Uncultured Bacteria Research Articles

Microbial community assembly of the hyperaccumulator plant Sedum plumbizincicola in two contrasting soil types with three levels of cadmium contamination

Microbial communities are closely related to plant performance and numerous studies have shown their involvement with the growth and development of host plants, resistance to pathogen invasion and adaptation to environmental stress. Here we described in detail the ecological process of the microbial community assembly in hyperaccumulator plant Sedum plumbizincicola. We divided the microbiota into four ecological compartments (bulk soil, rhizosphere, root endosphere and aboveground endosphere). The results showed that host selection strongly controlled the aggregation of microbial community. So that microbes occupied different niches from the bulk soil to the aboveground endosphere, and bacterial diversity and network complexity decreased gradually. Soil types were the second influencing factor, especially for the microbial community in the root endosphere. The SourceTracker analysis further confirmed the vertical migration of microbes from bulk soil to aboveground endosphere. In addition, under the condition of heavy metal pollution, the microbial community of S. plumbizincicola tended to form a microbial pool dominated by Proteobacteria and Actinobacteria. Ellin6067, Sphingomonas, Ralstonia, SC-I-84_uncultured bacterium, Burkholderiaceae_Undibacterium and Pedosphaeraceae_uncultured bacterium etc. were identified as the vital biomarker taxa. Among these genera, the relative abundance of last three was significantly positively correlated with the activation and transfer of cadmium, and they mainly enriched in paddy soil. This study provides evidence for the mechanism by which the microbial community assembly occurs and experience for regulating the microbial community and increasing the accumulation efficiency of potentially toxic metals in S. plumbizincicola.

Dietary Association with Midgut Microbiota Components of Eocanthecona furcellata (Wolff)

Eocanthecona furcellata is an important predatory stinkbug that attacks many lepidopteran pests. For mass-rearing, artificial diets are used to rear this predator in the laboratory; however, the fitness of the predators is reduced, and little is known about the cause. Since gut microbiota plays vital roles in the digestion and development of many hosts and can consequently affect host fitness, an understanding of the microbial community composition of E. furcellata may help to solve this unresolved problem. We compared the development and reproduction of E. furcellata reared on an artificial diet, and a natural (Spodoptera litura) or semi-natural (Tenebrio molitor) diet, and then the midgut microbiota were assessed using high-throughput 16S rRNA. The results of the high-throughput 16S rRNA show that the bacterial richness and diversity in the artificial diet gut samples increased considerably compared with the other samples. Proteobacteria and Firmicutes were the dominant phyla in E. furcellata. At the genus level, Serratia (however, the relative abundance was lower in the artificial diet gut samples), Enterococcus, and an uncultured bacterium genus of family Enterobacteriaceae, were dominant. The midgut microbiota components significantly differed among the diets, indicating that the gut bacteria had a dietary association with E. furcellata. This study provides a better understanding of midgut microbiota and the artificial diets that might affect them in E. furcellata.

Bacillus subtilis reduces antibiotic resistance genes of animal sludge in vermicomposting by improving heat stress tolerance of Eisenia foetida and bacterial community adjustment

Antibiotic resistance genes (ARGs) in livestock industry have been recognized as a kind of pollutant. The effect of Bacillus subtilis (B. subtilis) as an additive for the reduction of ARGs in animal sludge from livestock and poultry wastewater treatment plant during vermicomposting was investigated. We also evaluated the oxidative stress level and growth of earthworms, Eisenia foetida, bacterial community succession, and the quality of the end products. Two treatments were conducted using B. subtilis, one at 18 °C and another at 28 °C. Controls were setup without the bacteria. The results showed that inoculation of B. subtilis promoted the degradation of organics at 28 °C and increased the germination index to 236%. The increased activities of the superoxide dismutase (1.69 U/mg pr) and catalase (8.05 U/mg pr) and the decreased activity of malondialdehyde (0.02 nmol/mg pr) by B. subtilis at 28 °C showed that the earthworms were relieved of heat stress. The addition of B. subtilis reduced the abundance of 32 target ARGs, including integron (intI-1), transposase (IS613) and resistant genes, such as sulfonamide (sul2), quinolone (oprJ), macrolide-lincosamide-streptogramin group B (ermF, ermB), tetracycline (tetL-02, tetX), β-lactama (blaOXA10-01) and aminoglycoside [strB, aac(6′)-Ib(aka aacA4)-01, aac(6′)-Ib(aka aacA4)-02]. Organic matter degrading Membranicola, Paludisphaera, Sphingorhabdus and uncultured bacterium belonging to the order Chitinophagales, nitrifying and nitrogen-fixing Singulisphaera and Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, soil remediating Achromobacter, and plant growth promoting Kaistia, Galbibacter and Ilumatobacter were increased significantly (P < 0.05). However, the growth of harmful bacteria such as Burkholderiaceae was inhibited in the vermicompost. In earthworm guts, the probiotic Mesorhizobium was promoted, while the pathogenic uncultured bacterium belonging to the family Enterobacteriaceae was reduced. Besides, B. subtilis enhanced the host relationships between bacteria and ARGs. These findings might be helpful in the removal of ARGs in animal wastes and in understanding the synergy between earthworms and microorganisms.

Bioprospecting for Novel Bacterial Sources of Hydrolytic Enzymes and Antimicrobials in the Romanian Littoral Zone of the Black Sea.

Marine microorganisms have evolved a large variety of metabolites and biochemical processes, providing great opportunities for biotechnologies. In the search for new hydrolytic enzymes and antimicrobial compounds with enhanced characteristics, the current study explored the diversity of cultured and uncultured marine bacteria in Black Sea water from two locations along the Romanian coastline. Microbial cell density in the investigated samples varied between 65 and 12.7 × 103 CFU·mL-1. The total bacterial community identified by Illumina sequencing of 16S rRNA gene comprised 185 genera belonging to 46 classes, mainly Gammaproteobacteria, Alphaproteobacteria, Flavobacteriia, and 24 phyla. The 66 bacterial strains isolated on seawater-based culture media belonged to 33 genera and showed variable growth temperatures, growth rates, and salt tolerance. A great fraction of these strains, including Pseudoalteromonas and Flavobacterium species, produced extracellular proteases, lipases, and carbohydrases, while two strains belonging to the genera Aquimarina and Streptomyces exhibited antimicrobial activity against human pathogenic bacteria. This study led to a broader view on the diversity of microbial communities in the Black Sea, and provided new marine strains with hydrolytic and antimicrobial capabilities that may be exploited in industrial and pharmaceutical applications.

Residue dynamics of florpyrauxifen-benzyl and its effects on bacterial community structure in paddy soil of Northeast China

Florpyrauxifen-benzyl is an herbicide that has been developed in recent years. Its degradation mode in paddy soil environments is not clear. In this study, the degradation dynamics in paddy soil and water were studied by ultrahigh-performance liquid chromatography. Microbial degradation was the main degradation pathway. Using third-generation high-throughput sequencing technology, the changes in the soil bacterial community structure were studied. After 30 days of application, compared with the control group (F0), the abundance of Sphingomonas, Lysobacter, and Flavisolibacter in the recommended and repeated application groups (F1, F5 and F10) increased significantly, and uncultured bacterium and Terrimonas decreased significantly. Compared with the F0 and F1 groups, the species diversity of the F0 and F1 groups showed a significant increase over time. The species diversity of the F5 and F10 groups decreased significantly on Days 5 and 15. On Day 30, the recovery even exceeded that of the control group. Luteimonas and five other genera were positively correlated with herbicide residues, and Pseudolabrys and two other genera were negatively correlated. Repeated application showed a significant effect on the structure of the soil bacterial community, mainly showing a trend of a significant decrease in the initial stage and gradual recovery in the later stage. The results will guide the safe and rational use of florpyrauxifen-benzyl and provide a scientific basis for florpyrauxifen-benzyl dynamic supervision of environmental pollution and protection of black soil in Northeast China.

GgMOB: Elucidation of genomic conjugative features and associated cargo genes across bacterial genera using genus-genus mobilization networks.

Horizontal gene transfer mediated by conjugation is considered an important evolutionary mechanism of bacteria. It allows organisms to quickly evolve new phenotypic properties including antimicrobial resistance (AMR) and virulence. The frequency of conjugation-mediated cargo gene exchange has not yet been comprehensively studied within and between bacterial taxa. We developed a frequency-based network of genus-genus conjugation features and candidate cargo genes from whole-genome sequence data of over 180,000 bacterial genomes, representing 1,345 genera. Using our method, which we refer to as ggMOB, we revealed that over half of the bacterial genomes contained one or more known conjugation features that matched exactly to at least one other genome. Moreover, the proportion of genomes containing these conjugation features varied substantially by genus and conjugation feature. These results and the genus-level network structure can be viewed interactively in the ggMOB interface, which allows for user-defined filtering of conjugation features and candidate cargo genes. Using the network data, we observed that the ratio of AMR gene representation in conjugative versus non-conjugative genomes exceeded 5:1, confirming that conjugation is a critical force for AMR spread across genera. Finally, we demonstrated that clustering genomes by conjugation profile sometimes correlated well with classical phylogenetic structuring; but that in some cases the clustering was highly discordant, suggesting that the importance of the accessory genome in driving bacterial evolution may be highly variable across both time and taxonomy. These results can advance scientific understanding of bacterial evolution, and can be used as a starting point for probing genus-genus gene exchange within complex microbial communities that include unculturable bacteria. ggMOB is publicly available under the GNU licence at https://ruiz-hci-lab.github.io/ggMOB/.

Accurate identification of living Bacillus spores using laser tweezers Raman spectroscopy and deep learning

Accurately, rapidly, and noninvasively identifying Bacillus spores can greatly contribute to controlling a plenty of infectious diseases. Laser tweezers Raman spectroscopy (LTRS) has confirmed to be a powerful tool for studying Bacillus spores at a single cell level. In this study, we constructed a single-cell Raman spectra dataset of living Bacillus spores and utilized deep learning approach to accurately, nondestructively identify Bacillus spores. The trained convolutional neural network (CNN) could efficiently extract tiny Raman spectra features of five spore species, and provide a prediction accuracy of specie identification as high as 100 %. Moreover, the spectral feature differences in three Raman bands at 660, 826, and 1017 cm−1 were confirmed to mostly contribute to producing such high prediction accuracy. In addition, optimal CNN model was employed to monitor and identify sporulation process at different metabolic phases in one growth cycle. The obtained average prediction accuracy of metabolic phase identification was approximately 88 %. It can be foreseen that, LTRS combined with CNN approach have great potential for accurately identifying spore species and metabolic phases at a single cell level, and can be gradually extended to perform identification for many unculturable bacteria growing in soil, water, and food.

Hemotropic Mycoplasma spp. in Aquatic Mammals, Amazon Basin, Brazil.

Hemotropic Mycoplasma spp. (hemoplasmas) are uncultivable bacteria that infect mammals, including humans. We detected a potentially novel hemoplasma species in blood samples from wild river dolphins in the Amazon River Basin, Brazil. Further investigation could determine pathogenicity and zoonotic potential of the detected hemoplasma.

Detection of Colistin Sulfate on Piglet Gastrointestinal Tract Microbiome Alterations.

The gut microbiome exerts important functions on host health maintenance, whereas excessive antibiotic use may cause gut flora dysfunction resulting in serious disease and dysbiosis. Colistin is a broad-spectrum antibiotic with serious resistance phenomena. However, it is unclear whether colistin alters the gastrointestinal tract microbiome in piglets. In this study, 16s rDNA-based metagenome analyses were used to assess the effects of colistin on the modification of the piglet microbiome in the stomach, duodenum, jejunum, cecum, and feces. Both α- and β-diversity indices showed that colistin modified microbiome composition in these gastrointestinal areas. In addition, colistin influenced microbiome composition at the phylum and genus levels. At the species level, colistin upregulated Mycoplasma hyorhinis, Chlamydia trachomatis, Lactobacillus agilis, Weissella paramesenteroides, and Lactobacillus salivarius abundance, but downregulated Actinobacillus indolicus, Campylobacter fetus, Glaesserella parasuis, Moraxella pluranimalium, Veillonella caviae, Neisseria dentiae, and Prevotella disiens abundance in stomachs. Colistin-fed piglets showed an increased abundance of Lactobacillus mucosae, Megasphaera elsdenii DSM 20460, Fibrobacter intestinalis, and Unidentified rumen bacterium 12-7, but Megamonas funiformis, Uncultured Enterobacteriaceae bacterium, Actinobacillus porcinus, Uncultured Bacteroidales bacterium, and Uncultured Clostridiaceae bacterium abundance was decreased in the cecum. In feces, colistin promoted Mucispirillum schaedleri, Treponema berlinense, Veillonella magna, Veillonella caviae, and Actinobacillus porcinus abundance when compared with controls. Taken together, colistin modified the microbiome composition of gastrointestinal areas in piglets. This study provides new clinical rationalization strategies for colistin on the maintenance of animal gut balance and human public health.

The effect of salinity on soil chemical characteristics, enzyme activity and bacterial community composition in rice rhizospheres in Northeastern Thailand

Saline soil is one of the major problems limiting rice productivity in the Northeastern area of Thailand. Thus, the aims of this study were to determine soil physicochemical analysis and soil enzyme activities, and bacterial communities in the rhizosphere of ‘RD 6’ rice grown in salt-affected rice fields. The Ban Thum sample showed the highest electrical conductivity (EC; greater than 6 dS m−1) and total Na, while the EC in other fields were at non- or slightly saline levels. The principal component analysis revealed that soil chemical characteristics and soil enzymes activities explained 73.4% of total variation. Soil enzyme activities including dehydrogenase and fluorescein diacetate (FDA) hydrolysis, and soil characteristics including organic matter (OM) and organic carbon (OC) were significantly negatively correlated to EC. This indicated that these soil properties were adversely impacted by salts. Interestingly, activities of all hydrolytic enzymes were not affected by soil salinity. Bacteria that were able to colonize the rhizosphere soils were Achromobacter cholinophagum, Rhizobium tarimense, and unculturable bacteria. In this regard, study on the relationship of soil chemical characteristics and soil enzyme activities together with bacterial communities provided promising data for assessing rice field soil quality in the future.

The Dynamic Shift of Bacterial Communities in Hybrid Anaerobic Baffled Reactor (ABR)—Aerobic Granules Process for Berberine Pharmaceutical Wastewater Treatment

Because of its anticancer, anti-inflammatory, and antibiotic properties, berberine has been used extensively in medication. The extensive production of berberine results in the generation of wastewater containing concentrated residual berberine. However, to date, limited related studies on the biological treatment of berberine wastewaters have been carried out. A lab-scale anaerobic baffled reactor (ABR)–aerobic granular sludge (AGS) process was developed for berberine removal from synthetic wastewater. The system showed effective removal of the berberine. In order to better understand the roles of the bacterial community, the ABR–aerobic granular sludge system was operated in the state with the highest BBR removal rate in this study. The bacterial community dynamics were studied using the 16S rDNA clone library. The results showed that the hybrid ABR-AGS process achieved 92.2% and 94.8% overall removals of berberine and COD, respectively. Bacterium was dominant species in ABR, while the CFB group bacteria and Betaproteobacteria were dominant species in AGS process. The uncultured bacterium clone B135, Bacillus endophyticus strain a125, uncultured bacterium mle1-42, uncultured bacterium clone OP10D15, and uncultured bacterium clone B21.29F54 in ABR, and uncultured bacterium clone F54, uncultured bacterium clone ZBAF1-105, uncultured bacterium clone SS-9, and uncultured bacterium clone B13 in AGS process were identified as functional species in the biodegradation of berberine and/or its metabolites. Both anaerobic and aerobic bacterial communities could adapt appropriately to different berberine selection pressures because the functional species’ identical functions ensured comparable pollutant removal performances. The information provided in this study may help with future research in gaining a better understanding of berberine biodegradation.

The Gill Microbiota of Argopecten purpuratus Scallop Is Dominated by Symbiotic Campylobacterota and Upwelling Intensification Differentially Affects Their Abundance.

Despite the great importance of gills for bivalve mollusks (respiration, feeding, immunity), the microbiota associated with this tissue has barely been characterized in scallops. The scallop Argopecten purpuratus is an important economic resource that is cultivated in areas where coastal upwelling is intensifying by climate change, potentially affecting host-microbiota interactions. Thus, we first characterized the bacterial community present in gills from cultivated scallops (by 16S rRNA gene amplicon sequencing) and assessed their stability and functional potential in animals under farm and laboratory conditions. Results showed that under both conditions the gill bacterial community is dominated by the phylum Campylobacterota (57%), which displays a chemoautotrophic potential that could contribute to scallop nutrition. Within this phylum, two phylotypes, namely symbionts A and B, were the most abundant; being, respectively, taxonomically affiliated to symbionts with nutritional functions in mussel gills, and to uncultured bacteria present in coral mucus. Additionally, in situ hybridization and scanning electron microscopy analyses allowed us to detect these symbionts in the gills of A. purpuratus. Given that shifts in upwelling phenology can cause disturbances to ecosystems, affecting bacteria that provide beneficial functions to the host, we further assessed the changes in the abundance of the two symbionts (via qPCR) in response to a simulated upwelling intensification. The exposure to combined decreasing values in the temperature, pH, and oxygen levels (upwelling conditions) favored the dominance of symbiont B over symbiont A; suggesting that symbiont abundances are modulated by these environmental changes. Overall, results showed that changes in the main Campylobacterota phylotypes in response to upwelling intensification could affect its symbiotic function in A. purpuratus under future climate change scenarios. These results provide the first insight into understanding how scallop gill-microbial systems adapt and respond to climate change stressors, which could be critical for managing health, nutrition, and scallop aquaculture productivity.

Biosynthesis of coelulatin for the methylation of anthraquinone featuring HemN-like radical S-adenosyl-L-methionine enzyme.

Bacterial aromatic polyketides are usually biosynthesized by the type II polyketide synthase (PKS-II) system. Advances in deoxyribonucleic acid (DNA) sequencing, informatics, and biotechnologies have broadened opportunities for the discovery of aromatic polyketides. Meanwhile, metagenomics is a biotechnology that has been considered as a promising approach for the discovery of novel natural products from uncultured bacteria. Here, we cloned a type II polyketide biosynthetic gene cluster (BGC) from the soil metagenome, and the heterologous expression of this gene cluster in Streptomyces coelicolor M1146 resulted in the production of three anthraquinones, two of which (coelulatins 2 and 3) had special hydroxymethyl and methyloxymethyl modifications at C2 of the polyketide scaffold. Gene deletion and in vitro biochemical characterization indicated that the HemN-like radical S-adenosyl-L-methionine (SAM) enzyme CoeI exhibits methylation and is involved in C2 modification.

Novel database and cut-off value for bacterial amoA gene revealed a spatial variability pattern of the ammonia-oxidizing bacteria community from river to sea

Ammonia-oxidizing bacteria (AOB) catalyze the first step of nitrification, oxidizing ammonia to nitrite, and are characterized by amoA gene encoding ammonia monooxygenase. To analyze the AOB community effectively, an integral taxonomy database containing 14,058 amoA sequences and the optimal cut-off value at 95 % for OTU clustering were determined. This method was evaluated to be efficient by the analysis of environmental samples from the river, estuary, and sea. Using this method, a significant spatial variance of the AOB community was found. The diversity of AOB was highest in the estuary and lowest in the ocean. Nitrosomonas were the predominant AOB in the sediments of the freshwater river and estuary. Nearly all the AOB-amoA sequences belonged to uncultured bacterium in the sediments of deep sea. In general, an integral AOB taxonomic database and a suitable cut-off value were constructed for the comprehensive exploration of the diversity of AOB from river to sea.

Molecular Confirmation of Staphylococci Strain’s Identification Isolated in the Hospital and University Center of Brazzaville, Republic of Congo

Bacteria of the genus Staphylococcus are pathogenic Gram-positive bacteria responsible for various infections, including skin suppuration, which can be severe or chronic. The objective of this study was to confirm Staphylococci strain’s identification isolated by bacteriological methods from biological products of CHU-B patients, by molecular methods based on the analysis of the gene coding for 16S rRNA. In total, 30 strains of Staphylococci were isolated including 8 (26.66%) community strains, 22 (73.33%) hospital strains. The products of the amplification of gene fragments encoding 16S rRNA from 10 strains of Staphylococci including 6 strains of Staphylococcus aureus (S. aureus) and 4 Coagulase Negative Staphylococci (CNS) were sequenced. The sequences obtained were subjected to bioinformatics analysis to confirm the results of conventional bacteriological methods. Six (6) S. aureus strains, 2 Staphylococcus haemolyticus strains, 1 uncultured bacterium clone nbw618g09c1, and one Staphylococcus sp. have been identified. These results made it possible to confirm the effectiveness of the molecular method and to show the limits of traditional bacteriological methods in the complete identification of bacteria.

Metagenome-assembled genome extraction and analysis from microbiomes using KBase.

Uncultivated Bacteria and Archaea account for the vast majority of species on Earth, but obtaining their genomes directly from the environment, using shotgun sequencing, has only become possible recently. To realize the hope of capturing Earth's microbial genetic complement and to facilitate the investigation of the functional roles of specific lineages in a given ecosystem, technologies that accelerate the recovery of high-quality genomes are necessary. We present a series of analysis steps and data products for the extraction of high-quality metagenome-assembled genomes (MAGs) from microbiomes using the U.S. Department of Energy Systems Biology Knowledgebase (KBase) platform ( http://www.kbase.us/ ). Overall, these steps take about a day to obtain extracted genomes when starting from smaller environmental shotgun read libraries, or up to about a week from larger libraries. In KBase, the process is end-to-end, allowing a user to go from the initial sequencing reads all the way through to MAGs, which can then be analyzed with other KBase capabilities such as phylogenetic placement, functional assignment, metabolic modeling, pangenome functional profiling, RNA-Seq and others. While portions of such capabilities are available individually from other resources, the combination of the intuitive usability, data interoperability and integration of tools in a freely available computational resource makes KBase a powerful platform for obtaining MAGs from microbiomes. While this workflow offers tools for each of the key steps in the genome extraction process, it also provides a scaffold that can be easily extended with additional MAG recovery and analysis tools, via the KBase software development kit (SDK).

A Metabolomics-Based Toolbox to Assess and Compare the Metabolic Potential of Unexplored, Difficult-to-Grow Bacteria.

Novel high-throughput cultivation techniques create a demand to pre-select strains for in-depth follow-up studies. We report a workflow to identify promising producers of novel natural products by systematically characterizing their metabolomes. For this purpose, 60 strains from four phyla (Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes) comprising 16 novel species and six novel genera were cultivated from marine and terrestrial sources. Their cellular metabolomes were recorded by LC-MS/MS; data analysis comprised databases MS/MS matching, in silico compound assignment, and GNPS-based molecular networking. Overall, 1052 different molecules were identified from 6418 features, among them were unusual metabolites such as 4-methoxychalcone. Only a minor portion of the 755 features were found in all phyla, while the majority occurred in a single phylogroup or even in a single strain. Metabolomic methods enabled the recognition of highly talented strains such as AEG42_45, which had 107 unique features, among which a family of 28 potentially novel and related compounds according to MS/MS similarities. In summary, we propose that high-throughput cultivation and isolation of bacteria in combination with the presented systematic and unbiased metabolome analysis workflow is a promising approach to capture and assess the enormous metabolic potential of previously uncultured bacteria.

Role of gene sequencing for the diagnosis, tracking and prevention of bacterial infections

Gene sequencing is the inevitable future of diagnostic microbiology. Of the various molecular assays available, sequencing is the promising technique for detecting culture-negative infections due to uncultivable bacteria namely culture-negative endocarditis, meningitis, brain abscess, keratitis, urinary tract infections, empyema, septic arthritis and septicaemia. Sequencing also helps to predict full resistance profile of bacteria and its virulence traits. Sequencing is an emerging and powerful technique to perform the epidemiological studies in an outbreak situation. This review focuses on the common applications of sequencing in clinical bacteriology including isolate characterisation, antimicrobial resistance and virulence factor profiling, establishing the source of infections and tracking the disease transmission.

Validation of Propidium Monoazide-qPCR for Assessing Treatment Effectiveness against ‘Candidatus Liberibacter asiaticus’ in Citrus

Huanglongbing (HLB) is an important citrus disease associated with the phloem-limited, uncultured bacterium ‘Candidatus Liberibacter asiaticus’(CLas). Effective treatments against CLas have to be validated in the field, however, methods for the field assessment of treatment effectiveness are time-consuming, in part because DNA-based assays, including quantitative PCR (qPCR), cannot differentiate between live and dead bacterial DNA. The aim of this study was to develop a method for rapid the evaluation of HLB therapies in field experiments. To this aim, a DNA extraction method from citrus leaf tissues with propidum monoazide (PMA), a dye that binds covalently to dsDNA making it unavailable for amplification in subsequent qPCR reactions, was optimized. The results indicated that the efficacy of PMA-qPCR was highly dependent on the primer set used. Primers targeting the 16S region of CLas showed a clear distinction between qPCR from PMA-treated and non-treated samples, while the RNR and LJ900 primers did not show significant differences between the DNA extraction methods. The PMA-qPCR viability analysis of CLas from citrus cuttings treated with different ampicillin (Amp) concentrations showed that all concentrations reduced CLas titers significantly starting 4 days after the initial treatment, unlike the water treatment, which did not show any change. This method was used for assessing the antibacterial activity of Amp, Streptomycin, Oxytetracycline (OTC), and a water control in field tests. The PMA-qPCR results indicated that Amp and OTC displayed significant antibacterial activity against CLas by 8 days post-injection, which was not detected in the non-PMA qPCR analysis. This method could allow the rapid validation of treatments against CLas in field experiments and facilitate the implementation of effective management strategies against HLB.

Paracrocinitomix mangrovi gen. nov., sp. nov., isolated from a mangrove sediment: proposal of two new families, Phaeocystidibacteraceae fam. nov. and Owenweeksiaceae fam. nov., and emended description of the family Schleiferiaceae.

A Gram-stain-negative and short rod-shaped bacterial strain designated GM2-3-6-6T was obtained from a mangrove sediment. Cells were light yellow-pigmented, catalase-positive and oxidase-positive. Carotenoid pigment was produced. Phylogeny of the 16S rRNA gene showed that strain GM2-3-6-6T was affiliated to the family Crocinitomicaceae, sharing maximum sequence similarities with Crocinitomix algicola 0182T, C. catalasitica IFO 15977T, and Putridiphycobacter roseus SM1701T of 93.8%, 93.6%, and 92.5%, respectively. The average nucleotide identity values, digital DNA-DNA hybridization estimates and average amino acid identity values between strain GM2-3-6-6T and the three close relatives were 68.6-68.8%, 18.5-19.2%, and 59.0-62.3%, respectively. The complete circular genome of strain GM2-3-6-6T was 4,365,762bp in length with a DNA G + C content of 35.0%. The respiratory quinone was MK-7. The major polar lipids consisted of phosphatidylethanolamine, two unidentified phospholipids, one unidentified aminoglycolipid, one unidentified aminolipid and four other unidentified lipids. The major fatty acids were iso-C15:0, iso-C15:1 G, summed feature 3 (C16:1ω7c and/or C16:1ω6c), and iso-C17:0 3-OH. Based on genomic, phenotypic, and chemotaxonomic characterizations, strain GM2-3-6-6T represents a novel species of a novel genus, for which the name Paracrocinitomix mangrovi gen. nov., sp. nov. is proposed. The type strain is GM2-3-6-6T (= MCCC 1K04831T = KCTC 82931T). Additionally, phylogenomic analysis of the type strains of the family Schleiferiaceae and family Cryomorphaceae related members including uncultivated bacteria, was performed using the Genome Taxonomic Database toolkit (GTDB-Tk). Based on 16S rRNA gene phylogeny and genomic features, two novel families, Phaeocystidibacteraceae fam. nov. and Owenweeksiaceae fam. nov. are proposed. An emended description of the family Schleiferiaceae is also proposed.