Immortalized Canine Adipose-Derived Mesenchymal Stem Cells Maintain the Immunomodulatory Capacity of the Original Primary Cells.

Mesenchymal stem cells (MSCs) are a promising cell source for stem cell therapy of intractable diseases in veterinary medicine, but donor-dependent cellular heterogeneity is an issue that influences therapeutic efficacy. Thus, we previously established immortalized cells that maintain the fundamental properties of primary cells, but functional evaluation had not been performed. Therefore, we evaluated the immunomodulatory capacity of the immortalized canine adipose-derived MSCs (cADSCs) in vitro and in vivo to investigate whether they maintain primary cell functions. C57BL/6J mice were treated with dextran sulfate sodium (DSS) to induce colitis, injected intraperitoneally with immortalized or primary cADSCs on day 2 of DSS treatment, and observed for 10 days. Administration of immortalized cADSCs improved body weight loss and the disease activity index (DAI) in DSS-induced colitic mice by shifting peritoneal macrophage polarity from the M1 to M2 phenotype, suppressing T helper (Th) 1/Th17 cell responses and inducing regulatory T (Treg) cells. They also inhibited the proliferation of mouse and canine T cells in vitro. These immunomodulatory effects were comparable with primary cells. These results highlight the feasibility of our immortalized cADSCs as a cell source for stem cell therapy with stable therapeutic efficacy because they maintain the immunomodulatory capacity of primary cells.

Immortalized Canine Adipose-Derived Mesenchymal Stem Cells as a Novel Candidate Cell Source for Mesenchymal Stem Cell Therapy.

Mesenchymal stem cells are expected to be a cell source for stem cell therapy of various diseases in veterinary medicine. However, donor-dependent cell heterogenicity has been a cause of inconsistent therapeutic efficiency. Therefore, we established immortalized cells from canine adipose tissue-derived mesenchymal stem cells (ADSCs) to minimize cellular heterogeneity by reducing the number of donors, evaluated their properties, and compared them to the primary cells with RNA-sequencing. Immortalized canine ADSCs were established by transduction with combinations of the R24C mutation of human cyclin-dependent kinase 4 (CDKR24C), canine cyclin D1, and canine TERT. The ADSCs transduced with CDK4R24C, cyclin D1, and TERT (ADSC-K4DT) or with CDK4R24C and cyclin D1 (ADSC-K4D) showed a dramatic increase in proliferation (population doubling level >100) without cellular senescence compared to the primary ADSCs. The cell surface markers, except for CD90 of the ADSC-K4DT and ADSC-K4D cells, were similar to those of the primary ADSCs. The ADSC-K4DT and ADSC-K4D cells maintained their trilineage differentiation capacity and chromosome condition, and did not have a tumorigenic development. The ability to inhibit lymphocyte proliferation by the ADSC-K4D cells was enhanced compared with the primary ADSCs and ADSC-K4DT cells. The pathway analysis based on RNA-sequencing revealed changes in the pathways mainly related to the cell cycle and telomerase. The ADSC-K4DT and ADSC-K4D cells had decreased CD90 expression, but there were no obvious defects associated with the decreased CD90 expression in this study. Our results suggest that ADSC-K4DT and ADSC-K4D cells are a potential novel cell source for mesenchymal stem cell therapy.

Genetic variation of mitochondrial DNA in Phalacrocorax carbo in Japan.

A Japanese resident bird, Phalacrocorax carbo hanedae (Japanese name: Kawa-u), was threatened with extinction due to deterioration of its habitat in the 1970s, but the population has since recovered thanks to environmental protection measures. This study analyzed the genetic diversity of 18 Kawa-u individuals living in the basins of the Abe and Warashina rivers in Shizuoka Prefecture, Japan. We obtained seven haplotypes of mitochondrial D-loop sequences and compared them with 49 European P. carbo D-loop haplotypes. We identified four new haplotypes but no clear genetic evidence distinguishing the Kawa-u as a distinct subspecies of P. carbo. Our results suggest the need for further surveillance of the P. carbo genetic lineage, regardless of the geographical distribution.

Unique response characteristics in persistent strains of Listeria monocytogenes exposed to low pH.

Some Listeria monocytogenes strains are persistent in food processing environments, where this pathogen may be subjected to various stresses. This study aimed to elucidate the response of persistent strains of L. monocytogenes to low pH and H2O2 exposure. Almost all of the persistent strains examined were highly susceptible to low pH, whereas H2O2 susceptibility was comparable to that of control strains. Two persistent strains isolated from the same sample, however, exhibited lower susceptibility to low pH. These findings suggest an acid-susceptible phenotype predominates in the habitat, indicating that environmental conditions contribute to the establishment of persistence. Representative strains exhibiting acid-susceptible and less acid-susceptible phenotypes were further investigated regarding acid response characteristics. Less acid-susceptible strains exhibited increased survival in acidified brain heart infusion (BHI) broth compared with acidified phosphate-buffered saline (PBS). These strains also exhibited increased survival in acidified PBS containing glucose and glutamate, which are involved in acid response mechanisms, compared with acidified PBS alone. However, neither acidified BHI broth nor exogenous glucose and glutamate increased survival of acid-susceptible strains. An adaptive acid tolerance response of the acid-susceptible phenotype was observed, but this was limited compared with that of the less acid-susceptible phenotype.

Sequence-Based Characterization of Listeria monocytogenes Strains Isolated from Domestic Retail Meat in the Tokyo Metropolitan Area of Japan.

The level of Listeria monocytogenes contamination of domestic retail meat in Tokyo, Japan, was assessed by comparison of isolates from 2004 to 2007 with those isolated before 2003. The overall prevalence of L. monocytogenes among these samples significantly diminished over time (1998-2003, 28.0%; 2004-2007, 17.6%) reflecting a significant decrease in the frequency of contamination of beef. Serotype 1/2a was isolated most frequently, reflecting a change in the predominant serotype in pork from 1/2c to 1/2a. We performed a simple genetic subtyping method based on 3 genes, iap, sigB, and actA, as well as traditional multilocus sequence typing to classify the allele types (ATs). No extensive variation among sequence types was detected. However, increased genetic diversity among the ATs of the 3 genes in the 2004-2007 isolates was evident. We identified AT 26 of the iap gene, which was not previously reported in Japanese isolates, and 6 ATs of the sigB gene, including 4 with nonsense mutations not currently registered in L. monocytogenes DNA databases. sigB is an evolutionally conserved gene that plays a role in the stress response. Our results indicate that the sigB gene may be relatively unstable among L. monocytogenes strains circulating in Japan.

[Constrictive Pericarditis with Intractable Ascites after Aortic Valve Replacement;Report of a Case].

We report a 68-year-old man, who developed refractory ascites of unknown cause after aortic valve replacement. He was diagnosed with constrictive pericarditis because of "dip-and-plateau" waveform findings via cardiac catheterization and operated with cardiopulmonary bypass. Following waffle procedure, we incised pericardium for decompression, so that pericardial mobility and diastolic dysfunction was improved. Postoperative computed tomography (CT) image also showed decrease of ascites fluid. We concluded that pericardiotomy is an established surgical procedure and is excellent indication to constrictive pericarditis.

What Did Endovascular Aortic Repair Bring for the Treatment Strategy of Abdominal Aortic Aneurysm?

Objective: We examined the effects of the introduction of endovascular aortic repair (EVAR) on treatment for abdominal aortic aneurysms (AAAs). Subjects: We compared patients in the following three periods: period I (January 2002-December 2006, 105 patients), period II (January 2007-December 2011, 242 patients, duration of 5 years after the introduction of EVAR), and period III (January 2012-December 2016, 237 patients, duration of 5 years after period II). We used the American Society of Anesthesiologists (ASA) classification for risk assessment. Results: In the Open repair (OR) group, the incidences of ASA class 2 increased and classes 3 and 4 decreased significantly in periods II and III compared with period I. In all periods, there were no in-hospital deaths. Suprarenal aortic cross-clamping was required in 18 patients (19.1%) in period III and 5 patients (6.3) in period I, and the difference was significant (P<0.05). In the EVAR group, no differences in age, sex, or ASA classification class were observed between periods II and III. In period II, one patient died due to aneurysm rupture during surgery. Significant differences were observed when comparing both groups in periods II and III: patients in the EVAR group were older (P<0.01) and the OR group had a higher proportion of ASA class 2 patients and the EVAR group had a higher proportion of ASA class 3 or 4 patients (P<0.01). Among all AAA surgeries, rupture occurred in 25 patients (23.8%) in period I, 18 patients (7.4) in period II, and 16 patients (6.8) in period III. The number of ruptures was significantly lower in periods II and III than in period I (P<0.01). Conclusions: The findings of this study suggest that EVAR should be indicated for high-risk patients and had the good outcome of AAA treatment. (This is a translation of Jpn J Vasc Surg 2018; 27: 27-32.).

[Emergent Thoracic Endovascular Aortic Repair and Subxiphoid Pericardiotomy for Retrograde Type A Acute Aortic Dissection with Cardiac Tamponade;Report of a Case].

Type A acute aortic dissection has a high rate of mortality. Emergent surgical repair is the gold standard treatment, but some patients cannot tolerate the open surgery. Here, we report an 82-year-old patient with a history of cerebral infarction and cerebral bleeding who presented with a depressed level of consciousness and who was in a state of shock. A computed tomography (CT) scan showed cardiac tamponade associated with retrograde type A aortic dissection(RAAD), with a primary entry tear at a distal site of the left subclavian artery. We therefore performed emergent primary entry closure with stent grafting using the Conformable Gore Tag device and emergent drainage of the cardiac tamponade with subxiphoid pericardiotomy. Postoperative CT scan showed complete closure of the primary entry tear and a completely thrombosed false lumen. Primary entry closure with stent grafting could be an effective option for RAAD that meets anatomical criteria.

Sequential transition of the injury phenotype, temperature-dependent survival and transcriptional response in Listeria monocytogenes following lethal H2O2 exposure.

The food-borne pathogen Listeria monocytogenes is present persistently in food processing environments, where this bacterium is exposed to various stress factors, including oxidative stress. This study aimed to elucidate the temperature-dependent response of L. monocytogenes to H2O2 exposure and the phenotypic changes in colony formation by H2O2-treated bacteria. Survival curves indicated an increase in the resistance to H2O2 in L. monocytogenes as the temperature decreased during the stress exposure procedure. Transcriptional induction of genes with key roles in response to H2O2, including sigB and kat, was observed at 37°C, but not at 20°C, whereas other stress response genes were induced at both temperatures. Following H2O2 exposure, L. monocytogenes produced small colony phenotypes and the colony size decreased in a stress exposure duration-dependent manner. Resuscitated cells with no ability to form colonies in the absence of sodium pyruvate were also found. Our findings show the possibility that a sequential transition in the injury phenotype from small colony phenotype to resuscitated cells occurred during the course of exposure to H2O2. The higher H2O2 resistance at 20°C than 37°C suggests further investigation of the response to H2O2 exposure under the lower temperatures, including refrigeration temperature, which may contribute to elucidation of bacterial survival over extended time periods in food-processing environments.

Genetic subtyping of Listeria monocytogenes via multiple-locus sequence typing using iap, sigB and actA.

Pulse field gel electrophoresis (PFGE) is widely used for listeriosis surveillance. Although this technique is effective for epidemiology, the data among laboratories are inconsistent. We previously reported a method for Listeria monocytogenes subtyping combined with sequence analysis of partial iap and whole genome restriction fragment length polymorphism (RFLP) using XbaI, ClaI (BanIII) and PstI. However, distinguishing subtypes was challenging, because the output comprised complicated fragment patterns. In this study, we aimed to establish a simple genotyping method that does not depend on visual observation, rather it focuses on multi-locus sequence typing (MLST) using three genes, iap, sigB and actA. Sixty-eight strains of L. monocytogenes including EGD-e as a reference strain were investigated to ensure consistency with previous data on the genetic characterization. All strains were grouped into 29 types by both analyses. Although there are some differences in classification, major clades included the same strains. Simpson's indices of diversity (SID) by MLST and iap-RFLP-based typing were 0.967 (95% confidence interval [CI]: 0.955/0.978) and 0.967 (95% CI: 0.955/0.979), respectively. The discriminatory power of both methods can be considered almost identical. Compared with the results of 38 selected strains, the strains within the MLST clusters in this study coincided with those obtained using PFGE. Thus, the MLST strategy could help differentiate among L. monocytogenes isolates during epidemiological studies.

3ß-hydroxysterol δ24-reductase on the surface of hepatitis C virus-related hepatocellular carcinoma cells can be a target for molecular targeting therapy.

In our previous study, we demonstrated that 3ß-hydroxysterol Δ24-reductase (DHCR24) was overexpressed in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC), and that its expression was induced by HCV. Using a monoclonal antibody against DHCR24 (2-152a MAb), we found that DHCR24 was specifically expressed on the surface of HCC cell lines. Based on these findings, we aimed to establish a novel targeting strategy using 2-152a MAb to treat HCV-related HCC. In the present study, we examined the antitumor activity of 2-152a MAb. In the presence of complement, HCC-derived HuH-7 cells were killed by treatment with 2-152a MAb, which was mediated by complement-dependent cytotoxicity (CDC). In addition, the antigen recognition domain of 2-152a MAb was responsible for the unique anti-HCV activity. These findings demonstrate the feasibility of using 2-152a MAb for antibody therapy against HCV-related HCC. In addition, surface DHCR24 on HCC cells exhibited a functional property, agonist-induced internalization. We showed that 2-152a MAb-mediated binding of a cytotoxic agent (a saponin-conjugated secondary antibody) to surface DHCR24 led to significant cytotoxicity. This suggests that surface DHCR24 on HCC cells can function as a carrier for internalization. Therefore, surface DHCR24 could be a valuable target for HCV-related HCC therapy, and 2-152a MAb appears to be useful for this targeted therapy.

[Clinical Results of Thoracic Endovascular Aortic Repair (TEVAR) for the Treatment of Aortic Arch Aneruysm].

Thoracic endovascular aortic repair( TEVAR) combined of neck branch reconstruction( debranching TEVAR:dTEVAR) is applied to aortic arch aneurysm for minimally invasive reduction and improvement of treatment results. We report the initial and long-term results of dTEVAR for the treatment of aortic arch aneurysm. TEVAR for zone 0-2 area was applied in 49 cases (Zone 0:1:2;8:5:36 cases). Total dTEVAR was applied in zone 1 cases and 2 dTEVAR was performed in zone 2 cases. One dTEVAR or TEVAR with simple coverage of left subclavian artery was applied in zone 2 cases. Operative mortality within 30 days was 0% and there was 1 case in hospital death due to coronary arterial rupture after percutaneous coronary intervention. Perioperative morbidity included 1 case(2.0%) spinal cord ischemia, 4( 8.2%)arm claudication. In long-term follow-up, 5 cases died during the observation period (stroke in 1 case, heart failure in 1, neoplasma in 2, unknown in 1), but not in aortic rupture. Secondary type1 endoleak occurred in 2 cases(4.1%). Graft occlusion for neck branch reconstruction was complicated with 3 cases(6.1%), however no complications related to the graft occlusion. Our strategy of TEVAR provided durable results in patients treated for aortic arch aneurysm, with few adverse events.

Long-term results of open stent-grafting using a Matsui-Kitamura stent to treat thoracic aortic aneurysm.

PURPOSE: We describe a retrospective study of initial and long-term outcomes with an open stent grafting (OSG) with a Matsui-Kitamura stent for treating thoracic aortic aneurysm. METHODS: Between August 2005 and September 2013, 50 patients with aortic arch disease extending to the descending aorta underwent OSG. Circulatory arrest with total cardiopulmonary bypass and selective cerebral perfusion were used, and the aorta was transected between the brachiocephalic and left subclavian artery. The stent-graft was inserted, sutured to a transected aortic edge, and anastomosed to a four-branched arch graft. Preoperative, operative, and short- and long-term postoperative data were obtained from the patients' medical records. RESULTS: The perioperative (within 30 days) mortality rate was 8%. Two patients (4%) had a stroke and 5 patients (10%) had a spinal cord injury resulting in paraplegia or paraparesis (1 patient each) or transient paraplegia (3 patients). Actuarial survival rates at 1, 3, 5, and 7 years postoperatively were 87.8%, 78.3%, 70.7%, and 65.3%, respectively; the rates of freedom from an aortic event were 100%, 89.1%, 82.2%, and 74.7%. There were no complications related to use of the stent-graft. CONCLUSION: Our OSG method provided durable results in patients treated for thoracic aortic aneurysm, with few adverse events.

Genetic classification of Listeria monocytogenes serotype 4b strains, including epidemic clones, isolated from retail meat in the Tokyo metropolitan area.

A food-borne pathogen, Listeria monocytogenes serotype 4b, has been frequently isolated from patients with listeriosis, and numerous outbreaks of listeriosis are associated with this serotype. In the present study, we performed subtyping of L. monocytogenes serotype 4b strains on the basis of genetic analyses. Thirty-four isolates of serotype 4b were classified into 8 genotypes, namely genotypes 12, 15, 16, 17, 18, 23, 24, and 25, on the basis of the sequence for the partial iap gene. Genetic analyses revealed that genotype 16 and genotypes 24 and 25 belong to epidemic clone I (ECI) and ECII, respectively, which have been frequently associated with listeriosis outbreaks in the United States and Europe. The genotype isolated most frequently from retail meats in the Tokyo metropolitan area was genotype 12 (52%), followed by genotype 16 (29%), which belongs to ECI. We suggest that ECI is a common subtype of L. monocytogenes in retail meat in the area under investigation. On the other hand, ECII isolates were confirmed to be present in retail meat in Japan but were rare.

Establishment of a conditional transgenic system using the 2A peptide in the female mouse germline.

Transgenic mice are essential research tools in developmental biology studies. The 2A peptide allows multiple genes to be expressed simultaneously at comparable levels in somatic cells, but there are no reports of it being used successfully in germ cells. We constructed a Cre/loxP-based conditional vector containing the 2A peptide to significantly enhance the expression of a reporter and target gene from a constitutive promoter in oocytes. Mice with a transgene insertion containing the chicken ß-actin promoter, floxed EGFP-polyA cassette, mCherry reporter, 2A peptide and target gene DNA methyltransferase 3A2 (Dnmt3a2) were crossed with TNAP- or Vasa-Cre mice to produce offspring, in which mCherry and DNMT3A2 proteins were highly expressed in oocytes upon Cre-mediated removal of EGFP-polyA. This novel transgenic mouse line based on the 2A expression system can serve as a useful tool for examining gene function during oogenesis.

Forced expression of DNA methyltransferases during oocyte growth accelerates the establishment of methylation imprints but not functional genomic imprinting.

In mammals, genomic imprinting governed by DNA methyltransferase DNMT3A and its cofactor DNMT3L is essential for functional gametes. Oocyte-specific methylation imprints are established during oocyte growth concomitant with DNMT3A/DNMT3L expression, although the mechanisms of oocyte-specific imprinting are not fully understood. To determine whether the presence of DNMT3A/DNMT3L in oocytes is sufficient for acquisition of methylation imprints, we produced transgenic mice to induce DNMT3A/DNMT3L expression prematurely in oogenesis and analyzed DNA methylation imprints. The results showed that 2- to 4-fold greater expression of DNMT3A/DNMT3L was achieved in non-growing (ng) oocytes versus fully grown oocytes derived from wild-type mice, but the analyzed imprint domains were not methylated. Thus, the presence of DNMT3A/DNMT3L in ng oocytes is insufficient for methylation imprints, and imprinted regions are resistant to DNMT3A/DNMT3L in ng oocytes. In contrast, excess DNMT3A/DNMT3L accelerated imprint acquisition at Igf2r, Lit1, Zac1 and Impact but not Snrpn and Mest in growing oocytes. Therefore, DNMT3A/DNMT3L quantity is an important factor for imprint acquisition. Transcription at imprinted domains is proposed to be involved in de novo methylation; however, transcription at Lit1, Snrpn and Impact was observed in ng oocytes. Thus, transcription cannot induce DNMT3A catalysis at imprinted regions even if DNMT3A/DNMT3L is present. However, the accelerated methylation imprints in oocytes, with the exception of Igf2r, were erased during embryogenesis. In conclusion, a sufficient amount of DNMT3A/DNMT3L and a shift from the resistant to permissive state are essential to establish oocyte-specific methylation imprints and that maintenance of the acquired DNA methylation imprints is essential for functional imprinting.

Biofilm formation under different temperature conditions by a single genotype of persistent Listeria monocytogenes strains.

Some Listeria monocytogenes strains, termed persistent strains, originate from the same processing plant and have the ability to survive and grow over extended periods of time at contamination sources. In order to evaluate biofilm formation by such persistent strains, we isolated the pathogen from chicken samples collected from the same retail shop in repeated visits over 6 months. Strains that were of serotype 1/2b and were assigned to the same genotype by multi-virulence-locus sequence typing analysis were isolated on repeated occasions from December 1997 to June 1998 and thus were defined as persistent strains. In the present study, biofilm formation by the persistent strains was evaluated using microplates at 30 and 37°C. The biofilm-forming capability was measured after cells attaching to the microplate well were stained with crystal violet. Comparison of biofilm formation at 30°C among the persistent strains showed that a significantly higher amount of the stain was obtained from the persistent strains isolated from December to March than from those isolated from April to June. However, no significant difference in biofilm formation at 30°C was observed between persistent and nonpersistent groups of L. monocytogenes strains. In contrast, biofilm formation at 37°C was consistent among the persistent strains, and they produced significantly more biofilm at 37°C than did the nonpersistent strains. The persistent strains were also found to change their biofilm-forming ability in a temperature-dependent manner, which may suggest that the persistent strains alter their biofilm formation in response to changing environmental factors.

[Long-term results of open stent-grafting applied with a Matsui-Kitamura( MK) stent in treatment of arch aneurysm].

We report the initial and long-term results of open stent-grafting (OSG) applied with a Matsui-Kitamura (MK) stent in the treatment of thoracic aortic aneurysm (TAA). From August 2005 to March 2013, OSG for TAA was applied in 46 cases( male/female, 36/10, 54-86 years old, mean age 71). During deep hypothermic circulatory arrest with antegrade selective cerebral perfusion, stent graft was delivered through the transected proximal aortic arch, followed by arch replacement with a 4-branched prosthesis. Operative mortality within 30 days was 6.5%(respiratory failure in 1, multiple organ failure in 1and ischemic enteritis in 1) and there was 1 in hospital death due to brain stem infarction. Perioperative morbidity included 2( 4.3%) stroke, 5( 10.9%) spinal cord injuries( paraplegia in 1, paraparesis in 1 and transient paraparesis in 3). In long-term follow-up, survival was 86.8%, 77.2%, and 72.0% for 1, 3 and 5 years. Freedom from aortic event was 97.3%, 93.8% and 89.1% for 1, 3 and 5 years. Theseresults suggested the OSG method applied with a MK stent is a useful surgical procedure for the treatment of TAA.

Interaction between titanium and cadmium in various guinea pig organs.

Titanium (Ti) is used in many fields, while cadmium (Cd) is known to cause the itai-itai disease. In the present study, possible interactions between titanium and cadmium were investigated. Aorta, taenia coli, and liver were removed from male guinea pigs. Muscle tension was measured using intact aorta and taenia coli and using ß-escin-permeabilized taenia coli in a physiological salt solution and a hyperpotassium solution containing Cd and/or Ti. Cellular Cd contents were determined using all tissues after washout with EDTA solution. Cadmium-induced relaxation in the hyperpotassium solution recovered significantly (P < 0.01) following Ti treatment in taenia coli, but not in the aorta. In ß-escin-permeabilized taenia coli, the percentage recoveries after Cd treatment and after Ti plus Cd treatment were 67.3 ± 8.7 % (n = 4) and 87.7 ± 3.8 % (n = 4), respectively, compared with Ca-induced control contraction. Cellular Cd contents in taenia coli decreased significantly following treatment with Ti 10(-4) M. Although similar results were obtained using the aorta and the liver, there were no significant differences between the control and Ti 10(-5) M. High concentrations of Ti may reduce cellular Cd content.

Bacterial communities in fish sauce mash using culture-dependent and -independent methods.

In fish sauce production, microorganisms are associated with the fermentation process; however, the sequential changes in the bacterial communities have never been examined throughout the period of fermentation. In this study, we determined the bacterial floras in a fish sauce mash over 8 months, using three different culture media and 16S rRNA gene clone library analysis. During the first 4 weeks, viable counts of non-halophilic and halophilic bacteria decreased and were dominated by Staphylococcus species. Between 4 and 6 weeks, halophilic and highly halophilic bacterial counts markedly increased from 10(7) to 10(8) cfu/g, and the predominant species changed to Tetragenococcus halophilus. The occurrence of T. halophilus was associated with an increase of lactic acid and a reduction of pH values. In contrast, non-halophilic bacterial counts decreased to 10(6) cfu/g by 6 weeks with Bacillus subtilis as the dominant isolate. Clone library analysis revealed that the dominant bacterial group also changed from Staphylococcus spp. to T. halophilus, and the changes were consistent with those of the floras of halophilic and highly halophilic isolates. This is the first report describing a combination approach of culture and clone library methods for the analysis of bacterial communities in fish sauce mash.