Effect of organic acids on the formation of biogenic amines in fermented anchovy sauce comprising raw anchovy materials with different levels of freshness.

We investigated the effect of different levels of organic acids on the formation of biogenic amines in anchovy fish sauce. Fish sauce samples were prepared with fresh anchovies used immediately after being caught (F), and anchovies left at ambient temperature for 4 h (4 h), 18 h (18 h), or 24 h (24 h). Anchovies from each of the four groups were mixed with salt at a 4:1 ratio and then fermented at ambient temperature for varying periods of time. The F and 4 h anchovies contained higher levels of acetic acid, succinic acid, and lactic acid, and the levels increased during the fermentation process. The histamine content of the fish sauce samples prepared using F anchovies ranged from 44.0 to 9.2 mg/kg at 1 and 24 months of fermentation. The histamine content of fish sauce samples prepared using 4 h, 18 h, and 24 h anchovies was 111.5-37.0 mg/kg (4 h), 780.1-560.3 mg/kg (18 h), and 880.6-675.7 mg/kg (24 h). Our findings indicated that the histamine and other biogenic amine levels were closely associated with the degree of freshness of the raw anchovy material. These findings indicate that lower pH levels resulting from organic acids generated by the fresh raw material can effectively inhibit histamine formation.

Applicability Evaluation of Male-Specific Coliphage-Based Detection Methods for Microbial Contamination Tracking.

Outbreaks of food poisoning due to the consumption of norovirus-contaminated shellfish continue to occur. Male-specific (F+) coliphage has been suggested as an indicator of viral species due to the association with animal and human wastes. Here, we compared two methods, the double agar overlay and the quantitative real-time PCR (RT-PCR)-based method, for evaluating the applicability of F+ coliphage-based detection technique in microbial contamination tracking of shellfish samples. The RT-PCR-based method showed 1.6-39 times higher coliphage PFU values from spiked shellfish samples, in relation to the double agar overlay method. These differences indicated that the RT-PCR-based technique can detect both intact viruses and non-particle-protected viral DNA/RNA, suggesting that the RT-PCR based method could be a more efficient tool for tracking microbial contamination in shellfish. However, the virome information on F+ coliphage-contaminated oyster samples revealed that the high specificity of the RT-PCR- based method has a limitation in microbial contamination tracking due to the genomic diversity of F+ coliphages. Further research on the development of appropriate primer sets for microbial contamination tracking is therefore necessary. This study provides preliminary insight that should be examined in the search for suitable microbial contamination tracking methods to control the sanitation of shellfish and related seawater.

Abundance, antimicrobial resistance, and virulence of pathogenic Vibrio strains from molluscan shellfish farms along the Korean coast.

To reduce the outbreaks caused by the major pathogenic Vibrio species, V. parahaemolyticus, V. vulnificus, and V. cholerae, the distribution, antibiotic resistance, and virulence of these Vibrio strains were monitored in shellfish and seawater along the Korean coast. Among the Vibrio strains, V. parahaemolyticus was the most abundant species; during summer, this strain showed a substantial increase that correlated with the water temperature. Although >99.0% of the Vibrio species isolates were sensitive to seven antimicrobials recommended by the Center for Disease Control and Prevention for the treatment of Vibrio infections, multiple-antibiotic resistance to at least three antimicrobials was found in 14.3% to 50.0% of each Vibrio species. Among V. parahaemolyticus isolates, 14.3% were positive for the trh gene, whereas only 1% was positive for the tdh gene. These results should aid in implementing proper precautions to avoid potential human health risks associated with exposure to pathogenic Vibrio species.

Occurrence, virulence, and antimicrobial resistance of Vibrio parahaemolyticus isolated from bivalve shellfish farms along the southern coast of Korea.

Vibrio parahaemolyticus is the most common pathogen causing seafood-borne illnesses in Korea. The present study evaluated the occurrence, virulence, and antimicrobial resistance of V. parahaemolyticus in seawater and bivalves obtained in 2016 from the southern coast of Korea, an important region for commercial aquaculture industries, especially the Korean raw seafood culture. V. parahaemolyticus was detected in 87 of 160 (54.4%) bivalve samples and in 32 of 130 (24.5%) seawater samples. Especially high levels were detected during summer to early autumn. All the seawater and bivalves contained less than 2 and 5% of the tdh and trh genes of the isolates, respectively, and seawater isolates possessed two fewer genes than the bivalve isolates. Of 23 antimicrobials tested, three agents (ofloxacin, norfloxacin, and trimethoprim/sulfamethoxazole) effectively treated V. parahaemolyticus illness due to the sensitivity of the isolates. The isolates were highly resistant to ampicillin, however, excluding it as a treatment option. More than half of the isolates exhibited resistance to at least three antimicrobials. These findings indicate the importance of an integrated monitoring and surveillance program noting the occurrence, virulence, and antimicrobial resistance patterns of V. parahaemolyticus in various aquatic sources for preventing human health risks from seafood consumption.

Occurrence and virulence of Vibrio parahaemolyticus isolated from seawater and bivalve shellfish of the Gyeongnam coast, Korea, in 2004-2016.

We determined the abundance and virulence of Vibrio parahaemolyticus in seawater and bivalves from the Gyeongnam coast in Korea, a major area for the seafood industry, during 2004-2016. V. parahaemolyticus is one of the most common pathogen causing seafood-borne illnesses in Korea, and increases during the summer. Its occurrence in seawater and bivalve samples was seasonally dependent, with high levels during the summer to early autumn. There were more strains in the area of sea continually exposed to inland wastewater. Only 5.1% and 3.5% of V. parahaemolyticus isolates from seawater and bivalves, respectively, had the trh gene, and only the bivalve isolates produced the tdh gene at levels below 2%. Continuous monitoring is clearly needed to reduce seafood-borne outbreaks of disease caused by V. parahaemolyticus, and to reveal the occurrence patterns and the presence of toxic genes of the strains in different marine environments.

Comparison of bioaccumulation and elimination of Escherichia coli and male-specific bacteriophages by ascidians and bivalves.

Levels of Escherichia coli and male-specific bacteriophages (MSBs) were determined in the filter feeders obtained from retail markets, commercial farms, and wild beds in Korea. The accumulation and elimination of E. coli and MSBs were compared between ascidians and bivalves (oysters and mussels) during relaying and depuration. E. coli concentrations in ascidians from retail markets ranged between < 20 and 460 most probable number/100 g while MSBs were not detected. E. coli levels in bivalves from commercial farms and wild beds were not significantly different but bacterial levels in ascidians were consistently lower. Ascidians exhibited much lower ability than bivalves to accumulate E. coli and MSBs during relaying in a polluted coastal area. This study also shows that an equilibrium was developed between levels of microbes in water and ascidians and shellfish during relaying. E. coli and MSBs in ascidians decreased quickly during depuration in a clean seawater tank. However, after 1 day, E. coli in bivalves decreased by only 1.1-1.6 logs, and the elimination of MSBs was negligible. Therefore, depuration is an effective means to reduce the health risk of contaminated ascidians.

Distribution and antimicrobial susceptibility of Vibrio species associated with zooplankton in coastal area of Korea.

Vibrio parahaemolyticus and V. vulnificus are the most common pathogens causing seafood-borne illnesses in Korea. This study determines the abundance and antimicrobial resistance of pathogenic Vibrio species in seawater and zooplankton samples from the Geoje Island coast in Korea, which is an important area for coastal fisheries, the fishing industry, and tourism. The two Vibrio species were detected more in mesozooplankton samples than in seawater samples. V. parahaemolyticus isolates showed greater resistance than those of V. vulnificus for antimicrobials. Of V. parahaemolyticus isolates, 93.3% exhibited resistance to three or more antimicrobial agents. Conversely, more than 80% of V. vulnificus isolates showed susceptibility to all antimicrobials examined, with the exception of rifampicin. Our findings show that strong antimicrobial resistance of V. parahaemolyticus in the surveyed area was exposed to conventionally used antibiotics, therefore necessitating proper surveillance programs for the monitoring of antimicrobial resistance patterns in seawater bodies and aquatic animals.

Characterization of Vibrio parahaemolyticus isolated from oysters in Korea: Resistance to various antibiotics and prevalence of virulence genes.

Vibrio parahaemolyticus, found frequently in oysters, is the most prevalent gastroenteritis-causing pathogen in Korea and in several other Asian countries. This study monitored changes in the environmental parameters and occurrence of V. parahaemolyticus in oyster aquaculture sites. Of the 44 presumed V. parahaemolyticus isolates obtained, when tested against 16 antibiotics, 90.9, 86.4, and 75.0% of the 44 isolates exhibited resistance to vancomycin, ampicillin, and streptomycin, respectively. PCR analysis for the presence of the toxR gene confirmed 31 of the 44 isolates as being positive V. parahaemolyticus strains. The toxR positive isolates were tested for the presence of thermostable direct hemolysin (tdh) and tdh-related hemolysin (trh) virulence genes. Only 9.1% toxR positive isolate exhibit the trh gene and none of the isolates were tested positive for tdh. The occurrence of multi drug resistance strains in the environment could be an indication of excessive usage of antibiotics in agriculture and aquaculture fields.

Antimicrobial resistance of Vibrio parahaemolyticus and Vibrio alginolyticus strains isolated from farmed fish in Korea from 2005 through 2007.

The antimicrobial resistance patterns to 15 antimicrobial agents of Vibrio parahaemolyticus and Vibrio alginolyticus isolated from farmed fishes, including olive flounder (Paralichthys olivaceus), black rockfish (Sebastes schlegeli), red sea bream (Pagrus major), and sea bass (Lateolabrax japonicus), were investigated from 2005 through 2007. A total of 218 V. parahaemolyticus isolates and 153 V. alginolyticus isolates were obtained from the 180 fish samples collected from fish farms located along the southern coast of Korea. We found that 65.1% of V. parahaemolyticus and 85.6% of V. alginolyticus isolates showed antimicrobial resistance against more than one antimicrobial agent. The prevalence of resistance in V. parahaemolyticus isolates to ampicillin was highest (57.8%), followed by resistance to rifampin (11.9%), streptomycin (8.7%), and trimethoprim (6.4%). V. alginolyticus isolates were also most resistant to ampicillin (75.2%), followed by tetracycline (15.0%), trimethoprim (12.4%), and rifampin (9.8%). The prevalence of multiresistance to four or more antimicrobials was higher in V. alginolyticus (11.1%) than in V. parahaemolyticus (5%). Antimicrobial resistance rates per isolate of V. parahaemolyticus and V. alginolyticus possessing virulence genes were not different from those of the rest of the isolates.

Cloning and characterization of a catechol-degrading gene cluster from 3,4-dichloroaniline degrading bacterium Pseudomonas sp. KB35B.

We recently isolated a bacterium, Pseudomonas sp. KB35B, capable of growth on 3,4-dichloroaniline (DCA) as a sole carbon source. The isolated strain showed a high level of catechol 2,3-dioxygenase (CD-2,3) activity in the presence of 3,4-DCA. In an attempt to elucidate the relationship between biodegradation of 3,4-DCA and CD-2,3 activity, the genes encoding enzymes for the catabolic pathway of catechol were cloned and sequenced from the chromosomal DNA. The sequence analysis of the 10752 bp DNA fragment revealed 12 open reading frames in the order of nahRGTHINLOMKJX. Among the 12 genes, nahHINLOMK genes encode enzymes for the metabolism of catechol to TCA cycle intermediates. The nahR gene is the LysR type transcriptional regulator, and the nahH gene encodes CD-2,3 for meta-cleavages of catechol. 2-Hydroxymuconic semialdehyde hydrolase, 2-oxypent-4-dienoate hydratase, and 4-hydroxy-2-oxovalerate aldolase encoded by nahLMN genes are responsible for the three steps after meta-cleavages of catechol. The current results suggested that Pseudomonas sp. KB35B degrades 3,4-DCA via the meta-cleavage pathway of catechol.

Chlorothalonil-biotransformation by glutathione S-transferase of Escherichia coli.

It has recently been reported that one of the most important factors of yeast resistance to the fungicide chlorothalonil is the glutathione contents and the catalytic efficiency of glutathione S-transferase (GST) (Shin et al, 2003). GST is known to catalyze the conjugation of glutathione to a wide variety of xenobiotics, resulting in detoxification. In an attempt to elucidate the relation between chlorothalonil-detoxification and GST, the GST of Escherichia coli was expressed and purified. The drug-hypersensitive E. coli KAM3 cells harboring a plasmid for the overexpression of the GST gene can grow in the presence of chlorothalonil. The purified GST showed chlorothalonil-biotransformation activity in the presence of glutathione. Thus, chlorothalonil is detoxified by the mechanism of glutathione conjugation catalyzed by GST.

Glutathione-dependent biotransformation of the fungicide chlorothalonil.

A gene responsible for the chlorothalonil biotransformation was cloned from the chromosomal DNA of Ochrobactrum anthropi SH35B, capable of efficiently dissipating the chlorothalonil. The gene encoding glutathione S-transferase (GST) of O. anthropi SH35B was expressed in Escherichia coli, and the GST was subsequently purified by affinity chromatography. The fungicide chlorothalonil was rapidly transformed by the GST in the presence of glutathione. LC-MS analysis supported the formation of mono-, di-, and triglutathione conjugates of chlorothalonil by the GST. The monoglutathione conjugate was observed as an intermediate in the enzymatic reaction. The triglutathione conjugate has not been previously reported and seems to be the final metabolite in the biotransformation of chlorothalonil. The glutathione-dependent biotransformation of chlorothalonil catalyzed by the bacterial GST is reported.