| Background and ObjectiveSeafaring has a long story and is a main way to get resources and strengthen connection. However, sailors worked under the challenging conditions had increased disease morbidity compared with the land-based workers. Oral and skin directly exposed to external environment, are not only the first barrier of human body but also the mainly parts resided with microbes. Millions of microbial species including bacteria, virus and fungus colonize on or in the skin and oral mucosa; these organisms have particular species and form a microbial ecosystem. During the co-evolution process, commensal microbes participate in the host metabolic system and immune system to maintain the homeostasis with the host and resisting pathogen colonization. Clinical studies showed that abnormal composition of microbes correlated with host diseases, such as oral microbiome dysbiosis induced dental caries, periodontal disease and infectious diseases, while the skin microbiological anomaly associated with atopic dermatitis and psoriasis and other skin diseases. Special marine environment, such as poor air circulation in the narrow cabin, sultry and humid climate, shortage of fresh water supplement could alter the microbial community in vivo and on the surface of the host, thus influenced the metabolism and immunity ability of the host, and further induced decreased state of health and working ability of the seafarers. So we analysed the influence of the long sea voyage on the skin and oral microbial components based on the microbial community level, and expect to assess the human health status based on the microbial and achieve the purpose of health forecast and health prevention.Handelman et al were the first proposed the concept of metagenomics and used the term Metagenomics. The object of metagenomic study is the total genome of tiny organism in a certain environment, i.e., the sum of all the biological genetic material, including the culturable and unculturable microbial gene. The Metagenomic is based on the high-throughput DNA sequencing technology and bioinformatics methods to understand the population distribution and functional gene of microbial communities. This method can directly access to genetic information from nature and bypasses the obstacles that most microbe are difficult to culture, so has greatly expanded the utilization of microbial resource. The metagenomic method has become the new technology of microorganism study and was widely used in the research of composition and function of the environment, human oral cavity, skin and gut microbial community.In this study, we applied the metagenomic method to study the structure, quantity and diversity of buccal mucosa and belly button microbial population and further analysis the composition, metabolism function and the influence on health of commensal microbes to get further information about how the long sea voyage affected the seafarers. Combining with the use of molecular genetics, microbiology and bioinformatics, we studied the health problems of seafarers during the long sea voyage so as to provide new evidences for the research of microbial community, on this basis we analysed the correlation between microbial variation and the health status of long voyage seafarers and we might provide individualized treatment for the long voyage seafarers according to the change of Bacteria Spectrum.Materials and MethodsWe collected buccal mucosa and periumbilical microbial samples from 19 seafarers respectively before and after the long sea voyage, the microbial genomic DNA were extracted and furthered studied by sequencing methods. Among these microbial samples,6 volunteers and total 12 cases of buccal mucosa microbial sample were sequenced by Sanger sequencing method to learn the full-length 16S of rRNA gene,12 volunteers total 48 cases of buccal mucosa and belly button microbial sample were sequenced by Illumina Miseq sequencing platform to study the V4 segments of 16S rRNA gene, we also studied 2 cases of buccal mucosa and 2 cases belly button microbial samples from 1 volunteer by the advanced optical (post-light) DNA sequencing platform Ion-Torrent PGM system and Illumina Miseq system. In addition, the blood samples of long sea voyage seafarers were collected for blood routine and biochemical examination, the routine clinical examination method were used to detected the serum folate acid(FA), homocysteine(HCY) and vitamin B12 (VB12) content of seafarers during different batches of long sea voyage. The results of Sanger sequencing were submitted to the Genebank and the sequence similarity were compared with known by BLAST program, the 16S rRNA V4 segments gene sequencing results were analysed by QIIME software, while the metagenomic sequencing results were submitted to the mature general biological information software MG-RAST 3.3 for biological information analysis.Statistical analysis:Data were expressed as mean±standard deviation as appropriate. Paired samples t test was used for the mean comparison of paried samples. All data was analyzed using the Statistical Package for Social Science (version 19.0, SPSS Inc, Chicago, IL). Any p values (two tailed) below 0.05 were considered statistically significant.ResultsThe results of 16S rRNA and whole genome sequencing methods both showed that the Firmicutes were the main component of buccal mucosa microbes and the Streptococcus spp. were the main component at the genus level, in line with the previous reports. The abundance of Firmicutes and Streptococcus further increased after the long sea voyage. The microbial species and composition changed greatly after the long sea voyage compared with the early stage and the abundance of pathogen and opportunistic pathogen increased after the long sea voyage. The microbes from the two periods tended to divided into two groups and the later microbes were more concentrated. Both of the 16S rRNA and WGS results suggested that the microbial diversity reduced after the voyage. Analysis of microbial metabolic activity by projecting detected sequences to the Kyoto Encyclopedia of Genes and Genomes, KEGG map indicated that the number of gene involved in the metabolisms of carbohydrate, amino acid and lipid decreased after the voyage.The analysis results indicated that the abundance of serum folic acid decreased after the long sea voyage, in accordance to the decreased numbers of microbial enzymes involved in folate synthesis. The results of blood routine examination suggested the granulocyte and lymphocyte ratio (GRN/LYM) and the mean corpuscular haemoglobin concentration (MCHC) decreased, both of them were with statistical significance.The results of 16S rRNA and whole genome sequencing methods showed that the Proteobacteria were the main component of belly button microbes before the long sea voyage, while the Proteobacteria abundance decreased and the ratio of Firmicutes increased and become the main component. Staphylococcus and Corynebacterium were the dominant bacteria at the genus level before the long sea voyage, while the abundance of Staphylococcus increased and Corynebacterium decreased at the later period. In addition, the abundance of pathogen and opportunistic pathogen increased after the long sea voyage. The the microbes from the two periods tended to divided into two groups. In addition, KEGG analysis indicated that the microbial metabolic activity decreased after the long sea voyage which was consistent with these results of buccal mucosa analysis.ConclusionThe microbial species and abundance of buccal mucosa changed greatly between the two stage and the ratio of pathogens and opportunistic pathogen content increased after the long sea voyage; combining with the clinical examination index, we supposed the voyage environment affected the buccal mucosa microbial composition and further affect the oral cavity and systemic immune function of seafarers, and probably contributed to the increased morbidity of certain diseases. The decreased microbial diversity presumably induced by the monotonous marine environment which impeded the microbial diversity. Moreover, the discovery that the detected genes involved in the microbial folic acid synthesis metabolism decreased after the long sea voyage, which positively corralated to the observation that the serum folic acid concentration decreased after the voyage, indicates that commensal microbes may reflect the health condition of health.The tendency of the belly button microbial diversity, pathogen, opportunistic pathogen and metabolic activity variety consistent with the buccal mucosa microbial variety, further suggesting that the long sea environment could influence the commensal microbiome and finally affected the host health status.The main innovation1. In this study, we used the Next Generation DNA sequencing technology, Ion-Torrent and Illumina system for microbial genome sequencing, and used advanced bioinformatics analysis software MG-RAST to analyze the data, we got a much more complete microbial composition and distribution of the oral mucosa and the belly button surface.2. We discovered the difference of microbial diversity, the abundance variety of pathogen and conditional pathogenic bacteria and the microbial metabolic function before and after the long sea voyage. Based on these data, we preliminarily investigated the effect of long sea voyage environment on the commensal microbiome and accumulated scientific evidence for the health management of the seafarers.3. We discovered that sailing on voyage without touching more than 3 months, the folic acid metabolism pathway were blocked, consistenting with the tendency of the metabolic function of buccal mucosa microbiome. With Irregular touching arrangement and continuous supplements of compound vitamin and mineral supplements for more than three months, we found that the decreased tendency of serum folate metabolism was reversed. The concentration of serum folic acid, homocysteine and vitamins tend to be the same with the shore based personnel. These results indicated that through increasing the frequency of touch on the shore and supplements of compound vitamins and trace elements, the blocked phenomenon of folic acid was reserved. |
PDF Full Text Request