Haruo Sugita

The vitamin B12-producing ability of the intestinal microflora of freshwater fish

Abstract The vitamin B 12 -producing ability of the intestinal microflora in six freshwater fishes (Japanese eel, carp, goldfish, ayu, tilapia and channel catfish) was examined. Aeromonas , Enterobacteriaceae and Pseudomonas were widely distributed in the intestinal tract of the freshwater fish examined. An obligate anaerobe, Bacteroides type A, predominated in carp and tilapia (which do not require dietary vitamin B 12 ), along with goldfish and ayu. However, this bacterium was not detected in fish which require dietary vitamin B 12 (the eel and channel catfish). In general, the vitamin B 12 -producing ability of obligate anaerobes including Bacteroides type A, other Bacteroidaceae and Clostridium was much higher than aerobes and facultative anaerobes. It also was found that there is a close relationship between the amounts of vitamin B 12 and viable counts of Bacteroides type A in the intestinal contents of carp. These results strongly suggested that the Bacteroides type A is closely involved in vitamin B 12 production in the intestinal tract of freshwater fish.

Production of the antibacterial substance by Bacillus sp. strain NM 12, an intestinal bacterium of Japanese coastal fish

Intestinal bacteria isolated from seven coastal fish were examined for their antibacterial ability against Vibrio vulnificus RIMD 2219009 using a double-layer method. Of 1055 bacteria examined, 28 (2.7%) isolates could inhibit the growth of the target strain. High activities (≥20.0 mm) were only seen in 15 isolates (1.4%), which were composed of 11 Vibrionaceae, three coryneforms and one Bacillus isolates. Since Bacillus sp. strain NM 12 isolated from the intestine of a dragonets (Callionymus sp.) showed the highest activity, it was chosen as an active strain. This bacterium efficiently produced an antibacterial substance when incubated in 50% PYBG liquid medium (pH=6.5) prepared with 25% seawater for 48 h at 20°C. At least one of the antibacterial substances was a heat labile siderophore with a molecular mass of less than 5 kDa and had a wide antibacterial spectrum: it inhibited the growth of 227 (62.5%) of 363 intestinal bacteria from seven coastal fish. These results suggest that Bacillus sp. strain NM 12 may be a suitable strain as a biocontrol agent in fish intestines and culture water.

Vibrio alginolyticus, a tetrodotoxin-producing bacterium, in the intestines of the fish Fugu vermicularis vermicularis

To clarify the mechanism of toxification in animals contaminated with tetrodotoxin, the intestinal contents of the puffer Fugu vermicularis vermicularis were examined for bacterial flora in 1985. Twenty-six out of 33 strains belonged to the genus Vibrio. These bacteria were classified into Groups I to VII, based on biological and biochemical characters. High performance liquid chromatography and gas chromatography-mass spectrometry, together with mouse bioassay for toxicity, clearly demonstrated that Group I produced tetrodotoxin and anhydrotetrodotoxin under cultivation with a medium composed of Phytone peptone (BBL) and NaCl. Some other groups also produced this toxin and/or related substances to some extent. Strains of Group I were all identified as Vibrio alginolyticus. Two strains among four produced a detectable amount of tetrodotoxin and/or anhydrotetrodotoxin, as measured by all instrumental analyses applied. Our findings suggest that some strains of V. alginolyticus are closely related to the toxification of the puffer, and probably of other species.

Novel ecological niche of Cetobacterium somerae, an anaerobic bacterium in the intestinal tracts of freshwater fish

Aims:  This study was conducted to clarify the taxonomic status of Bacteroides type A strains with high vitamin B12‐producing ability that is widely distributed in the intestinal tracts of freshwater fish.

Production of amylase by the intestinal microflora in cultured freshwater fish

The amylase‐producing ability of the intestinal microflora in cultured specimens of ayu, carp, channel catfish, Japanese eel and tilapia was determined. Mean viable counts of aerobes and anaerobes ranged from 1·1×106 to 3·7×108 cfu g−1 and from 1·3×103 to 1·6×108 cfu g−1, respectively. Aeromonas spp. and Bacteroidaceae were predominant in four to five fish species. Of 206 strains examined, 65 (31·6%) produced ≥0·01 U amylase ml−1. The percentage of producers differed among families and genera of bacteria and fish species. While 56% of the anaerobes produced amylase, only 20% of the aerobes did. More than 50% of Aeromonas, Bacteroidaceae and Clostridium strains produced amylase efficiently while Acinetobacter, coryneforms, Enterobacteriaceae, Moraxella, Plesiomonas and Streptococcus strains did not. High amylase production (≥0·05 U ml−1) was found in 12 strains, 11 from Aeromonas and one Pseudomonas. The percentage of high amylase producers in Japanese eel was lower than the other four fish (2–30%). These results strongly suggest that the amylase produced by the intestinal microflora play an important role in the digestion of starch in freshwater fish to some extent.

Antibacterial abilities of intestinal bacteria in freshwater cultured fish

Abstract Intestinal bacteria isolated from seven freshwater cultured fish were examined for their antibacterial ability against 18 target strains, including members of the genus Aeromonas and human pathogenic bacteria, using a double-layer method. A total of 304 isolates, including aerobic and anaerobic bacteria, were classified into 18 taxonomic groups. All fish species harbored intestinal bacteria with antibacterial abilities. An average of 3.2% of tested strains exhibited antibacterial activity against 18 different species, including 12 Aeromonas species and pathogenic bacteria, but the activity varied with fish species, and taxonomic groups and strains of tested bacteria. All taxonomic groups tested, except for Acinetobacter spp., coryneforms and Staphylococcus spp., contained strains exhibiting antibacterial activity. In particular, 7–12 target strains were inhibited by strains of Aeromonas caviae, A. hydrophila, Pseudomonas spp. and Clostridium spp. These results suggest that the antibacterial ability of intestinal bacteria may protect host fish against pathogenic bacteria, to some extent.

The Establishment of an Intestinal Microflora in Developing Goldfish (Carassius auratus) of Culture Ponds

The bacterial flora in the intestinal tract of goldfish (Carassius auratus) was investigated at different stages of fish development. The floras of the diets and the water and sediment of a culture pond were also analyzed. The total counts in the intestine ranged from 2.2 × 106−2.1 × 108 cells g−1 wet weight.Aeromonas hydrophila, A. punctata, Pseudomonas, Bacteroidaceae andClostridium species were the common components in the intestinal tract of goldfish from larvae to adult stage.Bacteroides type A appeared at 44 days with a density of 103 cells g−1 and then predominated with densities of 105−107 cells g−1. The intestinal microflora of goldfish become relatively stable after 67 days of hatching. These observations suggest that the intestinal microflora of adult goldfish becomes established approximately 2 months after hatching.

Identification of intestinal bacteria from Japanese flounder (Paralichthys olivaceus) and their ability to digest chitin.

Aims:  The aim of the present study was to clarify the taxonomic status of intestinal bacteria isolated from Japanese flounder (Paralichthys olivaceus) and describe their ability to digest chitin.

Identification of Plesiomonas shigelloides isolated from freshwater fish with the microplate hybridization method

A total of 74 strains were isolated from the intestines of seven freshwater fish and 10 water samples, and identified to the species level by the conventional method based on morphological, physiological and biochemical properties, and DNA-DNA hybridization method. Achromopeptidase treatment was found to be useful for the recovery of DNAs from Plesiomonas shigelloides cells because the amount of DNA obtained with this treatment was 3.1-7.2 times greater than with lysozyme. All the strains were identified as P. shigelloides on the basis of the mol% G + C contents of DNAs and homology values against the type strain of this microorganism, along with morphological, physiological, and biochemical properties. The P. shigelloides was detected in 29 out of 51 fish specimens and four out of 10 water samples. Black bass, Japanese eel, ayu, and tilapia harbored high densities of P. shigelloides , ranging from 104-108 CFU/g, with high frequencies of occurrence (73-100%). These results strongly suggested that the microplate hybridization method is an excellent procedure for the ecological study of P. shigelloides .

Changes in the bacterial composition of water in a carp rearing tank

Abstract The bacteria from an 800-l carp rearing tank were isolated on five different media during a 45-day experimental period. Total viable counts and the variety of groups of bacteria detected in the water increased after the carp were stocked. The Vibrio-Aeromonas group and Bacteroides type A, which were major components in the intestinal tracts of carp, increased after fish were introduced, and decreased after fish were removed.