradation of organic carbon supports uncultured microbial populations in estuarine sediments.

BACKGROUND: A large proportion of prokaryotic microbes in marine sediments remains uncultured, hindering our understanding of their ecological functions and metabolic features. Recent environmental metagenomic studies suggested that many of these uncultured microbes contribute to the degradation of organic matter, accompanied by acetogenesis, but the supporting experimental evidence is limited. RESULTS: Estuarine sediments were incubated with different types of organic matters under anaerobic conditions, and the increase of uncultured bacterial populations was monitored. We found that (1) lignin stimulated the increase of uncultured bacteria within the class Dehalococcoidia. Their ability to metabolize lignin was further supported by the presence of genes associated with a nearly complete degradation pathway of phenolic monomers in the Dehalococcoidia metagenome-assembled genomes (MAGs). (2) The addition of cellulose stimulated the increase of bacteria in the phylum Ca. Fermentibacterota and family Fibrobacterales, a high copy number of genes encoding extracellular endoglucanase or/and 1,4-beta-cellobiosidase for cellulose decomposition and multiple sugar transporters were present in their MAGs. (3) Uncultured lineages in the order Bacteroidales and the family Leptospiraceae were enriched by the addition of casein and oleic acid, respectively, a high copy number of genes encoding extracellular peptidases, and the complete ß-oxidation pathway were found in those MAGs of Bacteroidales and Leptospiraceae, respectively. (4) The growth of unclassified bacteria of the order Clostridiales was found after the addition of both casein and cellulose. Their MAGs contained multiple copies of genes for extracellular peptidases and endoglucanase. Additionally, 13C-labeled acetate was produced in the incubations when 13C-labeled dissolved inorganic carbon was provided. CONCLUSIONS: Our results provide new insights into the roles of microorganisms during organic carbon degradation in anaerobic estuarine sediments and suggest that these macro and single molecular organic carbons support the persistence and increase of uncultivated bacteria. Acetogenesis is an additional important microbial process alongside organic carbon degradation. Video Abstract.

Screening and isolation of acid-tolerant bacteria using a novel pH shift culture method.

Acid-tolerant bacteria, which multiply under neutral pH and can survive under acidic pH conditions, have a potential role in various applications under acidic conditions. Despite higher biomass productivity, their isolation and utilisation are not sufficiently developed compared to those of acidophiles. It takes considerable effort to distinguish the acid-tolerant bacteria from the rest of the bacterial community using conventional screening methods. Thus, we developed a novel screening method for acid-tolerant bacteria, which involves shifting the pH between acidic and neutral conditions. With this method, the bacterium Enterobacter sp. AC06 was isolated. Based on comparisons with the results reported in previous studies, the strain can be classified as acid-tolerant bacteria. The decreases in the live cell concentrations were 3.87 and 6.16 log cycles after 3 h acid treatment under pH 3.0 and 2.5, respectively. These results suggest that it is possible to isolate acid-tolerant bacteria using the pH shift culture method. In summary, this is the first study on bacterial screening based on acid tolerance. Our novel method potentially contributes to the understanding and utilisation of acid-tolerant bacteria by enhancing screening efficiency. Furthermore, our novel concept shift culture is potentially valuable for screening previously uncultured bacteria tolerant to various selective stress conditions.

The status Candidatus for uncultured taxa of Bacteria and Archaea: SWOT analysis.

The status Candidatus was introduced to bacterial taxonomy in the 1990s to accommodate uncultured taxa defined by analyses of DNA sequences. Here I review the strengths, weaknesses, opportunities and threats (SWOT) associated with the status Candidatus in the light of a quarter century of use, twinned with recent developments in bacterial taxonomy and sequence-based taxonomic discovery. Despite ambiguities as to its scope, philosophical objections to its use and practical problems in implementation, the status Candidatus has now been applied to over 1000 taxa and has been widely adopted by journals and databases. Although lacking priority under the International Code for Nomenclature of Prokaryotes, many Candidatus names have already achieved de facto standing in the academic literature and in databases via description of a taxon in a peer-reviewed publication, alongside deposition of a genome sequence and there is a clear path to valid publication of such names on culture. Continued and increased use of Candidatus names provides an alternative to the potential upheaval that might accompany creation of a new additional code of nomenclature and provides a ready solution to the urgent challenge of naming many thousands of newly discovered but uncultured species.

Extensive microbial diversity within the chicken gut microbiome revealed by metagenomics and culture.

BACKGROUND: The chicken is the most abundant food animal in the world. However, despite its importance, the chicken gut microbiome remains largely undefined. Here, we exploit culture-independent and culture-dependent approaches to reveal extensive taxonomic diversity within this complex microbial community. RESULTS: We performed metagenomic sequencing of fifty chicken faecal samples from two breeds and analysed these, alongside all (n = 582) relevant publicly available chicken metagenomes, to cluster over 20 million non-redundant genes and to construct over 5,500 metagenome-assembled bacterial genomes. In addition, we recovered nearly 600 bacteriophage genomes. This represents the most comprehensive view of taxonomic diversity within the chicken gut microbiome to date, encompassing hundreds of novel candidate bacterial genera and species. To provide a stable, clear and memorable nomenclature for novel species, we devised a scalable combinatorial system for the creation of hundreds of well-formed Latin binomials. We cultured and genome-sequenced bacterial isolates from chicken faeces, documenting over forty novel species, together with three species from the genus Escherichia, including the newly named species Escherichia whittamii. CONCLUSIONS: Our metagenomic and culture-based analyses provide new insights into the bacterial, archaeal and bacteriophage components of the chicken gut microbiome. The resulting datasets expand the known diversity of the chicken gut microbiome and provide a key resource for future high-resolution taxonomic and functional studies on the chicken gut microbiome.

Enhancement of polychlorinated biphenyl biodegradation by resuscitation promoting factor (Rpf) and Rpf-responsive bacterial community.

The activities of indigenous bacterial communities in polychlorinated biphenyls (PCBs) contaminated environments is closely related to the efficiency of bioremediation processes. Using resuscitation promoting factor (Rpf) from Micrococcus luteus is a promising method for resuscitation and stimulation of functional bacterial populations under stressful conditions. This study aims to use the Rpf to accelerate the biodegradation of Aroclor 1242, and explore putative PCB degraders which were resuscitated by Rpf addition. The Rpf-responsive bacterial populations were investigated using culture-dependent and culture-independent approaches, respectively. The results confirm that Rpf was capable of enhancing PCB degradation of enriched cultures from PCB-contaminated soils, and improving the activities of cultures with low tolerance to PCBs. High-throughput 16S rRNA analysis displays that the Rpf greatly altered the composition and abundance of bacterial populations in the phylum Proteobacteria. Identification of the resuscitated strains further suggests that the Rpf-responsive population was mostly represented by Sphingomonas and Pseudomonas, which are most likely the key PCB-degraders for enhanced biodegradation of PCBs.

Opening up the Single-Cell Toolbox for Microbial Natural Products Research.

The diverse microbes that produce natural products represent an important source of novel therapeutics, drug leads, and scientific tools. However, the vast majority have not been grown in axenic culture and are members of complex communities. While meta-'omic methods such as metagenomics, -transcriptomics, and -proteomics reveal collective molecular features of this "microbial dark matter", the study of individual microbiome members can be challenging. To address these limits, a number of techniques with single-bacterial resolution have been developed in the last decade and a half. While several of these are embraced by microbial ecologists, there has been less use by researchers interested in mining microbes for natural products. In this review, we discuss the available and emerging techniques for targeted single-cell analysis with a particular focus on applications to the discovery and study of natural products.

Determination of available breaking stress of agar and gellan gum plate culture methods and the duration of bacterial culture under strong acidic conditions.

AIMS: Several acidophilic bacteria have not been cultured, primarily owing to the lack of suitable culture methods under strong acidic conditions. This study aimed to quantitatively evaluate the strengths of the agar plates (AP) and gellan gum plates (GP), and optimal culture periods under strong acidic conditions. METHODS AND RESULTS: To define the lower limit of plate strength for bacterial isolation culture, the diameter of Escherichia coli K12 colonies and the breaking stress of plates at different concentrations of gelling agents, medium composition and pH conditions were determined. The lower limit of available strength of AP and GP was 19·6 and 14·8 kPa, respectively. Medium composition slightly affected AP breaking stress, although GP with a high cationic concentration medium could not be prepared. CONCLUSIONS: Assessment of the strength limits of AP and GP revealed that AP is not suitable for prolonged bacterial culture (≥72 h). Furthermore, GP was completely ineffective for bacterial culture under highly acidic conditions (≤pH 1·0). SIGNIFICANCE AND IMPACT OF THE STUDY: Our quantitative evaluation method based on breaking stress is a potentially valuable tool to understand the state and the suitable limit of plate culture methods in more detail under various conditions.

Proposed nomenclature or classification changes for bacteria of medical importance: taxonomic update 5.

A key aspect of medical, public health, and diagnostic microbiology laboratories is the accurate identification and rapid reporting and communication to medical staff regarding patients with infectious agents of clinical importance. Microbial taxonomy continues to change at a very rapid rate in the era of molecular diagnostics including whole genome sequencing. This update focuses on taxonomic changes and proposals that may be of medical importance from 2018 to 2020.

Single-cell genomics of uncultured bacteria reveals dietary fiber responders in the mouse gut microbiota.

BACKGROUND: The gut microbiota can have dramatic effects on host metabolism; however, current genomic strategies for uncultured bacteria have several limitations that hinder their ability to identify responders to metabolic changes in the microbiota. In this study, we describe a novel single-cell genomic sequencing technique that can identify metabolic responders at the species level without the need for reference genomes, and apply this method to identify bacterial responders to an inulin-based diet in the mouse gut microbiota. RESULTS: Inulin-feeding changed the mouse fecal microbiome composition to increase Bacteroides spp., resulting in the production of abundant succinate in the mouse intestine. Using our massively parallel single-cell genome sequencing technique, named SAG-gel platform, we obtained 346 single-amplified genomes (SAGs) from mouse gut microbes before and after dietary inulin supplementation. After quality control, the SAGs were classified as 267 bacteria, spanning 2 phyla, 4 classes, 7 orders, and 14 families, and 31 different strains of SAGs were graded as high- and medium-quality draft genomes. From these, we have successfully obtained the genomes of the dominant inulin-responders, Bacteroides spp., and identified their polysaccharide utilization loci and their specific metabolic pathways for succinate production. CONCLUSIONS: Our single-cell genomics approach generated a massive amount of SAGs, enabling a functional analysis of uncultured bacteria in the intestinal microbiome. This enabled us to estimate metabolic lineages involved in the bacterial fermentation of dietary fiber and metabolic outcomes such as short-chain fatty acid production in the intestinal environment based on the fibers ingested. The technique allows the in-depth isolation and characterization of uncultured bacteria with specific functions in the microbiota and could be exploited to improve human and animal health. Video abstract.

Diversity of endophytic bacterial community inhabiting in tropical aerobic rice under aerobic and flooded condition.

The role and activity of bacterial endophytes remains largely unexplored and detail insight into Indian rice agro ecosystem is still little explored. In this study, we examined the diversity of endophytic bacteria in aerobic rice (variety ARB6) under aerobic and flooded field conditions. Based on 16S rRNA gene RFLP cloning sequencing, 900 clones with 144 representatives (72 aerobic and 72 flooded) revealed majority of clones affiliated to Gammaproteobacteria (64.58%), Betaproteobacteria (9.72%), Alphaproteobacteria (17.36), Firmicutes (6.26%) and Bacteroidetes (2.08). The study suggests that the aerobic rice variety harbours plant growth promoting (PGP) genera (viz. Pantoea, Enterobacter, Paenibacillus, etc). Investigations on aerobic rice under aerobic and flooded conditions revealed high richness and diversity of endophytic bacteria under aerobic condition inferring that the endophytic bacteria are beneficial for rice growth and productivity, and hence, would be helpful in designing better strategies for rice cultivation under drought or water scarce conditions.

Microbial community structure analysis in Acer palmatum bark and isolation of novel bacteria IAD-21 of the candidate division FBP.

BACKGROUND: The potential of unidentified microorganisms for academic and other applications is limitless. Plants have diverse microbial communities associated with their biomes. However, few studies have focused on the microbial community structure relevant to tree bark. METHODS: In this report, the microbial community structure of bark from the broad-leaved tree Acer palmatum was analyzed. Both a culture-independent approach using polymerase chain reaction (PCR) amplification and next generation sequencing, and bacterial isolation and sequence-based identification methods were used to explore the bark sample as a source of previously uncultured microorganisms. Molecular phylogenetic analyses based on PCR-amplified 16S rDNA sequences were performed. RESULTS: At the phylum level, Proteobacteria and Bacteroidetes were relatively abundant in the A. palmatum bark. In addition, microorganisms from the phyla Acidobacteria, Gemmatimonadetes, Verrucomicrobia, Armatimonadetes, and candidate division FBP, which contain many uncultured microbial species, existed in the A. palmatum bark. Of the 30 genera present at relatively high abundance in the bark, some genera belonging to the phyla mentioned were detected. A total of 70 isolates could be isolated and cultured using the low-nutrient agar media DR2A and PE03. Strains belonging to the phylum Actinobacteria were isolated most frequently. In addition, the newly identified bacterial strain IAP-33, presumed to belong to Acidobacteria, was isolated on PE03 medium. Of the isolated bacteria, 44 strains demonstrated less than 97% 16S rDNA sequence-similarity with type strains. Molecular phylogenetic analysis of IAD-21 showed the lowest similarity (79%), and analyses suggested it belongs to candidate division FBP. Culture of the strain IAD-21 was deposited in Japan Collection of Microorganisms (JCM) and Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) as JCM 32665 and DSM 108248, respectively. DISCUSSION: Our results suggest that a variety of uncultured microorganisms exist in A. palmatum bark. Microorganisms acquirable from the bark may prove valuable for academic pursuits, such as studying microbial ecology, and the bark might be a promising source of uncultured bacterial isolates.