una samples, various bacterial species were found, such as Brochothrix thermosphacta (Figure 2(a) band 4), Lactococcus plantarum (Figure 2(a) band 8) and Edwardsiella ictaluri (Figure 2(b) band 13). Acinetobacter baumannii (Figure 2(a) band 9; Figure 2(c) band 27; Figure 2(d) band 32), Oceanospirillum beijerinckii (Figure 2(b) band 14), Plesiomonas shigelloides (Figure 2(a) band 1) and various Shewanella species (Figure 2(a) band 7; Figure 2(b) band 11, band 15, band 17) were detected in the fresh salmon and the fresh tuna.
In general, frozen fish samples showed less bacterial diversity in DGGE analysis. The predominant bacteria in frozen salmon were Kluyvera intermedia (Figure 2(a) band 2), Serratia quinivorans (Figure 2(a) band 3), Pseudomonas vranovensis (Figure 2(c) band 24), and Aeromonas viridans (Figure 2(c) band 25). Serratia ureilytica (Figure 2(a) band 5), Carnobacterium viridans (Figure 2(a) band 6), and Vagococcus salmoninarum (Figure 2(d) band 29) were the most prevalent bacteria in frozen tuna.
4.1. Bacterial DNA Extraction
All the fish samples were collected from supermarkets and the bacterial load was expected to be low since the retailers should guarantee the safety of the products they sold. As expected, little bacterial DNA was obtained when extracted from the fish samples without any preenrichment. We used a simple enrichment step in order to increase bacterial quantity and obtain successful PCR products. It may favor the growth of certain group of bacteria . However, one of our aims was targeting bacterial community profile and bacteria of concern (e.g. pathogens) from a consumer’s perspective, not focusing
Figure 3. Banding pattern analysis of bacterial community by band-search algorithm and bands comparison among the same fish type. (a) Salmon; (b) Tuna.
on the ratio of bacterial species in the whole bacterial population.
Another challenge was to obtain bacterial DNA for successful PCR from a complex matrix, such as food and environmental samples, because there are inhibitory components which cannot be eliminated easily . Thus, extraction and separation of bacterial community DNA from the fish matrix is a critical step for successful amplification of bacterial DNA. Somatic cell releasing agent (SRA), which consists of non-ionic detergents and surface active agents, can lyse somatic cells without cleaving bacterial cells . SRA has been used to remove non-bacterial cells in environmental applications when determining bacterial loads . Results indicate that SRA is an effective reagent in eliminating fish cells, which was likely the primary source of contamination in total bacterial DNA extraction (Figure 1(A) Lanes 5 and 6; Figure 1(B) Lanes 5 and 6).
4.2. Bacterial Community Structure and Species Diversity in Fish
Overall, the fresh samples demonstrated more bacterial diversity than the frozen samples. The amplified PCR products from the fresh samples had more bands separated by DGGE. This indicates that some bacteria lost viability during freezing. Nevertheless, freezing, as a selective factor, was observed to favor the growth of other bacterial species (Aerococcus viridans, Kluyvera intermedia) or have little influence on the survival of some species (Carnobacterium viridans, Enterococcus faecalis, Enterococcus mundtii, Serratia ureilytica, Vagococcus salmoninarum). The fresh fish samples were not clustered according to fish type (Figure 4(a)), indicating that the fish samples could become contaminated with various bacteria through multiple pathways, which may be highly relevant to the cultivation or post-harvesting handling. This would result in random and nonspecific bac-
Figure 4. Banding pattern analysis of bacterial community by band-search algorithm and bands comparison among the same storage condition. (a) Fresh; (b) Frozen.
terial diversity. However, after freezing, some bacteria were inactivated by low temperature, leaving similar surviving bacteria to appear in the same fish type (Figure 3). Within the frozen samples, the bacterial identities were clustered by fish type (Figure 4(b)), except that only one frozen salmon sample was clustered with the frozen tuna samples. This may indicate that the composition of microbial communities were more influenced by after-harvest practices than the original environmental contamination and freezing reduced the species differences of bacterial community in the fish samples.
4.3. Bacterial Sources and Possible Health Risks
Several species found in this study are spoilage bacteria. Carnobacterium maltaromaticum, a lactic acid bacterium, is commonly distributed in marine or river water environments and can tolerate low temperature. C. maltaromaticum is also a fish pathogen and has been isolated from spoiled chilled seafood . Brochothrix thermosphacta is a common psychrotrophic spoilage microorganism, which has been found in meat, poultry, and fish products, and is recognized as the main bacteria causing “off-flavors” [20,47]. Many strains have been confirmed as human pathogens and/or aquatic life pathogens. Aerococcus viridans is a nosocomial pathogen, which may cause respiratory or urinary tract infections and fatal diseases, and is commonly penicillin-resistant [37,48]. Acinetobacter baumannii is an important opportunistic pathogen; multi-drug resistant strains frequently cause nosocomial outbreaks . Plesiomonas shigelloides, a
Table 1. Identification of bands obtained by PCR-DGGE based on the V3 region of 16S rRNA and the closest sequence match of known bacteria in other references.
gastrointestinal pathogen, may induce “travelers’ diarrhea” . Edwardsiella ictaluri, a fish pathogen, causes enteric septicemia of catfish (ESC) . Haemophilus piscium may lead to trout ulcer disease . Shewanella spp are commonly isolated from marine fish and the marine environment [23,29]. Shewanella putrefaciens is an opportunistic pathogen, which causes soft tissue infection and bacteremia in human . Serratia ureilytica, first isolated from water, can utilize urea and metabolize chitin-containing marine life by producing proteases and chitinase, leading to shellfish spoilage [21,52].
Harmful bacteria were also detected in frozen samples, though with less frequency than in fresh fish. Carnobacterium viridans, a facultative anaerobic psychrophilic bacteria, has been identified in packaged sausage in other studies and this bacterium causes spoilage and green discoloration . Vagococcus salmoninarum, a fish pathogen, may cause hyperaemia and haemorrhage in gills and viscera [40,53]. Enterococcus faecalis is a primary human fecal contamination indicator for microbial source tracking (MST). Enterococcus faecalis is also reported to induce most enterococcal infections, includeing endocarditis, bacteremia, urinary tract infections, and intra-abdominal infections .
Pseudomonas spp. are ubiquitous in the environment. Pseudomonas vranovensis has been isolated from soil and Pseudomonas veronii from water [36,38]. Several Pseudomonas species are spoilage bacteria or pathogens, but no cases indicated that the two species detected in this study have ever induced food spoilage or foodborne disease. Kluyvera intermedia is very common in surface water and soil, as well as from human sources .
Some waterborne bacteria are frequently found in fresh fish, such as C. maltaromaticum, P. shigelloides, E. ictaluri, O. beijerinckii, Shewanella spp., and S. ureilytica. Furthermore, microorganisms such as K. intermedia, Enterrococcus spp., Lactococcus spp. and Pseudomonas spp. are widely distributed in the environment. Thus, fish might have acquired these bacteria from various pathways: water, harvesting, transportation, storage, etc. Banding pattern analysis showed the bacterial community from frozen fish samples were clustered clearly in accordance with the fish types (Figure 4(b)), but the bacterial community from both types of the fresh fish were mixed together (Figure 4(a)). This implies that freezing acts as a selective pressure by inactivating some microorganisms while favoring others that are more resistant to temperature stress in each type of fish, which make the bacterial community of the frozen fish samples more dependent on the fish types. Some waterborne bacteria were still detectable in frozen fish, such as C. maltaromaticum, S. quinivorns, O. oncorhynchi subsp. Incaldanensis, S. japonica and V. salmoninarum. Others, such as A. viridans, Enterococcus spp., K. intermedia and M. hajekii may be from water or fish processing. As shown in the analysis of bacterial community, storage conditions appear to be highly significant in the shift of species composition of the bacterial community found in fish samples. Some pathogens, such as Vibrio, Salmonella, or Listeria, that usually cause seafoodborne outbreaks were not detected in this study. It may be because the samples were collected from supermarkets where good handling is practiced in most steps. Another reason might be the limitation of DGGE in detecting bacterial community from the food samples since not all the bands were successfully sequenced. Out of the 94 bands that were sequenced, some of them could not be accurately confirmed since the gene similarity was below 95% when compared with the Genebank database. The identification results from these bands were not included in this study.
Somatic release agent (SRA) was used to remove the fish matrix for efficient extraction of bacterial DNA and 16S rRNA gene amplification. This was much more effective than the traditional multiple centrifugation method. PCRDGGE was an easy and feasible technique to analyze the bacterial population of fish samples. Banding patterns and sequencing results indicated that the bacterial diversity differs between fish types and is affected by storage conditions. Spoilage bacteria and pathogens from fish samples were mainly from the environments (e.g. water or soil); however, human factors, such as post-harvesting processing, were considerable. Recommendations for food handlers and retailers are using appropriate sanitizers for minimizing microbial contamination from harvesting to retail chain.
The authors thank Jonathan Lutz for his help in editing the manuscript, Minseok Kim and Jill Stiverson for their kind support in experiments and data analysis.
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