Corynebacterium accolens Releases Antipneumococcal Free Fatty Acids from Human Nostril and Skin Surface Triacylglycerols.

UNLABELLED: Bacterial interspecies interactions play clinically important roles in shaping microbial community composition. We observed that Corynebacterium spp. are overrepresented in children free of Streptococcus pneumoniae (pneumococcus), a common pediatric nasal colonizer and an important infectious agent. Corynebacterium accolens, a benign lipid-requiring species, inhibits pneumococcal growth during in vitro cocultivation on medium supplemented with human skin surface triacylglycerols (TAGs) that are likely present in the nostrils. This inhibition depends on LipS1, a TAG lipase necessary for C. accolens growth on TAGs such as triolein. We determined that C. accolens hydrolysis of triolein releases oleic acid, which inhibits pneumococcus, as do other free fatty acids (FFAs) that might be released by LipS1 from human skin surface TAGs. Our results support a model in which C. accolens hydrolyzes skin surface TAGS in vivo releasing antipneumococcal FFAs. These data indicate that C. accolens may play a beneficial role in sculpting the human microbiome. IMPORTANCE: Little is known about how harmless Corynebacterium species that colonize the human nose and skin might impact pathogen colonization and proliferation at these sites. We show that Corynebacterium accolens, a common benign nasal bacterium, modifies its local habitat in vitro as it inhibits growth of Streptococcus pneumoniae by releasing antibacterial free fatty acids from host skin surface triacylglycerols. We further identify the primary C. accolens lipase required for this activity. We postulate a model in which higher numbers of C. accolens cells deter/limit S. pneumoniae nostril colonization, which might partly explain why children without S. pneumoniae colonization have higher levels of nasal Corynebacterium. This work narrows the gap between descriptive studies and the needed in-depth understanding of the molecular mechanisms of microbe-microbe interactions that help shape the human microbiome. It also lays the foundation for future in vivo studies to determine whether habitat modification by C. accolens could be promoted to control pathogen colonization.

Mucinivorans hirudinis gen. nov., sp. nov., an anaerobic, mucin-degrading bacterium isolated from the digestive tract of the medicinal leech Hirudo verbana.

Three anaerobic bacterial strains were isolated from the digestive tract of the medicinal leech Hirudo verbana, using mucin as the primary carbon and energy source. These strains, designated M3(T), M4 and M6, were Gram-stain-negative, non-spore-forming and non-motile. Cells were elongated bacilli approximately 2.4 µm long and 0.6 µm wide. Growth only occurred anaerobically under mesophilic and neutral pH conditions. All three strains could utilize multiple simple and complex sugars as carbon sources, with glucose fermented to acid by-products. The DNA G+C contents of strains M3(T), M4 and M6 were 44.9, 44.8 and 44.8 mol%, respectively. The major cellular fatty acid of strain M3(T) was iso-C15 : 0. Phylogenetic analysis of full-length 16S rRNA gene sequences revealed that the three strains shared >99 % similarity with each other and represent a new lineage within the family Rikenellaceae of the order Bacteroidales, phylum Bacteroidetes. The most closely related bacteria to strain M3(T) based on 16S rRNA gene sequences were Rikenella microfusus DSM 15922(T) (87.3 % similarity) and Alistipes finegoldii AHN 2437(T) (87.4 %). On the basis of phenotypic, genotypic and physiological evidence, strains M3(T), M4 and M6 are proposed as representing a novel species of a new genus within the family Rikenellaceae, for which the name Mucinivorans hirudinis gen. nov., sp. nov. is proposed. The type strain of Mucinivorans hirudinis is M3(T) ( = ATCC BAA-2553(T) = DSM 27344(T)).

Metagenomic analysis of the medicinal leech gut microbiota.

There are trillions of microbes found throughout the human body and they exceed the number of eukaryotic cells by 10-fold. Metagenomic studies have revealed that the majority of these microbes are found within the gut, playing an important role in the host's digestion and nutrition. The complexity of the animal digestive tract, unculturable microbes, and the lack of genetic tools for most culturable microbes make it challenging to explore the nature of these microbial interactions within this niche. The medicinal leech, Hirudo verbana, has been shown to be a useful tool in overcoming these challenges, due to the simplicity of the microbiome and the availability of genetic tools for one of the two dominant gut symbionts, Aeromonas veronii. In this study, we utilize 16S rRNA gene pyrosequencing to further explore the microbial composition of the leech digestive tract, confirming the dominance of two taxa, the Rikenella-like bacterium and A. veronii. The deep sequencing approach revealed the presence of additional members of the microbial community that suggests the presence of a moderately complex microbial community with a richness of 36 taxa. The presence of a Proteus strain as a newly identified resident in the leech crop was confirmed using fluorescence in situ hybridization (FISH). The metagenome of this community was also pyrosequenced and the contigs were binned into the following taxonomic groups: Rikenella-like (3.1 MB), Aeromonas (4.5 MB), Proteus (2.9 MB), Clostridium (1.8 MB), Eryspelothrix (0.96 MB), Desulfovibrio (0.14 MB), and Fusobacterium (0.27 MB). Functional analyses on the leech gut symbionts were explored using the metagenomic data and MG-RAST. A comparison of the COG and KEGG categories of the leech gut metagenome to that of other animal digestive-tract microbiomes revealed that the leech digestive tract had a similar metabolic potential to the human digestive tract, supporting the usefulness of this system as a model for studying digestive-tract microbiomes. This study lays the foundation for more detailed metatranscriptomic studies and the investigation of symbiont population dynamics.

Draft Genome Sequence of Aeromonas veronii Hm21, a Symbiotic Isolate from the Medicinal Leech Digestive Tract.

Aeromonas veronii strain Hm21 was isolated from the digestive tract of the medicinal leech Hirudo verbana and has been used to identify genes that are important for host colonization. This species is also a symbiont in the gut of zebrafish and is a pathogen of mammals and fish. We present here a 4.68-Mbp draft genome sequence for Hm21.

Investigation into the physiologies of Aeromonas veronii in vitro and inside the digestive tract of the medicinal leech using RNA-seq.

Host-associated microbial communities are widespread in nature and vital to the health and fitness of the host. Deciphering the physiology of the microbiome in vivo is critical to understanding the molecular basis of the symbiosis. Recently, the development and application of high-throughput sequencing techniques, particularly RNA-seq, for studying microbial communities has enabled researchers to address not only which microbes are present in a given community but also how the community functions. For microbes that can also be cultivated in the laboratory, RNA-seq provides the opportunity to identify genes that are differentially expressed during symbiosis by comparing in vitro to in vivo transcriptomes. In the current study, we used RNA-seq to identify genes expressed by the digestive-tract microbiome of the medicinal leech, Hirudo verbana, and by one of the two dominant symbionts, Aeromonas veronii, in a rich medium. We used a comparative approach to identify genes differentially expressed during symbiosis and gain insight into the symbiont's physiology in vivo. Notable findings include evidence for the symbionts experiencing environmental stress, performing arginine catabolism, and expressing noncoding RNAs that are implicated in stationary phase survival, a state in which A. veronii persists for months within the host.

Stratified bacterial community in the bladder of the medicinal leech, Hirudo verbana.

Most animals harbour symbiotic microorganisms inside their body, where intimate interactions occur between the partners. The medicinal leech, Hirudo verbana, possesses 17 pairs of excretory bladders that harbour a large number of intracellular and extracellular symbiotic bacteria. In this study, we characterized the bladder symbionts using molecular phylogenetic analyses, transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH). Restriction fragment length polymorphism (RFLP) and sequence analyses of 16S rRNA gene clone libraries suggested that six bacterial species co-colonize the leech bladders. Phylogenetic analyses revealed that these species belong to the alpha-Proteobacteria (Ochrobactrum symbiont), beta-Proteobacteria (Beta-1 and Beta-2 symbionts), delta-Proteobacteria (Bdellovibrio symbiont) and Bacteroidetes (Niabella and Sphingobacterium symbionts). Species-specific PCR detection and FISH confirmed the localization of the symbiotic bacteria in the bladders. The Ochrobactrum, Beta-1, Bdellovibrio and Sphingobacterium symbionts were consistently detected in 13 leeches from two populations, while infection rate of the other symbionts ranged between 20% and 100% in the two leech populations. Transmission electron microscopy observations of the bladders revealed epithelial cells harbouring a number of intracellular bacilli and an additional type of extracellular, rod-shaped bacteria in the luminal region. Fluorescence in situ hybridization with group-specific oligonucleotide probes revealed the spatial organization of the bacterial species in the bladder: the Ochrobactrum symbiont was located intracellularly inside epithelial cells; the Bacteroidetes were localized close to the epithelium in the lumen of the bladder; and the Bacteroidetes layer was covered with dense beta-proteobacterial cells. These results clearly demonstrate that a simple but organized microbial community exists in the bladder of the medicinal leech.