| Based on the cooperative project"Autochthonous phage-host systems in different aquatic habitats"(HB08302(1)) in the charge of Third Institute of Oceanography, State Oceanic Administration in China and Leibniz Institute for Baltic Sea Research Warnemünde in Germany, and the legⅢ-Ⅳof DY115-20 cruise of"Dayang Yihao"deep-sea research vessel, which was surveying the eastern area of Pacific Ocean, 13 samples were collected from coasts of Xiamen Harbor and Baltic Sea, and 29 samples were gathered from the hydrothermal vents area of East Pacific Rise and the marine area from East Pacific Rise to Auckland, New Zealand. The total bacteria and culturable bacteria numbers of the samples were investigated and the biodiversity of TCBS group bacteria was studied subsequently. The viral abundances in samples from coasts of Xiamen Harbor and Baltic Sea were enumerated and the comparison of relating methods for enumeration was done at the same time. On the basis of this, we focused on the isolation and characterization of relating phages to accumulate data for discussion of the phage-host interaction and phage therapy to pathogenic bacteria. The above studies showed that:1. The bacterial abundances of the samples were determined by epifluorescence microscopy. The numbers of culturable bacteria and TCBS group bacteria were estimated by Zobell 2216E medium and TCBS Agar with spread plate method and membrane filter method. The results showed: Bacterial abundances in seawater samples from Xiamen Harbor ranged from 0.63 to 0.80×10~9cells/L, with the geometric mean of 0.72×10~9cells/L (n=3); the percentages of culturable bacteria in total bacteria of the samples were floating from 0.03% to 0.85%; and the concentrations of TCBS group bacteria fluctuating between 20 and 35 CFU/mL, which accounted for 0.65% to 11.89% of the culturable bacteria. Bacterial numbers were ranging from 1.45 to 20.1×10~9cells/L with an average of 2.66×10~9cells/L (n=6) in seawater samples from Baltic Sea; the proportions of culturable bacteria in total bacteria of the samples were fluctuating between 0.08% and 0.86%; and the concentrations of TCBS group bacteria were floating from 11 to 95 CFU/mL, which accounted for 0.30% to 1.40% of the culturable bacteria. In eastern Pacific Ocean surveying area, bacterial abundances in surface seawater samples waved within 0.70 and 1.74×10~9cells/L, with the geometric mean of 1.10×10~9cells/L (n=14); and the concentrations of TCBS group bacteria varied from 5 to 150 CFU/mL. The range of bacterial abundances in sedimental samples of East Pacific Rise was from 4.99 to 209.51×10~6cells/g, and the geometric mean was 13.68×10~6cells/g (n=8); the concentrations of TCBS group bacteria varied from 2 to 184 CFU/g. With the exception of few special habitats, the bacterial abundances in surface seawater from coastal and oceanic environments were not obviously different; but the bacterial abundances in sediments of East Pacific Rise were closely related with the habitats and demonstrated great differences with each other. More than 99% of the total bacteria were in"Viable But Nonculturable State"in seawater samples from Xiamen Harbor and Baltic Sea coastal environments. The TCBS group bacteria numbers in samples from Xiamen Harbor, Baltic Sea and eastern Pacific Ocean indicated that the concentrations of TCBS group bacteria in different samples may be nearly the same, but their proportions in total bacteria were totally different and without regularity.2. The viral and bacterial abundances determined by epifluorescence microscopy indicated significant correlation between bacteria and virus-like particles in samples from both Xiamen Harbor and Baltic Sea, and generally the samples which contained a large number of bacteria also had much more virus-like particles at the same time with the virus-to-bacteria ratios ranging from 6.86 to 49.53. The viral numbers in seawater of Xiamen Harbor ranged from 0.51 to 1.15×10~10VLPs/L, with the geometric mean of 0.82×10~10VLPs/L (n=3), while ranging from 2.11 to 18.13×10~10VLPs/L with a geometric mean of 5.03×10~10VLPs/L (n=6) in seawater of Baltic Sea. Both epifluorescence microscopy and flow cytometry demonstrated similar numbers of virus-like particles in the samples from Baltic Sea. Because flow cytometry has the defects such as expensive price, need of high operation skill level and poor repeatability of results, epifluorescence microscopy is still the standard method for determination of bacterial and viral abundances in environmental samples now. 3. Identification of 203 TCBS group bacteria by 16SrDNA sequence analysis method showed that TCBS Agar was not sufficiently selective for vibrios in marine environmental samples, and many other bacteria could also grow on it. Therefore, the bacteria isolated by TCBS Agar should be described as"TCBS group bacteria"but not"culturable vibrios". The 203 TCBS group bacteria strains were divided into 18 genera, 49 (sub) species. The distribution of TCBS group bacteria was regional, and the species composition and distribution in different areas were obviously different and were closely related with their habitats. The bacteria of family Vibrionaceae were common in samples from Xiamen Harbor and Baltic Sea coastal environments, and could be detected in every sample. The vibrios isolated from Xiamen Harbor were identified as 6 species from genus Vibrio and Photobacterium, while the vibrios from Baltic Sea were identified as 9 species from genus Vibrio and Enterovibrio. Bacteria belonged to genus Vibrio could be detected in all the samples from the two coastal surveying areas, but the species composition was different and only V. azureus could be detected in both surveying areas. In eastern Pacific Ocean, only one strain of genus Vibrio was isolated. Most of the bacteria strains belonged to family Halomonadaceae, including 11 species from genus Halomonas, Cobetia and Chromohalobacter, and were detected in 23 of the total 24 samples. The bacteria from genus Halomonas should be ubiquitous in this surveying area because they were detected in either seawater or sediment samples and could be isolated from 22 samples.4. 7 phages of TCBS group bacteria were isolated from the seawater and biological samples by traditional double-layer plate method, and the five host strains were identified as members of genera Vibrio, Pseudoalteromonas and Klebsiella respectively according to the 16SrDNA sequence analysis method. The morphology under electron microscope divided the phages into family Corticoviridae, Myoviridae, Podoviridae and Siphoviridae respectively. The combination of phages morphology and restriction analysis by enzyme BglⅡand EcoRⅤshowed that the phages should be different, and the types of nucleic acids were all dsDNA.5. Determination of the host ranges of 7 phages by more than 40 different TCBS group bacteria showed that phage pY-2 and p2-4 were capable of lysing other bacteria from the same genus Vibrio as their host bacteria, and phage pYD-3, pAC-7, KP2 and KP3 had the ability to infect other bacteria from different genera. Phage KP1 was only specific to the original host strain AC-11. The subsequent results of extremely low EOP (efficiency of plating) indicated that the capability of phages to infect other bacteria was very weak, and the changing of morphology of phage plaques evinced that the infection features might also have a change.6. 3 lytic phages KP1, KP2 and KP3 of marine bacterium strain AC-11, which was identified as Klebsiella pneumoniae subsp. pneumoniae by phylogenetic analysis, had similar morphology under electron microscope. The biological characteristics of these phages such as the optimal multiplicity of infection, adsorption rates, one-step growth curve and the sensitivity to temperature, pH, UV light, ether and chloroform were studied. The acquired parameters indicated that they were different strain types in combination with the results of restriction analysis and host range.7. Preservation of phages by different methods such as stored at liquid nitrogen, -70℃, -20℃and 4℃conditions showed that 4℃preservation in the refrigerator was the best method during daily work.
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