Antibiofilm activity in the culture supernatant of a marine Pseudomonas sp. bacterium.

In the marine environment, most solid surfaces are covered by microbial biofilms, mainly composed of bacteria and diatoms. The negative effects of biofilms on materials and equipment are numerous and pose a major problem for industry and human activities. Since marine micro-organisms are an important source of bioactive metabolites, it is possible that they synthesize natural ecofriendly molecules that inhibit the adhesion of organisms. In this work, the antibiofilm potential of marine bacteria was investigated using Flavobacterium sp. II2003 as a target. This strain is potentially a pioneer strain of bacteria that was previously selected from marine biofilms for its strong biofilm-forming ability. The culture supernatants of 86 marine heterotrophic bacteria were tested for their ability to inhibit Flavobacterium sp. II2003 biofilm formation and the Pseudomonas sp. IV2006 strain was identified as producing a strong antibiofilm activity. The Pseudomonas sp. IV2006 culture supernatant (SNIV2006) inhibited Flavobacterium sp. II2003 adhesion without killing the bacteria or inhibiting its growth. Moreover, SNIV2006 had no effect on the Flavobacterium sp. II2003 cell surface hydrophilic/hydrophobic and general Lewis acid-base characteristics, but modified the surface properties of glass, making it on the whole more hydrophilic and more alkaline and significantly reducing bacterial cell adhesion. The glass-coating molecules produced by Pseudomonas sp. IV2006 were found to probably be polysaccharides, whereas the antibiofilm molecules contained in SNIV2006 and acting during the 2 h adhesion step on glass and polystyrene surfaces would be proteinaceous. Finally, SNIV2006 exhibited a broad spectrum of antibiofilm activity on other marine bacteria such as Flavobacterium species that are pathogenic for fish, and human pathogens in both the medical environment, such as Staphylococcus aureus and Pseudomonas aeruginosa, and in the food industry, such as Yersinia enterocolitica. Thus, a wide range of applications could be envisaged for the SNIV2006 compounds, both in aquaculture and human health.

Marine bacteria from the French Atlantic coast displaying high forming-biofilm abilities and different biofilm 3D architectures.

BACKGROUND: Few studies have reported the species composition of bacterial communities in marine biofilms formed on natural or on man-made existing structures. In particular, the roles and surface specificities of primary colonizers are largely unknown for most surface types. The aim of this study was to obtain potentially pioneering bacterial strains with high forming-biofilm abilities from two kinds of marine biofilms, collected from two different surfaces of the French Atlantic coast: an intertidal mudflat which plays a central role in aquaculture and a carbon steel structure of a harbour, where biofilms may cause important damages. RESULTS: A collection of 156 marine heterotrophic aerobic bacteria isolated from both biofilms was screened for their ability to form biofilms on polystyrene 96-well microtiter plates. Out of 25 strains able to build a biofilm in these conditions, only four bacteria also formed a thick and stable biofilm on glass surfaces under dynamic conditions. These strains developed biofilms with four different three-dimensional architectures when observed by confocal laser scanning microscopy: Flavobacterium sp. II2003 biofilms harboured mushroom-like structures, Roseobacter sp. IV3009 biofilms were quite homogeneous, Shewanella sp. IV3014 displayed hairy biofilms with horizontal fibres, whereas Roseovarius sp. VA014 developed heterogeneous and tousled biofilms. CONCLUSIONS: This work led for the first time to the obtaining of four marine bacterial strains, potentially pioneering bacteria in marine biofilms, able to adhere to at least two different surfaces (polystyrene and glass) and to build specific 3D biofilms. The four selected strains are appropriate models for a better understanding of the colonization of a surface as well as the interactions that can occur between bacteria in a marine biofilm, which are crucial events for the initiation of biofouling.

Characterization of the colistin (polymyxin E1 and E2) biosynthetic gene cluster.

Colistin is a mixture of polymyxin E1 and E2, bactericidal pentacationic lipopeptides used to treat infections caused by Gram-negative pathogens such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Industrial production of colistin is obtained by a fermentation process of the natural producer Paenibacillus polymyxa var colistinus. NonRibosomal peptide synthetases (NRPS) coding the biosynthesis of polymyxins A, B and P have been recently described, rendering thereof the improvement of their production possible. However, the colistin biosynthesis pathway was not published so far. In this study, a Paenibacillus alvei has been identified by biochemical (Api 50 CH system) and molecular (16S rDNA sequencing) methods. Its culture supernatant displayed inhibitory activity against Gram-negative bacteria (P. aeruginosa, K. pneumoniae, Salmonella spp.). Two polymyxins, E1 and E2, were recovered from the supernatant and were characterized by high resolution LC-MS. A genomic library (960 clones) was constructed to identify the gene cluster responsible for biosynthesis of polymyxins. Selection of the clones harbouring the sequences of interest was obtained by a simple PCR-based screening. We used primers targeting NRPS sequences leading to the incorporation of amino acids present in polymyxins E. The sequences from three clones of interest were assembled on 50.4 kb. Thus, five open reading frames corresponding to a new NRPS gene cluster of 41 kb were identified. In silico, analyses revealed the presence of three NRPS implicated in the biosynthesis of polymyxins E. This work provides insightful information on colistin biosynthesis and might contribute to future drug developments in this group of antibiotics.

New Biocalcifying Marine Bacterial Strains Isolated from Calcareous Deposits and Immediate Surroundings.

Marine bacterial biomineralisation by CaCO3 precipitation provides natural limestone structures, like beachrocks and stromatolites. Calcareous deposits can also be abiotically formed in seawater at the surface of steel grids under cathodic polarisation. In this work, we showed that this mineral-rich alkaline environment harbours bacteria belonging to different genera able to induce CaCO3 precipitation. We previously isolated 14 biocalcifying marine bacteria from electrochemically formed calcareous deposits and their immediate environment. By microscopy and µ-Raman spectroscopy, these bacterial strains were shown to produce calcite-type CaCO3. Identification by 16S rDNA sequencing provided between 98.5 and 100% identity with genera Pseudoalteromonas, Pseudidiomarina, Epibacterium, Virgibacillus, Planococcus, and Bhargavaea. All 14 strains produced carbonic anhydrase, and six were urease positive. Both proteins are major enzymes involved in the biocalcification process. However, this does not preclude that one or more other metabolisms could also be involved in the process. In the presence of urea, Virgibacillus halodenitrificans CD6 exhibited the most efficient precipitation of CaCO3. However, the urease pathway has the disadvantage of producing ammonia, a toxic molecule. We showed herein that different marine bacteria could induce CaCO3 precipitation without urea. These bacteria could then be used for eco-friendly applications, e.g., the formation of bio-cements to strengthen dikes and delay coastal erosion.

Lactococcus lactis Diversity Revealed by Targeted Amplicon Sequencing of purR Gene, Metabolic Comparisons and Antimicrobial Properties in an Undefined Mixed Starter Culture Used for Soft-Cheese Manufacture.

The undefined mixed starter culture (UMSC) is used in the manufacture of cheeses. Deciphering UMSC microbial diversity is important to optimize industrial processes. The UMSC was studied using culture-dependent and culture-independent based methods. MALDI-TOF MS enabled identification of species primarily from the Lactococcus genus. Comparisons of carbohydrate metabolism profiles allowed to discriminate five phenotypes of Lactococcus (n = 26/1616). The 16S sequences analysis (V1-V3, V3-V4 regions) clustered the UMSC microbial diversity into two Lactococcus operational taxonomic units (OTUs). These clustering results were improved with the DADA2 algorithm on the housekeeping purR sequences. Five L. lactis variants were detected among the UMSC. The whole-genome sequencing of six isolates allowed for the identification of the lactis subspecies using Illumina® (n = 5) and Pacbio® (n = 1) technologies. Kegg analysis confirmed the L. lactis species-specific niche adaptations and highlighted a progressive gene pseudogenization. Then, agar spot tests and agar well diffusion assays were used to assess UMSC antimicrobial activities. Of note, isolate supernatants (n = 34/1616) were shown to inhibit the growth of Salmonella ser. Typhimurium CIP 104115, Lactobacillus sakei CIP 104494, Staphylococcus aureus DSMZ 13661, Enterococcus faecalis CIP103015 and Listeria innocua CIP 80.11. Collectively, these results provide insightful information about UMSC L. lactis diversity and revealed a potential application as a bio-protective starter culture.

One-pot synthesis of 5-phenylimino, 5-thieno or 5-oxo-1,2,3-dithiazoles and evaluation of their antimicrobial and antitumor activity.

We here report the synthesis and biological evaluation of rare 4-substituted-5-phenylimino, 5-thieno- and 5-oxo-1,2,3-dithiazoles. Dithiazoles were selectively obtained in moderate to high yields (25-73%) via a one-pot reaction from various ethanoneoximes with sulfur monochloride, pyridine in acetonitrile followed by treatment by corresponding nucleophiles (aniline, thioacetamide and formic acid). All the synthesized compounds were screened for their antibacterial (against bacteria Escherichia coli, Salmonellaenterica serovar Typhimurium, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Bacillus cereus and Listeria inocua), antifungal (against pathogenic strains Candida albicans, Candida glabrata, Candida tropicalis and Issatchenkia orientalis) and antitumor (on human cell lines MCF-7 and MDA-MB-231) activity. 4-(2-Pyridinyl)-5H-1,2,3-dithiazole-5-thione and 4-ethylcarboxyl-5H-1,2,3-dithiazole-5-thione (5d, 5h) that are active against Gram-positive bacteria are significantly active against fungi. 4-(2-Benzofuranyl)-5-phenylimino-5H-1,2,3-dithiazole (4e) exerts antiproliferative activity.

Complete Genome Sequence of the Polymyxin E (Colistin)-Producing Paenibacillus sp. Strain B-LR.

Paenibacillus bacteria are recovered from varied niches, including human lung, rhizosphere, marine sediments, and hemolymph. Paenibacilli can have plant growth-promoting activities and be antibiotic producers. They can produce exopolysaccharides and enzymes of industrial interest. Illumina and PacBio reads were used to produce a complete genome sequence of the colistin producer Paenibacillus sp. strain B-LR.

Novel nonribosomal peptide synthetase (NRPS) genes sequenced from intertidal mudflat bacteria.

Nonribosomal peptide synthetases (NRPS) are actively sought out, due to pharmacologically important activities of their metabolites. In marine environment, the most prevalent nonribosomal peptide antibiotic producers are sponges inhabiting microorganisms. Conversely, strains from marine sediments and more especially from intertidal mudflats have not been extensively screened for the presence of new NRPS. In this study, for the first time, a collection of one hundred intertidal mudflat bacterial isolates (Marennes-Oléron Bay, France) was assessed for (1) the presence of NRPS genes by degenerated PCR targeting conserved adenylation domains and (2) for their production of antimicrobial molecules. (1) Bacteria with adenylation domains (14 strains) were identified by 16S rRNA gene sequence analysis and grouped into Firmicutes (one strain) and Proteobacteria (13 strains). In silico analysis of the NRPS amino acid sequences (n = 7) showed 41-58% ID with sequences found in the NCBI database. Three new putative adenylation domain signatures were found. (2) The culture supernatant of one of these strains, identified as a Bacillus, was shown to strongly inhibit the growth of Staphylococcus aureus, S. epidermidis, and Enterococcus faecalis. This study portends that the intertidal mudflat niche could be of interest for the discovery of new NRPS genes and antimicrobial producing strains.

A Novel Depsipeptide Produced by Paenibacillus alvei 32 Isolated from a Cystic fibrosis Patient.

The important viscosity of the respiratory tract mucus of Cystic fibrosis (CF) patients impairs the mucociliary transport system and allows the growth of numerous micro-organisms. Among them, Pseudomonas aeruginosa and Staphylococcus aureus are known to be responsible for pulmonary infections. We imagined that CF microflora could also harbour micro-organisms naturally equipped to compete with these pathogens. A method was developed to recover these antibiotic-producing strains within 20 CF sputum. Using this approach, we have isolated an unusual Gram-positive bacterium identified as Paenibacillus alvei by Api galleries and 16S rRNA gene sequence analysis. This strain has inhibited the growth of P. aeruginosa, S. aureus and Klebsiella pneumoniae, in co-cultures. A liquid mineral medium named MODT50 was designed and optimised for the production and the recovery of the antimicrobial compounds. The supernatant has inhibited the growth of all Gram-positive strains tested, even Methicillin-resistant S. aureus. One antimicrobial compound with a peptide structure (mainly active against S. aureus, Micrococcus luteus, and Pseudomonas stutzeri) has been purified and characterised by liquid chromatography-mass spectrometry. The new active molecule (m/z 786.6) named depsipeptide L possesses a 15-guanidino-3-hydroxypentadecanoic acid side chain (m/z 298) linked on a cyclic part of four amino acids residues (Ser, two Leu/Ile, Arg). This work reports for the first time the production of such a molecule by a P. alvei strain in a mineral medium. The CF lung microflora might represent a valuable source for the discovery of new antimicrobial-producing strains.

Megamonas rupellensis sp. nov., an anaerobe isolated from the caecum of a duck.

We report here the identification, characterization and culture of a Gram-negative to Gram-variable, rod-shaped, non-spore-forming anaerobic bacterium (strain FM1025(T)) isolated from the caecum of a duck. Phylogenetic analysis based on comparative 16S rRNA gene sequencing showed that this strain clustered with species of the family 'Acidaminococcaceae', with 94.9 % similarity to Megamonas hypermegale DSM 1672(T) and less than 91 % similarity with type strains of Pectinatus species. Sequence similarities of at least 98-99 % were observed with numerous sequences deposited in GenBank of uncultured strains from human and chicken caecal contents, but this strain is the first isolate of this taxon to be cultivated and described. On the basis of morphological, physiological and phylogenetic features, this strain should be assigned to a novel species in the genus Megamonas, for which the name Megamonas rupellensis sp. nov. is proposed. The type strain is strain FM1025(T) (=DSM 19944(T) =CIP 109788(T)).