Isolation and whole-genome sequencing analysis of Escherichia fergusonii harboring a heat-labile enterotoxin gene from retail chicken meat in Okinawa, Japan.

This study aimed to reveal the prevalence of heat-labile enterotoxin (LT) gene-positive Escherichia fergusonii in retail chicken meat and genetically characterize these strains. E. fergusonii harboring LT gene was isolated from 6 out of 60 (10%) retail chicken samples in Okinawa, Japan. Whole-genome sequencing analysis revealed that LT gene-positive E. fergusonii from chicken meat and feces contain an IncFII plasmid harboring elt1AB, and suggested to spread clonally to retail chicken through fecal contamination. Additionally, it was found that these strains harbor multidrug-resistant genes on their plasmids. Their pathogenicity and continuous monitoring are required for confirmation.

The Spread of Heat-Labile Enterotoxin-Harboring Escherichia fergusonii in Broiler Chickens and Pigs in Okinawa, Japan.

In 2012, Escherichia fergusonii harboring a heat-labile enterotoxin (LT) was isolated from healthy chickens in South Korea. However, little is known regarding the prevalence, spread, and pathogenicity of these strains in humans and animals. This study aimed to understand the public health threats, such as the distribution, antimicrobial resistance, and genetic diversity of E. fergusonii carrying LTs. E. fergusonii containing LT was isolated from 15.0% (52/346) of chicken fecal samples from all three tested chicken farms but not from 360 pig fecal samples. The antimicrobial susceptibility testing revealed that over 75% of strains were resistant to ampicillin, kanamycin, nalidixic acid, streptomycin, or tetracycline; additionally, 71.2% (37/52) of strains were resistant to all five of these antimicrobials. The 52 strains were clustered into eight pulsed-field gel electrophoresis (PFGE) types, with types V and type VI accounting for 84.6% (44/52). In the present study, multiple chicken farms harbored E. fergusonii with similar antimicrobial resistance patterns and genetic clonality. Since the pathogenicity of LT-bearing E. fergusonii in humans and animals, such as food poisoning and sporadic diarrhea via meat, the transmission of the strains, and the dissemination of antimicrobial resistance genes are unknown, additional research is required.

Correlation between antibiotic use and antibiotic resistance: A multicenter study using the Japan Surveillance for Infection Prevention and Healthcare Epidemiology (J-SIPHE) system in Hokkaido, Japan.

BACKGROUND: The Japan Surveillance for Infection Prevention and Healthcare Epidemiology (J-SIPHE) system aggregates information related to antimicrobial resistance (AMR) measures in participating medical institutions nationwide and is intended to be used for promotion of AMR measures in participating facilities and their communities. This multicenter study aimed to determine the usefulness of the J-SIPHE system for evaluating the correlation between antibiotic use and antibiotic resistance in Hokkaido, Japan. METHODS: Data on antibiotic use and detection rate of major resistant Gram-negative bacteria at 19 hospitals in 2020 were collected from the J-SIPHE system, and data correlations were analyzed using JMP Pro. RESULTS: The detection rate of carbapenem-resistant Pseudomonas aeruginosa was significantly positively correlated with carbapenem use (Spearman's ρ = 0.551; P = .015). There were significant positive correlations between the detection rate of fluoroquinolone-resistant Escherichia coli and the use of piperacillin/tazobactam, carbapenems, and quinolones [ρ = 0.518 (P = .023), ρ = 0.76 (P < .001), and ρ = 0.502 (P = .029), respectively]. CONCLUSIONS: This is the first multicenter study to investigate the correlation between antibiotic use and antibiotic resistance using the J-SIPHE system. The results suggest that using this system may be beneficial for promoting AMR measures.

A Case of Hepatitis E Virus Infection: One of Differential Diagnosis of Liver Injury in Patients Undergoing Peritoneal Dialysis.

Background: There are numerous causes of liver function disorder in patients undergoing peritoneal dialysis (PD). Infection with the Hepatitis E virus (HEV) is a rare cause of liver injury, and the behavior of HEV in patients with PD is unclear. Since patients undergoing dialysis are frequently polypharmatic, liver injury caused by HEV infection may be misdiagnosed as drug-induced liver injury. Case Presentation: A 61-year-old woman with PD developed abrupt elevation of blood transaminase levels on a routine outpatient session. Since the patient has been receiving tolvaptan as the only new medication, we suspected tolvaptan induced liver injury. In further investigating the cause of liver injury, the blood screening test was found to be positive for HEV-IgA. The patient was diagnosed with HEV infection, and had a self-limited course. Conclusion: When encountered with patients developing liver injury during PD, HEV infection should be included in the differential diagnosis.

Analysis of human clinical and environmental Leptospira to elucidate the eco-epidemiology of leptospirosis in Yaeyama, subtropical Japan.

BACKGROUND: Leptospirosis, a zoonosis caused by species in the spirochete genus Leptospira, is endemic to the Yaeyama region in Okinawa, subtropical Japan. Species of the P1 subclade "virulent" group, within the genus Leptospira, are the main etiological agents of leptospirosis in Okinawa. However, their environmental persistence is poorly understood. This study used a combination of bacterial isolation and environmental DNA (eDNA) metabarcoding methods to understand the eco-epidemiology of leptospirosis in this endemic region. FINDINGS: Polymerase chain reaction (PCR) characterized twelve human clinical L. interrogans isolates belonging to the P1 subclade "virulent" subgroup and 11 environmental soil isolates of the P1subclade "low virulent" subgroup (genetically related to L. kmetyi, n = 1; L. alstonii, n = 4; L. barantonii, n = 6) from the Yaeyama region targeting four virulence-related genes (lipL32, ligA, ligB and lpxD1). Clinical isolates were PCR positive for at least three targeted genes, while all environmental isolates were positive only for lipL32. Analysis of infected renal epithelial cells with selected clinical and environmental strains, revealed the disassembly of cell-cell junctions for the Hebdomadis clinical strain serogroup. Comparison of leptospiral eDNA during winter and summer identified operational taxonomic units corresponding to the species isolated from soil samples (L. kmetyi and L. barantonii) and additional P2 subclade species (L. licerasiae, L. wolffii-related, among others) that were not detected by soil cultivation. Total Leptospira read counts were higher in summer than in winter and the analysis of leptospiral/animal eDNA relationship suggested Rattus spp. as a potential reservoir animal. CONCLUSION: Our study demonstrated high environmental Leptospira diversity in the Yaeyama region, particularly during summer, when most of the leptospirosis cases are reported. In addition, several Leptospira species with pathogenic potential were identified that have not yet been reported in Yaeyama; however, the environmental persistence of P1 subclade species previously isolated from human clinical cases in this region was absent, suggesting the need of further methodology development and surveillance.

Transmission of SARS-CoV-2 during a 2-h domestic flight to Okinawa, Japan, March 2020.

BACKGROUND: Coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2), has rapidly spread globally. Potentially infected individuals travel on commercial aircraft. Thus, this study aimed to investigate and test the association between the use of face masks, physical distance, and COVID-19 among passengers and flight attendants exposed to a COVID-19 passenger in a domestic flight. METHODS: This observational study investigated passengers and flight attendants exposed to COVID-19 on March 23, 2020, on board a flight to Naha City, Japan. Secondary attack rates were calculated. Whole-genome sequencing of SARS-CoV-2 was used to identify the infectious linkage between confirmed cases in this clustering. The association between confirmed COVID-19 and proximity of passengers' seats to the index case and/or the use of face masks was estimated using logistic regression. RESULTS: Fourteen confirmed and six probable cases were identified among passengers and flight attendants. The secondary attack rate was 9.7%. Twelve of 14 SARS-CoV-2 genome sequences in confirmed cases were identical to that of the index case or showed only one nucleotide mutation. Risk factors for infection included not using a face mask (adjusted odds ratio [aOR]: 7.29, 95% confidence interval [95% CI]: 1.86-28.6), partial face mask use (aOR: 3.0, 95% CI: 0.83-10.8), and being seated within two rows from the index patient (aOR: 7.47, 95% CI: 2.06-27.2). CONCLUSION: SARS-CoV-2 was transmitted on the airplane. Nonuse of face masks was identified as an independent risk factor for contracting COVID-19 on the airplane.

Laboratory diagnostic, epidemiological, and clinical characteristics of human leptospirosis in Okinawa Prefecture, Japan, 2003-2020.

BACKGROUND: Leptospirosis is considered an endemic disease among agricultural workers in Okinawa Prefecture, which is the southernmost part of Japan and has a subtropical climate, but data on the current status and trend of this disease are scarce. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a retrospective study of clinically suspected leptospirosis patients whose sample and information were sent to the Okinawa Prefectural Institute of Health and Environment from November 2003 to December 2020. Laboratory diagnosis was established using culture, nested polymerase chain reaction (PCR), and/or microscopic agglutination test (MAT) with blood, cerebrospinal fluid, and/or urine samples. Statistical analyses were performed to compare the epidemiological information, clinical features, and sensitivities of diagnostic methods among laboratory-confirmed cases. Serogroups and the species of Leptospira isolates were determined by MAT using 13 antisera and flaB sequencing. A total of 531 clinically suspected patients were recruited, among whom 246 (46.3%) were laboratory confirmed to have leptospirosis. Among the confirmed cases, patients aged 20-29 years (22.4%) and male patients (85.7%) were the most common. The most common estimated sources of infection were recreation (44.5%) and labor (27.8%) in rivers. Approximately half of the isolates were of the L. interrogans serogroup Hebdomadis. The main clinical symptoms were fever (97.1%), myalgia (56.3%), and conjunctival hyperemia (52.2%). Headache occurred significantly more often in patients with Hebdomadis serogroup infections than those with other serogroup infections. The sensitivities of culture and PCR exceeded 65% during the first 6 days, while the sensitivity of MAT surpassed that of culture and PCR in the second week after onset. PCR using blood samples was a preferable method for the early diagnosis of leptospirosis. CONCLUSIONS/SIGNIFICANCE: The results of this study will support clinicians in the diagnosis and treatment of undifferentiated febrile patients in Okinawa Prefecture as well as patients returning from Okinawa Prefecture.

Sublethal and antioxidant effects of six ichthyotoxic algae on early-life stages of the Japanese pearl oyster.

Several species of harmful algae form blooms that are detrimental to aquatic organisms worldwide with severe economic loss to several industries. The cosmopolitan ichthyotoxic dinoflagellates and raphidophytes Karenia spp., Chattonella spp., Heterosigma spp., and Margalefidinium (Cochlodinium) polykrikoides are known to cause mass mortalities of fish and invertebrates, and the dinoflagellates Heterocapsa spp. are known to cause mass mortalities of shellfish, notably bivalve molluscs. The species K. mikimotoi, K. papilionacea, H. circularisquama, H. akashiwo, M. polykrikoides, and C. marina form recurrent harmful algal blooms (HAB) in coastal aquaculture areas of shellfish, coinciding with the reproduction seasons of natural and farmed bivalve molluscs. In the present study, their effects on eggs, fertilization, embryos, and three larval stages (D-shaped, umbo and pre-settling larvae) of a model bivalve species, the Japanese pearl oyster, Pinctada fucata martensii, are reported. The harmful algae had differential negative effects on each developmental stage, and had differential effects on larvae depending on their growth stage. Eggs were more affected by M. polykrikoides, K. mikimotoi and H. circularisquama than H. akashiwo and K. papilionacea. Fertilized eggs and developing embryos were more affected by M. polykrikoides and H. circularisquama than K. mikimotoi, K. papilionacea and H. akashiwo. Mortalities as well as abnormalities were not observed in any larval stage; however, motility of d-larvae and umbo larvae was more reduced by H. circularisquama and C. marina, than M. polykrikoides. In elder, 16 day-old larvae, all harmful algae induced a significant decrease in motility with the most severe effect observed during exposures to H. circularisquama, C. marina, H. akashiwo and M. polykrikoides. The superoxidase dismutase activity in larvae was not affected by exposure to any harmful alga; however, 6- and 16-day old larvae experienced a significant increase in GST activity following 48 h of exposures, with higher sensitivity of the elder larvae to C. marina, K. mikimotoi and M. polykrikoides. These results indicate that all tested harmful algae are differentially detrimental to the early-life development of the Japanese pearl oyster, with involvement of oxidative stress. Both M. polykrikoides and H. circularisquama were the most toxic followed by C. marina, K. mikimotoi, H. akashiwo and K. papilionacea. In addition, more developed larvae were most sensitive to these harmful algae in terms of motility-avoidance behavior and oxidative stress response, suggesting that ingestion of the harmful algae might enhance the toxicity of contact-dependent effects and dissolved extracellular compounds. The results also showed that superoxide anions were not associated with effects observed in larvae. Instead cellular detoxification was induced. The differential, stage-specific and growth-specific sublethal effects on bivalve development and recruitment also warrant further investigations of the oxidative stress and antioxidant enzyme activities in larvae of bivalves, to better address the toxicity mechanisms of ichthyotoxic HAB and their impacts on the reproduction, recruitment, and fitness of bivalve molluscs. Summary: The harmful algae Heterocapsa circularisquama, Chattonella marina, Hetrosigma akashiwo, Karenia mikimotoi, K. papilionacea, Margalefidinium (Cochlodinium) polykrikoides differentially affect early life stages of Japanese pearl oyster and activate detoxification enzymes in feeding larvae.

Molecular and serological epidemiology of Leptospira infection in cats in Okinawa Island, Japan.

Leptospirosis is a zoonosis caused by pathogenic Leptospira spp. Cats have been reported to be infected with Leptospira spp. and shed the bacteria in the urine. However, the importance of cats as an infection source for humans remains unclear. In this study, Leptospira infection in cats in Okinawa Prefecture, Japan, where leptospirosis is endemic, was investigated by leptospiral antibody and DNA detection using microscopic agglutination test and nested PCR, respectively. Moreover, multilocus sequence typing (MLST) and whole genome sequencing (WGS) were conducted on the Leptospira borgpetersenii serogroup Javanica isolated from cats, black rats, a mongoose, and humans. Anti-Leptospira antibodies were detected in 16.6% (40/241) of the cats tested, and the predominant reactive serogroup was Javanica. The leptospiral flaB gene was detected in 7.1% (3/42) of cat urine samples, and their sequences were identical and identified as L. borgpetersenii. MLST and WGS revealed the genetic relatedness of L. borgpetersenii serogroup Javanica isolates. This study indicated that most seropositive cats had antibodies against the serogroup Javanica and that cats excreted L. borgpetersenii in the urine after infection. Further, genetic relatedness between cat and human isolates suggests that cats may be a maintenance host for L. borgpetersenii serogroup Javanica and a source for human infection.

Childhood leptospirosis in an industrialized country: Population-based study in Okinawa, Japan.

Leptospirosis is considered underdiagnosed because of its nonspecific presentation and lack of proper understanding of its epidemiology. Early diagnosis and treatment are crucial. However, few data are available on confirmed leptospirosis cases in children in industrialized countries. We therefore aimed to describe epidemiologic and clinical characteristics of laboratory-confirmed childhood leptospirosis in Okinawa, Japan. We reviewed the national surveillance data of pediatric leptospirosis in Okinawa, Japan from January 2003 through December 2015. The database included all of laboratory-confirmed leptospirosis diagnosed at the only central laboratory for leptospirosis in the region. There were 44 children (0-20 years of age) with laboratory-confirmed leptospirosis. Of these, 90% were male, 91% were 10-20 years of age, and 96% of cases occurred in August and September. The number of laboratory-confirmed patients ranged from 0 to 11 per year (mean: 3.3 per year), and the estimated annual rate was 1.0 per 100,000 pediatric populations. In all cases, the presumed infection route was recreational exposure to river water. Commonly observed manifestations include fever (95%), myalgia (52%), and conjunctival suffusion (52%). Childhood leptospirosis in Okinawa, Japan occurred predominantly in teenage boys after freshwater exposure in summer, and most patients had characteristic conjunctival suffusion. Cohort studies would be helpful to better understand more detailed clinical manifestations in association with prognosis.

Molecular Epidemiological Study of Mumps Epidemics of 2015 in Okinawa, Japan.

Although major mumps epidemics occurred every 4-5 years in Okinawa Prefecture in Japan, no laboratory diagnoses were conducted. A mumps epidemic started in Okinawa in October 2014, and we collected clinical samples from 31 patients in 4 areas (Hokubu, Nanbu, Miyako, and Yaeyama) from July to December 2015, for virus isolation and RT-PCR, whose positive ratios were 52% and 87%, respectively. Phylogenetic analyses showed that all isolates were classified into genotype G, and with one exception, consisted of 2 subgenotypes, Ge (55.6%) and Gw (40.7%), which have been prominent in Japan recently. One isolate was classified in another lineage, which was detected in Japan for the first time, and was similar to a Hong Kong isolate from 2014. Remarkably, the geographic distributions of the 2 major lineages were separated. The Ge viruses were isolated from the main island of Okinawa and the Yaeyama Islands, whereas the Gw isolates were mainly detected from the Miyako Islands. These results suggest that the Ge and Gw mumps viruses mainly caused the mumps epidemics of 2015 in Okinawa, and that they spread independently in separate regions. This is the first report describing the molecular epidemiology of mumps epidemics in Okinawa Prefecture.

An Epidemiological Analysis of Summer Influenza Epidemics in Okinawa.

Objective This study evaluates the difference between winter influenza and summer influenza in Okinawa. Methods From January 2007 to June 2014, weekly rapid antigen test (RAT) results performed in four acute care hospitals were collected for the surveillance of regional influenza prevalence in the Naha region of the Okinawa Islands. Results An antigenic data analysis revealed that multiple H1N1 and H3N2 viruses consistently co-circulate in Okinawa, creating synchronized seasonal patterns and a high genetic diversity of influenza A. Additionally, influenza B viruses play a significant role in summer epidemics, almost every year. To further understand influenza epidemics during the summer in Okinawa, we evaluated the full genome sequences of some representative human influenza A and influenza B viruses isolated in Okinawa. Phylogenetic data analysis also revealed that multiple H1N1 and H3N2 viruses consistently co-circulate in Okinawa. Conclusion This surveillance revealed a distinct epidemic pattern of seasonal and pandemic influenza in this subtropical region.

Lethal effects of ichthyotoxic raphidophytes, Chattonella marina, C. antiqua, and Heterosigma akashiwo, on post-embryonic stages of the Japanese pearl oyster, Pinctada fucata martensii.

The inimical effects of the ichthyotoxic harmful algal bloom (HAB)-forming raphidophytes Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua on the early-life stages of the Japanese pearl oyster Pinctada fucata martensii were studied. Fertilized eggs and developing embryos were not affected following exposure to the harmful raphidophytes; however, all three algal species severely affected trochophores and D-larvae, early-stage D-larvae, and late-stage pre-settling larvae. Exposure to C. marina (5×102cellsml-1), C. antiqua (103cellsml-1), and H. akashiwo (5×103cellsml-1) resulted in decreased success of metamorphosis to the trochophore stage. A complete inhibition of trochophore metamorphosis was observed following exposure to C. antiqua at 5×103cellsml-1 and C. marina at 8×103cellsml-1. In all experiments, more than 80% of newly formed trochophores were anomalous, and in the case of exposure to H. akashiwo at 105cellsml-1 more than 70% of D-larvae were anomalous. The activity rates of D-larvae (1-day-old) were significantly reduced following exposure to C. antiqua (8×103cellsml-1, 24h), C. marina (8×103cellsml-1, 24h), and H. akashiwo (104cellsml-1, 24h). The activity rates of pre-settling larvae (21-day-old) were also significantly reduced following exposure to C. antiqua (103cellsml-1, 24h),C. marina (8×103cellsml-1, 24h), and H. akashiwo (5×104cellsml-1, 24h). Significant mortalities of both larval stages were induced by all three raphidophytes, with higher mortality rates registered for pre-settling larvae than D-larvae, especially following exposure to C. marina (5×102-8×103cellsml-1, 48-86h) and C. antiqua (103-8×103cellsml-1, 72-86h). Contact between raphidophyte cells and newly metamorphosed trochophores and D-larvae, 1-day-old D-larvae, and 21-day-old larvae resulted in microscopic changes in the raphidophytes, and then, in the motile early-life stages of pearl oysters. Upon contact and physical disturbance of their cells by larval cilia, H. akashiwo, C. marina and C. antiqua became immotile and shed their glycocalyx. The trochophores and larvae were observed trapped in a conglomerate of glycocalyx and mucus, most probably a mixture of larval mucous and raphidophyte tricosyts and mucocytes. All motile stages of pearl oyster larvae showed a typical escape behavior translating into increased swimming in an effort to release themselves from the sticky mucous traps. The larvae subsequently became exhausted, entrapped in more heavy mucous, lost their larval cilia, sank, become immotile, and died. Although other toxic mediators could have been involved, the results of the present study indicate that all three raphidophytes were harmful only for motile stages of pearl oysters, and that the physical disturbance of their cells upon contact with the ciliary structures of pearl oyster larvae initiated the harmful mechanism. The present study is the first report of lethal effects of harmful Chattonella spp. towards larvae of a bivalve mollusc. Blooms of H. akashiwo, C. antiqua and C. marina occur in all major cultivation areas of P. fucata martensii during the developmental period of their larvae. Therefore, exposure of the motile early-life stages of Japanese pearl oysters could adversely affect their population recruitment. In addition, the present study shows that further research with early-life development of pearl oysters and other bivalves could contribute to improving the understanding of the controversial harmful mechanisms of raphidophytes in marine organisms.

Molecular evolution of VP3, VP1, 3C(pro) and 3D(pol) coding regions in coxsackievirus group A type 24 variant isolates from acute hemorrhagic conjunctivitis in 2011 in Okinawa, Japan.

A large acute hemorrhagic conjunctivitis (AHC) outbreak occurred in 2011 in Okinawa Prefecture in Japan. Ten strains of coxsackievirus group A type 24 variant (CA24v) were isolated from patients with AHC and full sequence analysis of the VP3, VP1, 3C(pro) and 3D(pol) coding regions performed. To assess time-scale evolution, phylogenetic analysis was performed using the Bayesian Markov chain Monte Carlo method. In addition, similarity plots were constructed and pairwise distance (p-distance) and positive pressure analyses performed. A phylogenetic tree based on the VP1 coding region showed that the present strains belong to genotype 4 (G4). In addition, the present strains could have divided in about 2010 from the same lineages detected in other countries such as China, India and Australia. The mean rates of molecular evolution of four coding regions were estimated at about 6.15 to 7.86 × 10(-3) substitutions/site/year. Similarity plot analyses suggested that nucleotide similarities between the present strains and a prototype strain (EH24/70 strain) were 0.77-0.94. The p-distance of the present strains was relatively short (<0.01). Only one positive selected site (L25H) was identified in the VP1 protein. These findings suggest that the present CA24v strains causing AHC are genetically related to other AHC strains with rapid evolution and emerged in around 2010.

Detection of chromosomal blaCTX-M-15 in Escherichia coli O25b-B2-ST131 isolates from the Kinki region of Japan.

Escherichia coli O25b-B2-ST131 isolates harbouring bla(CTX-M-15) are distributed worldwide. The bla(CTX-M-15) transposition unit has often been found on plasmids and the genetic contexts have been examined; however, less is known about the frequency and contexts of the bla(CTX-M-15) transposition unit on the chromosome. This study was performed to determine the chromosomal location of the bla(CTX-M-15) transposition unit and to analyse the molecular structure of the region surrounding the bla(CTX-M-15) transposition unit in E. coli O25b-B2-ST131 isolates. Twenty-two E. coli O25b-B2-ST131 strains harbouring bla(CTX-M-15) that had been isolated from university hospital patients and nursing home residents in the Kinki region of Japan were examined. Inverse PCR (iPCR) targeting bla(CTX-M-15) was performed to classify the molecular structure of the region surrounding the bla(CTX-M-15) transposition unit. The isolates were classified into nine types (types A-I) considering the iPCR results; type A was the most prevalent type (13/22 isolates). Sequences of the iPCR-amplified DNA fragments showed that the bla(CTX-M-15) transposition unit consisted of ISEcp1, bla(CTX-M-15) and orf477Δ. A homology search of the obtained sequences showed that the bla(CTX-M-15) transposition unit was inserted into different chromosomal regions in eight of the nine classified types. Although 21 of the 22 E. coli isolates possessed chromosomally located bla(CTX-M-15) transposition units, clonal spread was not evident on pulsed-field gel electrophoresis (PFGE) analysis. Taken together, these data indicate that certain E. coli O25b-B2-ST131 strains harbouring chromosomal bla(CTX-M-15) have emerged and spread in the Kinki region of Japan.

Evaluation of the petrifilm aerobic count plate for enumeration of aerobic marine bacteria from seawater and Caulerpa lentillifera.

The enumeration and evaluation of the activity of marine bacteria are important in the food industry. However, detection of marine bacteria in seawater or seafood has not been easy. The Petrifilm aerobic count plate (ACP) is a ready-to-use alternative to the traditional enumeration media used for bacteria associated with food. The purpose of this study was to evaluate the usefulness of a simple detection and enumeration method utilizing the Petrifilm ACP for enumeration of aerobic marine bacteria from seawater and an edible seaweed, Caulerpa lentillifera. The efficiency of enumeration of total aerobic marine bacteria on Petrifilm ACP was compared with that using the spread plate method on marine agar with 80 seawater and 64 C. lentillifera samples. With sterile seawater as the diluent, a close correlation was observed between the method utilizing Petrifilm ACP and that utilizing the conventional marine agar (r=0.98 for seawater and 0.91 for C. lentillifera). The Petrifilm ACP method was simpler and less time-consuming than the conventional method. These results indicate that Petrifilm ACP is a suitable alternative to conventional marine agar for enumeration of marine microorganisms in seawater and C. lentillifera samples.

[Investigation and culture of microbial contaminants of Caulerpa lentillifera (Sea Grape)].

Caulerpa lentillifera is a kind of edible seaweed, known as 'sea grape' or 'green caviar'. It is used in fresh salads. However, it is sensitive to low temperature and osmotic pressure, and is easily spoilt by storage in a refrigerator or washing with tap water. That is the reason why it is difficult to prevent food poisoning, especially due to Vibrio parahaemolyticus. In this study we investigated of marine bacteria and V. parahaemolyticus in C. lentillifera and cultured them in order to develop effective control of bacteria in commercial farms. The sixteen farms in the Okinawa Islands were investigated from August to September in 2006. A total of 176 samples were collected from eleven points during the cultivation processes and from the products. About 10(3) cfu/mL of marine bacteria were detected in the seawater used in the tank culture, but after cultivation of C. lentillifera the number had increased to about 10(6) cfu/mL. The number of marine bacteria in C. lentillifera did not change significantly through the process of planting to the final product (about 10(7) cfu/g). V. parahaemolyticus was detected in seawater from all processes and C. lentillifera was isolated from 56% of seawater, 25% of seed-stocks, and 18.8% of product samples, though but thermostable direct hemolysin gene was not detected from enrichment cultures or isolated V. parahaemolyticus strains. These results indicate that for prevention of food poisoning by V. parahaemolyticus in C. lentillifera, it is important to establish a suitable sterilization procedure for each process.