Identification of Shewanella baltica as the most important H2S-producing species during iced storage of Danish marine fish

Abstract

Shewanella putrefaciens has been considered the main spoilage bacteria of low-temperature stored marine seafood. However, psychrotropic Shewanella have been reclassified during recent years, and the purpose of the present study was to determine whether any of the new Shewanella species are important in fish spoilage. More than 500 H2S-producing strains were isolated from iced stored marine fish (cod, plaice, and flounder) caught in the Baltic Sea during winter or summer time. All strains were identified as Shewanella species by phenotypic tests. Different Shewanella species were present on newly caught fish. During the warm summer months the mesophilic human pathogenic S. algae dominated the H2S-producing bacterial population. After iced storage, a shift in the Shewanella species was found, and most of the H2S-producing strains were identified as S. baltica. The 16S rRNA gene sequence analysis confirmed the identification of these two major groups. Several isolates could only be identified to the genus Shewanella level and were separated into two subgroups with low (44%) and high (47%) G+C mol%. The low G+C% group was isolated during winter months, whereas the high G+C% group was isolated on fish caught during summer and only during the first few days of iced storage. Phenotypically, these strains were different from the type strains of S. putrefaciens, S. oneidensis, S. colwelliana, and S. affinis, but the high G+C% group clustered close to S. colwelliana by 16S rRNA gene sequence comparison. The low G+C% group may constitute a new species. S. baltica, and the low G+C% group of Shewanella spp. strains grew well in cod juice at 0 degrees C, but three high G+C Shewanella spp. were unable to grow at 0 degrees C. In conclusion, the spoilage reactions of iced Danish marine fish remain unchanged (i.e., trimethylamine-N-oxide reduction and H2S production); however, the main H2S-producing organism was identified as S. baltica.

FIG. 1.

Changes in H₂S counts and aerobic counts of five fish (four cod and one plaice) during storage in ice. Fish were sampled in summer 1995 or winter 1996.

FIG. 2.

Changes in H₂S counts and aerobic counts of four fish (two cod, one plaice, and one flounder) during storage in ice. Fish were sampled in summer 2001.

FIG. 3.

Phylogenetic tree based on 16S rRNA gene sequence comparison, showing the taxonomic positions of 16 selected strains representing each subgroup of the different phenotypic traits (Table 1) within the genus Shewanella. The branching pattern was generated by neighbor-joining methods, and bootstrap values were calculated from 1,000 trees. Bar, sequence similarity of 0.01.

FIG. 4.

Growth of three S. baltica strains and six Shewanella spp. strains in cod juice at 0°C. Shewanella spp. strains belonging to groups C3, C4, and C5 had G+C mol% values of 43.1 to 44.2%, whereas strains belonging to groups C1 and C2 had G+C mol% values of 47.1 to 47.5%. G+C mol% of S. baltica were 46.4 to 46.8%. A1 and A2 and C1 and C2 correspond to the phenotypic trait codes in Table 1.