Kaistella rhinocerotis sp. nov., isolated from the faeces of rhinoceros and reclassification of Chryseobacterium faecale as Kaistella faecalis comb. nov.

Strain Ran72T, a novel Gram-stain-negative, obligately aerobic, non-motile, and rod-shaped bacterium, was isolated from the faeces of the rhinoceros species Ceratotherium simum. The novel bacterial strain grew optimally in Reasoner's 2A medium under the following conditions: 0 % (w/v) NaCl, pH 7.5, and 30 °C. Based on phylogenetic analysis using 16S rRNA gene sequencing, strain Ran72T was found to be most closely related to Chryseobacterium faecale F4T (98.4 %), Kaistella soli DKR-2T (98.0 %), and Kaistella haifensis H38T (97.4 %). A comprehensive genome-level comparison between strain Ran72T with C. faecale F4T, K. soli DKR-2T, and K. haifensis H38T revealed average nucleotide identity, digital DNA-DNA hybridization, and average amino acid identity values of ≤74.9, ≤19.3, and ≤78.7 %, respectively. The major fatty acids were anteiso-C15 : 0 (22.3 %), with MK-6 being the predominant respiratory quinone. The major polar lipids of strain Ran72T were phosphatidylethanolamine, four unidentified aminolipids, and two unidentified lipids. Based on our chemotaxonomic, genotypic, and phenotype characterizations, strain Ran72T was identified as representing a novel species in the genus Kaistella, for which the name Kaistella rhinocerotis sp. nov. is proposed, with the type strain Ran72T (=KACC 23136T=JCM 36038T). Based on the outcomes of our phylogenomic study, Chryseobacterium faecale should be reclassified under the genus Kaistella as Kaistella faecalis comb. nov.

Sustainable control of Microcystis aeruginosa, a harmful cyanobacterium, using Selaginella tamariscina extracts.

Eco-friendly reagents derived from plants represent a promising strategy to mitigate the occurrence of toxic cyanobacterial blooms. The use of an amentoflavone-containing Selaginella tamariscina extract (STE) markedly decreased the number of Microcystis aeruginosa cells, thus demonstrating significant anti-cyanobacterial activity. In particular, the Microcystis-killing fraction obtained from pulverized S. tamariscina using hot-water-based extraction at temperatures of 40 °C induced cell disruption in both axenic and xenic M. aeruginosa. Liquid chromatographic analysis was also conducted to measure the concentration of amentoflavone in the STE, thus supporting the potential M. aeruginosa-specific killing effects of STE. Bacterial community analysis revealed that STE treatment led to a reduction in the relative abundance of Microcystis species while also increasing the 16S rRNA gene copy number in both xenic M. aeruginosa NIBR18 and cyanobacterial bloom samples isolated from a freshwater environment. Subsequent testing on bacteria, cyanobacteria, and algae isolated from freshwater revealed that STE was not toxic for other taxa. Furthermore, ecotoxicology assessment involving Aliivibrio fischeri, Daphnia magna, and Danio rerio found that high STE doses immobilized D. magna but did not impact the other organisms, while there was no change in the water quality. Overall, due to its effective Microcystis-killing capability and low ecotoxicity, aqueous STE represents a promising practical alternative for the management of Microcystis blooms.

Biological and Chemical Approaches for Controlling Harmful Microcystis Blooms.

The proliferation of harmful cyanobacterial blooms dominated by Microcystis aeruginosa has become an increasingly serious problem in freshwater ecosystems due to climate change and eutrophication. Microcystis-blooms in freshwater generate compounds with unpleasant odors, reduce the levels of dissolved O2, and excrete microcystins into aquatic ecosystems, potentially harming various organisms, including humans. Various chemical and biological approaches have thus been developed to mitigate the impact of the blooms, though issues such as secondary pollution and high economic costs have not been adequately addressed. Red clays and H2O2 are conventional treatment methods that have been employed worldwide for the mitigation of the blooms, while novel approaches, such as the use of plant or microbial metabolites and antagonistic bacteria, have also recently been proposed. Many of these methods rely on the generation of reactive oxygen species, the inhibition of photosynthesis, and/or the disruption of cellular membranes as their mechanisms of action, which may also negatively impact other freshwater microbiota. Nevertheless, the underlying molecular mechanisms of anticyanobacterial chemicals and antagonistic bacteria remain unclear. This review thus discusses both conventional and innovative approaches for the management of M. aeruginosa in freshwater bodies.

Lineage-specific evolution of Aquibium, a close relative of Mesorhizobium, during habitat adaptation.

The novel genus Aquibium that lacks nitrogenase was recently reclassified from the Mesorhizobium genus. The genomes of Aquibium species isolated from water were smaller and had higher GC contents than those of Mesorhizobium species. Six Mesorhizobium species lacking nitrogenase were found to exhibit low similarity in the average nucleotide identity values to the other 24 Mesorhizobium species. Therefore, they were classified as the non-N2-fixing Mesorhizobium lineage (N-ML), an evolutionary intermediate species. The results of our phylogenomic analyses and the loss of Rhizobiales-specific fur/mur indicated that Mesorhizobium species may have evolved from Aquibium species through an ecological transition. Halotolerant and alkali-resistant Aquibium and Mesorhizobium microcysteis belonging to N-ML possessed many tripartite ATP-independent periplasmic transporter and sodium/proton antiporter subunits composed of seven genes (mrpABCDEFG). These genes were not present in the N2-fixing Mesorhizobium lineage (ML), suggesting that genes acquired for adaptation to highly saline and alkaline environments were lost during the evolution of ML as the habitat changed to soil. Land-to-water habitat changes in Aquibium species, close relatives of Mesorhizobium species, could have influenced their genomic evolution by the gain and loss of genes. Our study indicated that lineage-specific evolution could have played a significant role in shaping their genome architecture and conferring their ability to thrive in different habitats.IMPORTANCEPhylogenetic analyses revealed that the Aquibium lineage (AL) and non-N2-fixing Mesorhizobium lineage (N-ML) were monophyletically grouped into distinct clusters separate from the N2-fixing Mesorhizobium lineage (ML). The N-ML, an evolutionary intermediate species having characteristics of both ancestral and descendant species, could provide a genomic snapshot of the genetic changes that occur during adaptation. Genomic analyses of AL, N-ML, and ML revealed that changes in the levels of genes related to transporters, chemotaxis, and nitrogen fixation likely reflect adaptations to different environmental conditions. Our study sheds light on the complex and dynamic nature of the evolution of rhizobia in response to changes in their environment and highlights the crucial role of genomic analysis in understanding these processes.

Antagonistic actions of Paucibacter aquatile B51 and its lasso peptide paucinodin toward cyanobacterial bloom-forming Microcystis aeruginosa PCC7806.

Superior antagonistic activity against axenic Microcystis aeruginosa PCC7806 was observed with Paucibacter sp. B51 isolated from cyanobacterial bloom samples among 43 tested freshwater bacterial species. Complete genome sequencing, analyzing average nucleotide identity and digital DNA-DNA hybridization, designated the B51 strain as Paucibacter aquatile. Electron and fluorescence microscopic image analyses revealed the presence of the B51 strain in the vicinity of M. aeruginosa cells, which might provoke direct inhibition of the photosynthetic activity of the PCC7806 cells, leading to perturbation of cellular metabolisms and consequent cell death. Our speculation was supported by the findings that growth failure of the PCC7806 cells led to low pH conditions with fewer chlorophylls and down-regulation of photosystem genes (e.g., psbD and psaB) during their 48-h co-culture condition. Interestingly, the concentrated ethyl acetate extracts obtained from B51-grown supernatant exhibited a growth-inhibitory effect on PCC7806. The physical separation of both strains by a filter system led to no inhibitory activity of the B51 cells, suggesting that contact-mediated anti-cyanobacterial compounds might also be responsible for hampering the growth of the PCC7806 cells. Bioinformatic tools identified 12 gene clusters that possibly produce secondary metabolites, including a class II lasso peptide in the B51 genome. Further chemical analysis demonstrated anti-cyanobacterial activity from fractionated samples having a rubrivinodin-like lasso peptide, named paucinodin. Taken together, both contact-mediated inhibition of photosynthesis and the lasso peptide secretion of the B51 strain are responsible for the anti-cyanobacterial activity of P. aquatile B51.

The ß-Lactamase Activity at the Community Level Confers ß-Lactam Resistance to Bloom-Forming Microcystis aeruginosa Cells.

Many freshwater cyanobacteria, including Microcystis aeruginosa, lack several known antibiotic resistance genes; however, both axenic and xenic M. aeruginosa strains exhibited high antibiotic resistance against many antibiotics under our tested concentrations, including colistin, trimethoprim, and kanamycin. Interestingly, axenic PCC7806, although not the xenic NIBR18 and NIBR452 strains, displayed susceptibility to ampicillin and amoxicillin, indicating that the associated bacteria in the phycosphere could confer such antibiotic resistance to xenic strains. Fluorescence and scanning electron microscopic observations revealed their tight association, leading to possible community-level ß-lactamase activity. Combinatory treatment of ampicillin with a ß-lactamase inhibitor, sulbactam, abolished the ampicillin resistance in the xenic stains. The nitrocefin-based assay confirmed the presence of significant community-level ß-lactamase activity. Our tested low ampicillin concentration and high ß-lactamase activity could potentially balance the competitive advantage of these dominant species and provide opportunities for the less competitive species, thereby resulting in higher bacterial diversity under ampicillin treatment conditions. Non-PCR-based metagenome data from xenic NIBR18 cultures revealed the dominance of blaOXA-related antibiotic resistance genes followed by other class A ß-lactamase genes (AST-1 and FAR-1). Alleviation of ampicillin toxicity could be observed only in axenic PCC7806, which had been cocultured with ß-lactamase from other freshwater bacteria. Our study suggested M. aeruginosa develops resistance to old-class ß-lactam antibiotics through altruism, where associated bacteria protect axenic M. aeruginosa cells.

Morphological and physiological adaptations of psychrophilic Pseudarthrobacter psychrotolerans YJ56 under temperature stress.

Both culture-independent and culture-dependent analyses using Nanopore-based 16S rRNA sequencing showed that short-term exposure of Antarctic soils to low temperature increased biomass with lower bacterial diversity and maintained high numbers of the phylum Proteobacteria, Firmicute, and Actinobacteria including Pseudarthrobacter species. The psychrophilic Pseudarthrobacter psychrotolerans YJ56 had superior growth at 13 °C, but could not grow at 30 °C, compared to other bacteria isolated from the same Antarctic soil. Unlike a single rod-shaped cell at 13 °C, strain YJ56 at 25 °C was morphologically shifted into a filamentous bacterium with several branches. Comparative genomics of strain YJ56 with other genera in the phylum Actinobacteria indicate remarkable copy numbers of rimJ genes that are possibly involved in dual functions, acetylation of ribosomal proteins, and stabilization of ribosomes by direct binding. Our proteomic data suggested that Actinobacteria cells experienced physiological stresses at 25 °C, showing the upregulation of chaperone proteins, GroEL and catalase, KatE. Level of proteins involved in the assembly of 50S ribosomal proteins and L29 in 50S ribosomal proteins increased at 13 °C, which suggested distinct roles of many ribosomal proteins under different conditions. Taken together, our data highlights the cellular filamentation and protein homeostasis of a psychrophilic YJ56 strain in coping with high-temperature stress.

AamA-mediated epigenetic control of genome-wide gene expression and phenotypic traits in Acinetobacter baumannii ATCC 17978.

Individual deletions of three genes encoding orphan DNA methyltransferases resulted in the occurrence of growth defect only in the aamA (encoding AcinetobacterAdenine Methylase A) mutant of A. baumannii strain ATCC 17978. Our single-molecule real-time sequencing-based methylome analysis revealed multiple AamA-mediated DNA methylation sites and proposed a potent census target motif (TTTRAATTYAAA). Loss of Dam led to modulation of genome-wide gene expression, and several Dam-target sites including the promoter region of the trmD operon (rpsP, rimM, trmD, and rplS) were identified through our methylome and transcriptome analyses. AamA methylation also appeared to control the expression of many genes linked to membrane functions (lolAB, lpxO), replication (dnaA) and protein synthesis (trmD operon) in the strain ATCC 17978. Interestingly, cellular resistance against several antibiotics and ethidium bromide through functions of efflux pumps diminished in the absence of the aamA gene, and the complementation of aamA gene restored the wild-type phenotypes. Other tested phenotypic traits such as outer-membrane vesicle production, biofilm formation and virulence were also affected in the aamA mutant. Collectively, our data indicated that epigenetic regulation through AamA-mediated DNA methylation of novel target sites mostly in the regulatory regions could contribute significantly to changes in multiple phenotypic traits in A. baumannii ATCC 17978.

Rheinheimera faecalis sp. nov., isolated from Ceratotherium simum feces.

A novel strain YR1T, Gram-stain-negative, rod-shaped, catalase- and oxidase-positive, and aerobic bacterium, was isolated from the feces of Ceratotherium simum. The strain grew at 9-42 °C (optimal temperature, 30 °C), at pH 6.0-10.0 (optimal pH, 7.0), and in the presence of 0-3% (w/v) NaCl (optimal salinity, 0%). Phylogenetic analyses based on 16S rRNA gene sequencing indicated that strain YR1T was most closely related to Rheinheimera soli BD-d46T (98.6%), R. riviphila KYPC3T (98.6%), and R. mangrovi LHK 132T (98.1%). Moreover, the average nucleotide identity, average amino acid identity, and digital DNA-DNA hybridization values between strain YR1T and R. mangrovi LHK 132 T were 88.3%, 92.1%, and 35.3%, respectively, indicating that strain YR1T is a novel species in the genus Rheinheimera. The genome size and genomic DNA G + C content of strain YR1T were 4.5 Mbp and 46.37%, respectively. The major polar lipids were phosphatidylethanolamine and phosphatidylglycerol, while the predominant respiratory quinone was Q-8. Summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), C16: 0, and summed feature 8 (C18:1 ω7c) were the primary cellular fatty acids (> 16%). Based on these genotypic and phenotypic characteristics, strain YR1T was identified as a novel species in the genus Rheinheimera, for which the name Rheinheimera faecalis sp. nov. is proposed, with the type strain is YR1T (= KACC 22402T = JCM 34823T).

CDK7 controls E2F- and MYC-driven proliferative and metabolic vulnerabilities in multiple myeloma.

Therapeutic targeting of CDK7 has proven beneficial in preclinical studies, yet the off-target effects of currently available CDK7 inhibitors make it difficult to pinpoint the exact mechanisms behind MM cell death mediated by CDK7 inhibition. Here, we show that CDK7 expression positively correlates with E2F and MYC transcriptional programs in cells from patients with multiple myeloma (MM); its selective targeting counteracts E2F activity via perturbation of the cyclin-dependent kinases/Rb axis and impairs MYC-regulated metabolic gene signatures translating into defects in glycolysis and reduced levels of lactate production in MM cells. CDK7 inhibition using the covalent small-molecule inhibitor YKL-5-124 elicits a strong therapeutic response with minimal effects on normal cells, and causes in vivo tumor regression, increasing survival in several mouse models of MM including a genetically engineered mouse model of MYC-dependent MM. Through its role as a critical cofactor and regulator of MYC and E2F activity, CDK7 is therefore a master regulator of oncogenic cellular programs supporting MM growth and survival, and a valuable therapeutic target providing rationale for development of YKL-5-124 for clinical use.

Unlocking the mystery of lysine toxicity on Microcystis aeruginosa.

Lysine toxicity on certain groups of bacterial cells has been recognized for many years, but the detailed molecular mechanisms that drive this phenomenon have not been elucidated. Many cyanobacteria including Microcystis aeruginosa cannot efficiently export and degrade lysine, although they have evolved to maintain a single copy of the lysine uptake system through which arginine or ornithine can also be transported into the cytoplasm. Autoradiographic analysis using 14C-l-lysine confirmed that lysine was competitively uptaken into cells with arginine or ornithine, which explained the arginine or ornithine-mediated alleviation of lysine toxicity in M. aeruginosa. A relatively non-specific MurE amino acid ligase could incorporate l-lysine into the 3rd position of UDP-N-acetylmuramyl-tripeptide by replacing meso-diaminopimelic acid during the stepwise addition of amino acids on peptidoglycan (PG) biosynthesis. However, further transpeptidation was blocked because lysine substitution at the pentapeptide of the cell wall inhibited the activity of transpeptidases. The leaky PG structure caused irreversible damage to the photosynthetic system and membrane integrity. Collectively, our results suggest that a lysine-mediated coarse-grained PG network and the absence of concrete septal PG lead to the death of slow-growing cyanobacteria.

Blue Light Sensing BlsA-Mediated Modulation of Meropenem Resistance and Biofilm Formation in Acinetobacter baumannii.

The presence or absence of BlsA, a protein with a blue light-sensing flavin domain in the genomes of Acinetobacter species has aroused curiosity about its roles in the regulation of bacterial lifestyle under light. Genomic and transcriptomic analyses revealed the loss of BlsA in several multidrug-resistant (MDR) A. baumannii strains as well as the light-mediated induction of blsA, along with a possible BlsA-interacting partner BipA. Their direct in vivo interactions were verified using a bacterial two-hybrid system. The results demonstrated that the C-terminal region of BipA could bind to the C-terminal residues of BlsA under blue light at 23°C but not at 37°C. Genetic manipulations of blsA and bipA revealed that the coexistence of BlsA and BipA was required to induce the light-dependent expression of ompA in A. baumannii ATCC 17978 at 23°C. The same phenomenon occurred in the BlsA-deficient MDR strain in our functional complementation assay; however, the underlying molecular mechanism remains poorly understood. BlsA-modulated amounts of OmpA, the most abundant porin, in the outer membrane affected the membrane integrity and permeability of small molecules. Dark conditions or the deletion of ompA made the membrane more permeable to lipophilic ethidium bromide (EtBr) but not to meropenem. Interestingly, light illumination and low temperature conditions made the cells more sensitive to meropenem; however, this bactericidal effect was not noted in the blsA mutant or in the BlsA-deficient MDR strains. Light-mediated cell death and the reduction of biofilm formation at 23°C were abolished in the blsA mutant strain, suggesting multifaceted roles of BlsA in A. baumannii strains. IMPORTANCE Little is known about the functional roles of BlsA and its interacting partners in Acinetobacter species. Intriguingly, no BlsA homolog was found in several clinical isolates, suggesting that BlsA was not required inside the host because of the lack of blue light and the warm temperature conditions. As many chromophore-harboring proteins interact with various partners to control light-dependent cellular behaviors, the maintenance of blsA in the genomes of many Acinetobacter species during their evolution may be beneficial when fluctuations occur in two important environmental factors: light and temperature. Our study is the first to report the novel protein partner of BlsA, namely, BipA, and its contribution to multiple phenotypic changes, including meropenem resistance and biofilm formation. Rapid physiological acclimation to changing light or temperature conditions may be possible in the presence of the light-sensing BlsA protein, which may have more interacting partners than expected.

Zoo animal manure as an overlooked reservoir of antibiotic resistance genes and multidrug-resistant bacteria.

Animal fecal samples collected in the summer and winter from 11 herbivorous animals, including sable antelope (SA), long-tailed goral (LTG), and common eland (CE), at a public zoo were examined for the presence of antibiotic resistance genes (ARGs). Seven antibiotics, including meropenem and azithromycin, were used to isolate culturable multidrug-resistant (MDR) strains. The manures from three animals (SA, LTG, and CE) contained 104-fold higher culturable MDR bacteria, including Chryseobacterium, Sphingobacterium, and Stenotrophomonas species, while fewer MDR bacteria were isolated from manure from water buffalo, rhinoceros, and elephant against all tested antibiotics. Three MDR bacteria-rich samples along with composite samples were further analyzed using nanopore-based technology. ARGs including lnu(C), tet(Q), and mef(A) were common and often associated with transposons in all tested samples, suggesting that transposons carrying ARGs may play an important role for the dissemination of ARGs in our tested animals. Although several copies of ARGs such as aph(3')-IIc, blaL1, blaIND-3, and tet(42) were found in the sequenced genomes of the nine MDR bacteria, the numbers and types of ARGs appeared to be less than expected in zoo animal manure, suggesting that MDR bacteria in the gut of the tested animals had intrinsic resistant phenotypes in the absence of ARGs.

A MIR17HG-derived long noncoding RNA provides an essential chromatin scaffold for protein interaction and myeloma growth.

Long noncoding RNAs (lncRNAs) can drive tumorigenesis and are susceptible to therapeutic intervention. Here, we used a large-scale CRISPR interference viability screen to interrogate cell-growth dependency to lncRNA genes in multiple myeloma (MM) and identified a prominent role for the miR-17-92 cluster host gene (MIR17HG). We show that an MIR17HG-derived lncRNA, named lnc-17-92, is the main mediator of cell-growth dependency acting in a microRNA- and DROSHA-independent manner. Lnc-17-92 provides a chromatin scaffold for the functional interaction between c-MYC and WDR82, thus promoting the expression of ACACA, which encodes the rate-limiting enzyme of de novo lipogenesis acetyl-coA carboxylase 1. Targeting MIR17HG pre-RNA with clinically applicable antisense molecules disrupts the transcriptional and functional activities of lnc-17-92, causing potent antitumor effects both in vitro and in vivo in 3 preclinical animal models, including a clinically relevant patient-derived xenograft NSG mouse model. This study establishes a novel oncogenic function of MIR17HG and provides potent inhibitors for translation to clinical trials.

Constitutive Phenotypic Modification of Lipid A in Clinical Acinetobacter baumannii Isolates.

The degree of polymyxin B (PMB) resistance was measured in 40 clinical Acinetobacter baumannii isolates obtained from health care facilities. All of the tested isolates possessed a multidrug-resistant (MDR) phenotype against four classes of antibiotics (meropenem, doxycycline, gentamicin, and erythromycin), except for PMB. The blaOXA-23 gene was detected throughout the genetic analysis and experimental assay, indicating that all of the MDR strains were carbapenem-resistant A. baumannii strains. Multilocus sequence typing-based genotyping revealed that nine selected strains belonged to the international clone II lineage. When matrix-assisted laser desorption ionization-time of flight mass spectrometry was performed, intrinsic lipid A modification by phosphoethanolamine (PEtN) incorporation was noticeable only in the PMB-resistant (PMBR) strains. However, the presence of hexa- and penta-acylated lipid A due to the loss of the laurate (C12) acyl chain was noted in all PMB-susceptible strains but not in the PMBR strains. The reduction of negative surface charges in the PMBR strains was assessed by zeta potential analysis. Fluorescence imaging using dansyl-PMB revealed that, in the PMBR strains, PMB was less likely to bind to the cell surface. IMPORTANCE The widespread presence of MDR pathogens, including A. baumannii, is causing serious hospital-acquired infections worldwide. Extensive surveillance of MDR clinical A. baumannii isolates has been conducted, but the underlying mechanisms for their development of MDR phenotypes are often neglected. Either lipid A modification or loss of lipopolysaccharide in Gram-negative bacteria leads to PMBR phenotypes. The prevalence of intrinsic lipid A modification in PMBR clinical strains was attributed to high levels of basal expression of pmrC and eptA-1. Our findings suggest that new therapeutic strategies are warranted to combat MDR pathogens due to the emergence of many PMBR clinical strains.

Chryseobacterium faecale sp. nov., isolated from camel feces.

Strain F4T (=KACC 22401T=JCM 34836T), a novel Gram-stain-negative, aerobic, non-spore-forming, non-motile and rod-shaped bacterium, was isolated from camel (Camelus bactrianus) faeces. The newly identified bacterial strain F4T was grown in Reasoner's 2A medium [0-2 % (w/v) NaCl (optimum, 0 %), pH 7.0-8.0 (optimum, pH 7.0), and 18-40 °C (optimum, 30 °C)]. Phylogenetic analysis based on 16S rRNA gene sequencing confirmed that strain F4T belonged to the genus Chryseobacterium, with its closest neighbours being Chryseobacterium haifense DSM 19056T (98.0 %), Chryseobacterium anthropi CCUG 52764T (97.3 %), Chryseobacterium montana WG4T (95.7 %) and Chryseobacterium koreensis Chj70T (94.7 %). Complete genome sequence of strain F4T was obtained using a hybrid assembly pipeline integrating sequences obtained using both the Oxford Nanopore and Illumina platforms. Genomic comparisons of strain F4T with type species in the genus Chryseobacterium were conducted using digital DNA-DNA hybridization, average nucleotide identity and average amino acid identity, resulting in values of ≤20.5, ≤77.9 and ≤80.8 %, respectively. The genomic DNA G+C content of type strain F4T was 39.7 mol%. The major fatty acids of the strain F4T were anteiso-C15 : 0 and iso-C18 : 3, and MK-6 was its major respiratory quinone. Moreover, the major polar lipid of strain F4T was phosphatidylethanolamine. The genome of strain F4T harbours only one antibiotic resistance gene (blaCME-1) encoding a ß-lactamase, which attributes ß-lactam antibiotic resistance. Based on the results of our chemotaxonomic, genotypic and phenotype analyses, strain F4T is identified as a novel species of the genus Chryseobacterium, for which the name Chryseobacterium faecale sp. nov. is proposed.

6-Bromo-2-naphthol from Silene armeria extract sensitizes Acinetobacter baumannii strains to polymyxin.

The overuse of antibiotics has led to the emergence of multidrug-resistant bacteria, which are resistant to various antibiotics. Combination therapies using natural compounds with antibiotics have been found to have synergistic effects against several pathogens. Synergistic natural compounds can potentiate the effects of polymyxins for the treatment of Acinetobacter baumannii infection. Out of 120 types of plant extracts, only Silene armeria extract (SAE) showed a synergistic effect with polymyxin B (PMB) in our fractional inhibitory concentration and time-kill analyses. The survival rate of G. mellonella infected with A. baumannii ATCC 17978 increased following the synergistic treatment. Interestingly, the addition of osmolytes, such as trehalose, canceled the synergistic effect of SAE with PMB; however, the underlying mechanism remains unclear. Quadrupole time-of-flight liquid chromatography-mass spectrometry revealed 6-bromo-2-naphthol (6B2N) to be a major active compound that exhibited synergistic effects with PMB. Pretreatment with 6B2N made A. baumannii cells more susceptible to PMB exposure in a time- and concentration-dependent manner, indicating that 6B2N exhibits consequential synergistic action with PMB. Moreover, the exposure of 6B2N-treated cells to PMB led to higher membrane leakage and permeability. The present findings provide a promising approach for utilizing plant extracts as adjuvants to reduce the toxicity of PMB in A. baumannii infection.

Rhizorhabdus phycosphaerae sp. nov., isolated from the phycosphere of Microcystis aeruginosa.

A novel bacterial strain, designated MK52T, was isolated from the phycosphere of Microcystis aeruginosa. Strain MK52T is a Gram-stain-negative, pink-pigmented, rod-shaped, strictly aerobic bacterium. In 16S rRNA phylogenetic analysis, the MK52T strain was most closely related to Rhizorhabdus wittichii RW1T (98.66 %) and Rhizorhabdus histidinilytica UM2T (98.51 %). The genomic DNA G+C content of strain MK52T was calculated to be 65.5 mol%. The average nucleotide identity values of strain MK52T with R. wittichii RW1T and R. histidinilytica UM2T were 80.35 and 80.23 %, respectively, with digital DNA-DNA hybridization values of 23.6 and 22.9 %, respectively, and average amino acid identities of 75.59 and 75.79 %, respectively. The major isoprenoid quinone was Q-10, and the predominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and sphingoglycolipid. Fatty acid methyl ether analysis showed that summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) was the main cellular fatty acid in strain MK52T. Strain MK52T cells grew at 21-34 °C (optimum, 30 °C), pH 5-8 (optimum, pH 7) and with 0-2 % (w/v) NaCl (optimum, 0.5 % NaCl). Rhizorhabdus phycosphaerae sp. nov. is proposed as a new species (=KCTC 72877T=DSM 111424T) based on its genotypic and phenotypic characteristics.

Aquibium microcysteis gen. nov., sp. nov., isolated from a Microcystis aeruginosa culture and reclassification of Mesorhizobium carbonis as Aquibium carbonis comb. nov. and Mesorhizobium oceanicum as Aquibium oceanicum comb. nov.

A novel bacterial strain, NIBR3T, was isolated from a Microcystis aeruginosa culture. Strain NIBR3T was characterized as Gram-negative, rod-shaped, catalase- and oxidase-positive, and aerobic. The 16S rRNA gene sequence analysis showed that strain NIBR3T was most closely related to Mesorhizobium carbonis B2.3T (=KCTC 52461), Mesorhizobium oceanicum B7T (=KCTC 42783) and Mesorhizobium qingshengii CCBAU 33460T (=HAMBI 3277), at 98.7, 97.2 and 97.2% similarity, respectively. Our phylogenetic analyses revealed that three strains [strain NIBR3T with the previously reported two Mesorhizobium species (M. carbonis B2.3T and M. oceanicum B7T)] formed a distinct cluster from other Mesorhizobium type strains. The average nucleotide identity of strain NIBR3T relative to M. carbonis B2.3T , M. oceanicum B7T, and M. qingshengii CCBAU 33460T was found to be 84.3, 79.4 and 75.8 %, with average amino-acid identities of 85.1, 74.8 and 64.3 %, and digital DNA-DNA hybridization values of 27.6, 22.6 and 20.7 %, respectively. The genome size and genomic DNA G+C content of NIBR3T were 6.1 Mbp and 67.9 mol%, respectively. Growth of strain NIBR3T was observed at 23-45 °C (optimum, 33 °C), at pH 6-11 (optimum, 8) and in the presence of 0-4 % (w/v) NaCl (optimum, 0 %). The major polar lipids in this novel strain were phosphatidylethanolamine, phosphatidylcholine and phosphatidylmethylethanolamine. The predominant respiratory quinone was Q-10. Summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) was the most abundant cellular fatty acid in strain NIBR3T. Based on genotypic characteristics using our genomic data, strain NIBR3T was identified as a member of new genus, Aquibium gen. nov., with the two aforementioned stains. The type strain f the novel species, Aquibium microcysteis sp. nov., is NIBR3T (=KACC 22092T=HAMBI 3738T). We also reclassified Mesorhizobium carbonis and M. oceanicum as Aquibium carbonis comb. nov. and A. oceanicum comb. nov., respectively.

Occurrence of antibiotic resistance genes and multidrug-resistant bacteria during wastewater treatment processes.

Wastewater treatment plants (WWTPs) constantly receive a wide variety of contaminants, including pharmaceuticals, and are potential reservoirs of antibiotic resistance genes (ARGs). This favors the development of multidrug-resistant bacteria (MRB) through horizontal gene transfer. Samples from five different WWTP processes were collected in September 2020 and January 2021 to monitor ARG resistomes and culturable MRB in the presence of eight different antibiotics. Nanopore-based ARG abundance and bacterial community analyses suggested that ARG accumulation favors the generation of MRB. Activated and mixed sludges tended to have lower bacterial diversity and ARG abundance because of selective forces that favored the growth of specific microorganisms during aeration processes. Escherichia strains enriched in WWTPs (up to 71%) were dominant in all the samples, whereas Cloacamonas species were highly abundant only in anaerobically digested sludge samples (60%-79%). Two ARG types [sulfonamide resistance genes (sul1) and aminoglycoside resistance genes (aadA1, aadA13, and aadA2)] were prevalent in all the processes. The total counts of culturable MRB, such as Niabella, Enterococcus, Bacillus, and Chryseobacterium species, gradually increased during aerobic WWTP processes. Genomic analyses of all MRB isolated from the samples revealed that the resistome of Enterococcus species harbored the highest number of ARGs (7-18 ARGs), commonly encoding ant(6)-la, lnu(B), erm(B), and tet(S/M). On the other hand, Niablella strains possibly had intrinsic resistant phenotypes without ARGs. All MRB possessed ARGs originating from the same mobile genetic elements, suggesting that WWTPs are hotspots for the migration of ARGs and emergence of MRB.