MzmL, a novel marine derived N-acyl homoserine lactonase from Mesoflavibacter zeaxanthinifaciens that attenuates Pectobacterium carotovorum subsp. carotovorum virulence.

Quorum sensing (QS) is a conserved cell-cell communication mechanism widely distributed in bacteria, and is oftentimes tightly correlated with pathogen virulence. Quorum quenching enzymes, which interfere with QS through degrading the QS signaling molecules, could attenuate virulence instead of killing the pathogens, and thus are less likely to induce drug resistance. Many Gram-negative bacteria produce N-acyl homoserine lactones (AHLs) for interspecies communication. In this study, we isolated and identified a bacterial strain, Mesoflavibacter zeaxanthinifaciens XY-85, from an Onchidium sp. collected from the intertidal zone of Dapeng Reserve in Shenzhen, China, and found it had strong AHL degradative activity. Whole genome sequencing and blast analysis revealed that XY-85 harbors an AHL lactonase (designated MzmL), which is predicted to have an N-terminal signal peptide and share the "HXHXDH" motif with known AHL lactonases belonging to the Metallo-ß-lactamase superfamily. Phylogenetic studies showed MzmL was closest to marine lactonase cluster members, MomL and Aii20J, instead of the AiiA type lactonases. Ultra performance liquid chromatography-mass spectrometry analysis confirmed that MzmL functions as an AHL lactonase catalyzing AHL degradation through lactone hydrolysis. MzmL could degrade both short- and long-chain AHLs with or without a substitution of oxo-group at the C-3 position, and retained full bioactivity under a wide range of temperatures (28-100°C) and pHs (4-11). Furthermore, MzmL significantly reduced Pectobacterium carotovorum subsp. carotovorum virulence factor production in vitro, such as biofilm formation and plant cell wall degrading enzyme production, and inhibited soft rot development on potato slices. These results demonstrated that MzmL may be a novel type of AHL lactonase with good environmental stability, and has great potential to be developed into a novel biological control agent for bacterial disease management.

Pacificoceanicola onchidii gen. nov., sp. nov., isolated from a marine invertebrate from the South China Sea.

A Gram-stain-negative, facultative anaerobic, non-flagellated and oval-shaped (0.77-0.98 µm wide and 0.74-1.21 µm long) bacterial strain, designated XY-301T, was isolated from a marine invertebrate collected from the South China Sea. Strain XY-301T grew at 15-37 °C (optimum, 30-35 °C) and at pH 7.0-8.5 (optimum, pH 8.0). The strain was slightly halophilic and it only grew in the presence of 0.5-6.5 % (w/v) NaCl (optimum, 2.5-3.5 %). Its predominant fatty acid (>10 %) was C18 : 1 ω7c. The predominant polar lipids of XY-301T were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, six unidentified aminolipids, three unidentified phospholipids and two unknown polar lipids. The respiratory quinone was Q-10. The genome of XY-301T was 4 979 779 bp in size, with a DNA G+C content of 61.3 mol%. The average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity values between XY-301T and Pseudoprimorskyibacter insulae SSK3-2T were 73.3, 14.5 and 53.5 %, respectively. Based on the results of phylogenetic, phenotypic, chemotaxonomic and genomic analyses, strain XY-301T is considered to represent a novel species and a new genus of the family Roseobacteraceae, for which the name Pacificoceanicola onchidii gen. nov., sp. nov. is proposed. The type strain is XY-301T (=KCTC 72212T=MCCC 1K03614T).

Prodigiosin as an antibiofilm agent against multidrug-resistant Staphylococcus aureus.

Staphylococcus aureus is known for forming bacterial biofilms that confer increased antimicrobial resistance. Combining antibiotics with antibiofilm agents is an alternative approach, but the antibiofilm ability of prodigiosin (PG), a potential antibiotic synergist, against antimicrobial-resistant (AMR) S. aureus remains to be understood. The antibiofilm activity of PG against 29 clinical AMR S. aureus strains was evaluated using crystal violet staining, and its synergistic effects with vancomycin (VAN) was confirmed using the checkerboard test. The viability and metabolic activity of biofilms and planktonic cells were also assessed. The results revealed that PG exhibited promising inhibitory activity against biofilm formation and synergistic activity with VAN. It effectively reduced the metabolic activity of biofilms and suppressed the production of exopolysaccharides, which might be attributed to the downregulation of biofilm-related genes such as sarA, agrA, and icaA. These findings suggest that PG could be used as a preventive coating or adjuvant against biofilms in clinical settings.

A novel yeast strain Geotrichum sp. CS-67 capable of accumulating heavy metal ions.

Bioremediation, which has several advantages over traditional methods, represents an alternative means of dealing with heavy metal pollution. We screened for microorganisms showing heavy metal tolerance in polluted mangrove soils. A novel yeast, Geotrichum sp. CS-67, was discovered and tested for tolerance of Cu2+, Zn2+, and Ni2+. Zn2+ was the most efficiently sequestered by Geotrichum sp. CS-67 followed by Ni2+ and Cu2+. Zn2+ and Ni2+ were actively taken up into the cell, while Cu2+ was adsorbed to the cell wall. We used RNA-Seq to show that a large number of genes involved in the physiological and biochemical processing of heavy metals were differentially expressed in this yeast when it was subjected to Zn2+ and Ni2+ stress. From this panel, we selected the SED1, GDI1 and ZRT1 genes for validation by qRT-PCR and discovered that, during Zn2+ and Ni2+ stress, SED1 and GDI1 were upregulated, while ZRT1 was downregulated, which was consistent with the RNA-Seq results and the biochemical function of these genes. In conclusion, the novel yeast Geotrichum sp. CS-67 has a marked ability to accumulate heavy metal ions, making it of great interest as a possible microbial agent for heavy metal pollution remediation in the future.

Algibacter onchidii sp. nov., a symbiotic bacterium isolated from a marine invertebrate.

A novel symbiotic bacterium, designated strain XY-114T, was isolated from the cerata of an Onchidium marine invertebrate species collected in the South China Sea. Strain XY-114T was an aerobic, Gram-stain-negative, non-motile and short rod-shaped bacterium (0.5-0.8 µm wide and 1.0-1.5 µm long) without flagellum. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain XY-114T belonged to the genus Algibacter with the highest similarity of 97.2 % to the closest phylogenetic relative Algibacter aestuarii KYW371T. Cells grew at 15-37 °C (optimum, 30 °C), at pH 5.5-9.0 (optimum 7.0-8.0) and at NaCl concentrations of 0.5-5.0 % (w/v; optimum 1.5-3.0 %). The major fatty acids (>10 %) were summed feature 3 (comprising C16 : 1 ω7c and/or C16 : 1 ω6c), iso-C15 : 0, iso-C15 : 1 G and iso-C17 : 0 3-OH. The predominant polar lipid was phosphatidylethanolamine. The predominant respiratory quinone was MK-6. Flexirubin-type pigments were absent. The genome size of strain XY-114T was 3.4 Mbp, with 34.9 mol% of DNA G+C content. The average nucleotide identity, digital DNA-DNA hybridization and amino acid identity values between strain XY-114T and A. aestuarii KYW371T were 74.5 %, 17.0±1.8 % and 73.9 %. Characterization based on phylogenetic, phenotypic, chemotaxonomic and genomic evidence demonstrated that strain XY-114T represents a novel species of the genus Algibacter, for which the name Algibacter onchidii sp. nov. is proposed. The type strain is XY-114T (=KCTC 72217T=MCCC 1K03606T).

Muricauda onchidii sp. nov., isolated from a marine invertebrate from South China Sea, and transfers of Flagellimonas algicola, Flagellimonas pacifica and Flagellimonas maritima to Muricauda algicola comb. nov., Muricauda parva nom. nov. and Muricauda aurantiaca nom. nov., respectively, and emended description of the genus Muricauda.

An aerobic, Gram-stain-negative, rod-shaped and non-motile strain (XY-359T) was isolated from the mouth of a marine invertebrate Onchidium species from the South China Sea. It grew at pH 6.0-8.5 (optimum, pH 7.5), at 15-37 °C (optimum, 30 °C) and in the presence of 0.5-4.5 % (w/v) NaCl (optimum, 2.5 %). It could not hydrolyse Tweens 20, 40, 60 or 80 and no flexirubin-type pigments were produced. The major polar lipids were phosphatidylethanolamine, one unidentified aminolipid, six unidentified phospholipids and two unidentified polar lipids. The major fatty acids were iso-C17:0 3-OH, iso-C15:1 G and iso-C15:0 3-OH. The respiratory quinone was MK-6. Strain XY-359T showed the greatest degree of 16S rRNA sequence similarity to Flagellimonas algicola AsT0115T (96.54 %), followed by Muricauda flava DSM 22638T (96.27 %). Phylogenetic analysis based on 16S rRNA gene sequences and 31 core genes indicated that strain XY-359T belongs to the genus Muricauda. The genome size of strain XY-359T was 4 207 872 bp, with 39.1 mol% of DNA G+C content. The average nucleotide identity and digital DNA-DNA hybridization values between strain XY-359T and F. algicola AsT0115T were 74.58 % and 18.5 %, respectively, and those between strain XY-359T and M. flava DSM 22638T were 74.2 % and 18.3 %. The combined phenotypic, chemotaxonomic and phylogenetic data suggest that strain XY-359T represents a novel species of the genus Muricauda, for which the name Muricauda onchidii sp. nov. is proposed. The type strain is XY-359T (=MCCC 1K03658T =KCTC 72218T). Moreover, based on the proposal of nesting Spongiibacterium and Flagellimonas within Muricauda by García (Validation List No. 193) and the analyses of phylogenetic trees and average amino acid identities in this study, the transfers of F. algicola, F. pacifica and F. maritima to the genus Muricauda as Muricauda algicola comb. nov., Muricauda parva nom. nov. and M. aurantiaca nom. nov., respectively, are proposed, with an emended description of the genus Muricauda.

Pseudooceanicola onchidii sp. nov., isolated from a marine invertebrate from the South China Sea.

A novel bacterial strain, XY-99T, was isolated from the epidermis of a marine invertebrate of the genus Onchidium from seawater of the South China Sea. The cells of the strain were aerobic, Gram-stain-negative, non-motile, and oval-shaped (0.8-1.0 µm wide and 1.0-1.5 µm long) without a flagellum. The strain grew at temperatures of 15-37 °C (optimum, 35-37 °C), at pH 5.5-9.5 (optimum 7.5), and at NaCl concentrations of 0-12.0 % (w/v) (optimum 1.5-3.0 %). The major fatty acids (>10 %) were summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1ω6c), C16 : 0 and 11-methyl C18 : 1ω7c. The predominant polar lipid was diphosphatidylglycerol. The respiratory quinone was Q-10. The closet phylogenetic neighbours were Pseudooceanicola flagellatus DY470T and Pseudooceanicola nitratireducens JLT1210T, showing 97.5 and 97.3 % of 16 s rRNA gene sequence similarity. The genome size of XY-99T was 3 673 499 bp, with 64.5 % DNA G+C content. The average nucleotide identity and digital DNA-DNA hybridization values between XY-99T and Pseudooceanicola flagellatus DY470T were 72.8 and 14.0 %, respectively, while they were 79.3 and 22.3 % between XY-99T and Pseudooceanicola nitratireducens JLT1210T. Characterization based on phylogenetic, phenotypic, chemotaxonomic and genomic evidence demonstrated that XY-99T represents a novel species of the genus Pseudooceanicola, for which the name Pseudooceanicola onchidii sp. nov. is proposed. The type strain is XY-99T (=KCTC 72211T=MCCC 1K03607T).

Butenolide, a Marine-Derived Broad-Spectrum Antibiofilm Agent Against Both Gram-Positive and Gram-Negative Pathogenic Bacteria.

Bacterial biofilm can cause nosocomial recurrent infections and implanted device secondary infections in patients and strongly promotes development of pathogenic drug resistance in clinical treatments. Butenolide is an effective anti-macrofouling compound derived from a marine Streptomyces sp., but its antibiofilm efficacy remains largely unexplored. In the present study, the antibiofilm activities of butenolide were examined using biofilms formed by both Gram-positive and Gram-negative pathogenic model species. Four Escherichia coli strains, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus (MRSA) were used as targets in antibiofilm assays that examined the effects of butenolide, including the following: (i) on bacterial growth; (ii) in inhibiting biofilm formation and eradicating mature biofilm; (iii) on biofilm structures. In addition, the synergistic effect between butenolide with tetracycline was also examined. Butenolide not only effectively inhibited the biofilm formation but also eradicated pre-formed biofilms of tested bacteria. Fractional inhibitory concentration index (FICI) indicated that butenolide was a potential tetracycline enhancer against E. coli, P. aeruginosa, and MRSA. These results indicated that butenolide may hold a great potential as an effective antibiofilm agent to control and prevent biofilm-associated infections in future clinical treatments.

Acuticoccus kandeliae sp. nov., isolated from rhizosphere soil of the mangrove plant Kandelia, and emended description of Acuticoccus yangtzensis.

A novel bacterial strain, J103T, was isolated from rhizosphere soil of the mangrove plant Kandelia in Mai Po Inner Deep Bay Ramsar Site, Hong Kong. The strain was aerobic, Gram-stain-negative, oval-shaped with folds in the middle, non-motile and non-spore-forming. It grew at temperatures of 20-30 °C (optimum, 25-30 °C), at pH 6.0-9.0 (optimum pH 6.0) and at NaCl concentrations of 0.5-5.0 % (w/v) (optimum 1.0-2.0 %). Strain J103T was able to reduce nitrate to nitrite, and hydrolyse urea, Tween 40 and Tween 60. The major polar lipids were aminolipid, glycolipid, phosphatidylcholine and phosphatidylglycerol. The major fatty acids were C18 : 1ω7c and C19 : 0 cyclo ω8c. The respiratory quinone was Q-10. The DNA G+C content was 68.5 mol%. Sequence analysis of the 16S rRNA gene indicated that strain J103T belongs to the genus Acuticoccus, within the family Rhodobacteraceae. The closest phylogenetic neighbour was Acuticoccus yangtzensis JL1095T, showing 96.2 % 16S rRNA gene sequence similarity. The genome size of strain J103T was 6 478 100 bp. The average nucleotide identity and digital DNA-DNA hybridization values between strain J103T and Acuticoccus yangtzensis JL1095T were 75.44 and 16.43 %, respectively. Characterization based on phylogenetic, phenotypic, chemotaxonomic and genomic evidence demonstrated that strain J103T represents a novel species of the genus Acuticoccus, for which the name Acuticoccus kandeliae sp. nov. is proposed. The type strain is J103T (=DSM 104434T=MCCC 1K03288T).

Ruegeria kandeliae sp. nov., isolated from the rhizosphere soil of a mangrove plant Kandelia candel.

A Gram-negative, rod-shaped and motile bacterium, designated strain J95T, was isolated from the rhizosphere soil of a mangrove plant Kandeliacandel (L.) Druce in Mai Po Nature Reserve, Hong Kong. Growth of strain J95T was observed at pH 5.0-8.5 (optimum, 6.0-7.0), between 10-40 °C (30-37 °C) and in the presence of 0-9 % (w/v) NaCl (0.5-3 %). Chemotaxonomic analysis showed ubiquinone-10 as the predominant respiratory quinone and C18 : 1ω7c and C19 : 0 cycloω8c as the major fatty acids. The major polar lipids were lipid, aminolipid, phospholipid, phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine. The genomic contained a circular chromosome of 5.48 Mb with a DNA G+C content of 65.7 mol%. The genome included 5299 genes. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain J95T belongs to the genus Ruegeria with highest sequence similarity (96.8 %) to the type strain Ruegeria marina ZH17T. The combined phenotypic, chemotaxonomic and phylogenetic data suggested that strain J95T represents a novel species of the genus Ruegeria, for which the name Ruegeria kandeliae sp. nov. is proposed. The type strain is J95T (=MCCC 1K03284T=DSM 104293T).