Steric microstructure of mixed-species biofilm formed by interaction between Lactobacillus plantarum ML11-11 and Saccharomyces cerevisiae.

The mixed-species biofilm of Lactobacillus plantarum ML11-11 (LAB) and yeast had a double-layered structure with the ground layer composed of LAB cells, and the upper layer composed of coaggregates of LAB and yeast cells. The ability of LAB to adhere to both, the solid surface and the yeast cells, enabled the formation and maintenance of the biofilm as an ecosystem for LAB and yeast.

Studies on formation, control and application of biofilm formed by food related microorganisms.

Biofilms are sessile microbial aggregates on the interfaces, and they were usually considered as microbial contamination sources in medical care and various industries. We studied the control and application of biofilms formed by food-related microorganisms, and mechanism of the biofilm formation was also investigated. We studied the biofilm formation in mixed cultures using various combinations of two strains of food-related microorganisms. There were various microorganisms that showed decreased or increased biofilm formation in the mixed culture in comparison with that in a single culture. Biofilm formed by lactic acid bacteria and yeast isolated from traditional fermented food, Fukuyama pot vinegar, exhibited unique feature in that structure and formation mechanism, and expected to be used as an immobilized microorganism in fermentation production. Here our studies on the control and application of biofilms and the mechanisms of its formation were described.

Screening of lactic acid bacteria that can form mixed-species biofilm with Saccharomyces cerevisiae.

The abilities of lactic acid bacteria (LAB) to form mixed-species biofilm with Saccharomyces cerevisiae in a static co-culture were investigated out of 168 LAB stock cultures, and two Lactobacillus plantarum strains (D71 and E31) and one Leuconostoc mesenteroides strain K01 were found to form mixed-species biofilm with S. cerevisiae BY4741. SEM observation showed that there was no significant difference in morphological properties among these three mixed-species biofilms and they resembled that formed by S. cerevisiae with L. plantarum ML11-11 previously isolated from a brewing sample of Fukuyama pot vinegar. The co-aggregation assays showed that L. plantarum D71 and L. plantarum E31 could co-aggregate with S. cerevisiae similarly to L. plantarum ML11-11, while L. mesenteroides K01 had no ability to co-aggregate with yeast. The above results indicate that aggregation followed by direct cell-to-cell contact is required for mixed-species biofilm formation between these L. plantarum strains and S. cerevisiae, though some different mechanism may be involved in biofilm formation between L. mesenteroides strain and S. cerevisiae.

Acid resistance contributes to the high-pressure carbon dioxide resistance of Escherichia coli K-12.

Effect of deletion of acid resistant genes of E. coli on the high-pressure carbon dioxide (HPC) resistance was investigated. Genes coding amino acid decarboxylases, such as lysine, arginine, and glutamate decarboxylase, were found to contribute to HPC resistance. Protonophore-treated cells showed hypersensitivity to HPC, confirming that HPC induced cytoplasm acidification and exerted severe damage on cells by intrusion of gaseous carbon dioxide into cytoplasm.

Fimbriae and lipopolysaccharides are necessary for co-aggregation between Lactobacilli and Escherichia coli.

Cells of Lactobacilli co-aggregated with Escherichia coli K-12 cells to form co-aggregates under mixed-culture conditions at 37 °C for 24 h. Co-aggregation was inhibited by sodium dodecyl sulfate but not by protease. E. coli deletion mutants of fimbriae formation and lipopolysaccharide (LPS) formation did not co-aggregate with Lactobacilli. These results showed that fimbriae and LPS are necessary for co-aggregation between Lactobacilli and E. coli.

Sublethal high hydrostatic pressure treatment reveals the importance of genes coding cytoskeletal protein in Escherichia coli morphogenesis.

We studied morphologic changes after sublethal high hydrostatic pressure treatment (HPT) of Escherichia coli K-12 strains in which genes related to the cytoskeleton, cell wall, and cell division had been deleted. Some long filamentous and swelling cells were observed in wild-type bacteria, while some spherical, branched, or collapsed cells were observed in deletion mutants. In particular, ΔzapA and ΔrodZ showed distinguished morphologies. ZapA supports FtsZ, a cytoskeletal protein, forming ring with ZapB. RodZ, a cytoskeletal protein, interacts with MreB, also a cytoskeletal protein, and both factors are necessary for maintaining the rod shape of the cell. These results showed that insufficient formation of FtsZ rings induced cell elongation and that insufficient formation of MreB induced a branched and collapsed cell shape. Therefore, the correct formation of the bacteria cytoskeleton by FtsZ rings and MreB is important for keeping normal cell shape during growth after HPT, and the polymerization of cytoskeletal proteins was a critical target of sublethal HPT. These results indicate that sublethal HPT induces bacterial cell morphologic change and provide important information on the role of genes involved in morphogenesis. Therefore, sublethal HPT may be a good tool for studying the morphogenesis of bacterial cells.

External Ear Melanoma Treated with Auricular Reconstruction Using Four Different Tissues in a 16-Year-Old Patient.

The auricular region is a common site of cutaneous malignancies, most of which are nonmelanoma skin cancers, such as basal cell carcinoma and squamous cell carcinoma, in older patients. They are often treated with limited surgery that can be performed under local anesthesia. We report the case of a young patient with external ear melanoma who underwent reconstruction for defects of more than one-half of the helix and concha using four types of tissues: a rib cartilage graft, temporoparietal fascia flap, full-thickness skin graft, and retroauricular flap. Extending the retroauricular flap posteriorly to the entire hairless area allowed us to cover the anterior surface of the rib cartilage framework, which helped effectively achieve aesthetics. In auricle reconstruction, it is crucial to determine how well the anterior surface of the auricle is created.

Yeast mannan structure necessary for co-aggregation with Lactobacillus plantarum ML11-11.

Lactic acid bacteria (LAB) Lactobacillus plantarum ML11-11, an isolate from Fukuyama pot vinegar, and yeast Saccharomyces cerevisiae form significant mixed-species biofilm with direct cell-cell contact. Co-aggregation of L. plantarum ML11-11 and S. cerevisiae cells, mediated by the interaction between surface protein(s) on L. plantarum ML11-11 cells and surface mannan of S. cerevisiae cells, contributes significantly to mixed-species biofilm formation. In this study, co-aggregation activities of yeast mutants that were deleted of genes related to mannan biosynthesis were investigated to clarify the mannan structures essential for interaction with L. plantarum ML11-11. Among the 12 deletion mutants which had various incomplete mannan structures, only the mnn2 mutant lost the co-aggregation activity. In the mnn2 mutant, the gene coding the activity of attaching first branching mannose residue to mannan main chain is deleted and therefore the mnn2 mutant has unbranched mannan. From this result, it is clarified that the specific structure, consisted of mannan main chain to which are attached side chains containing one or more mannose residues, is critical for co-aggregation with L. plantarum ML11-11.

L-Tryptophan prevents Escherichia coli biofilm formation and triggers biofilm degradation.

The effect of deletion of trp operon and tna operon on the Escherichia coli biofilm formation was investigated in order to elucidate the role of L-tryptophan metabolism in biofilm formation. trp operon deletion mutants ΔtrpC, ΔtrpD and ΔtrpE deficient in L-tryptophan biosynthesis showed higher biofilm formation. In addition, ΔtnaC with increased L-tryptophan degradation activity showed higher biofilm formation. On the contrary, ΔtnaA deletion mutant which lost L-tryptophan degradation activity showed low biofilm formation. From these results, it was suggested that decrease of intracellular L-tryptophan level induced biofilm formation and increase of L-tryptophan repressed biofilm formation. So the effect of the addition of L-tryptophan to the medium on the E. coli biofilm formation was investigated. L-Tryptophan addition at starting culture decreased biofilm formation and furthermore L-tryptophan addition after 16 h culture induced the degradation of preformed biofilm. From the above results, it was suggested that maintenance of high intracellular L-tryptophan concentration prevents E. coli biofilm formation and elevation of intracellular L-tryptophan concentration triggers degradation of matured biofilm.

Mutants of Lactobacillus plantarum ML11-11 deficient in co-aggregation with yeast exhibited reduced activities of mixed-species biofilm formation.

Lactic acid bacteria (LAB) mutants deficient in inter-species co-aggregation with yeast were spontaneously derived from Lactobacillus plantarum ML11-11, a significant mixed-species biofilm former in static co-cultures with budding yeasts. These non-co-aggregative mutants also showed significant decreases in mixed-species biofilm formation. These results suggest the important role of co-aggregation between LAB and yeast in mixed-species biofilm formation. Cell surface proteins obtained by 5 M LiCl extraction from the wild-type cells and non-co-aggregative mutant cells were analyzed by SDS-PAGE. There was an obvious difference in protein profiles. The protein band at 30 kDa was present abundantly in the wild-type cell surface fraction but was significantly decreased in the mutant cells. This band assuredly corresponded to the LAB surface factors that contribute to co-aggregation with yeasts.

[Two cases of hyperammonemic patients treated by chemotherapy for colorectal cancer].

We report two patients having hyperammonemic encephalopathy while being treated with chemotherapy for colorectal cancer. The first patient was a 69-year-old man with sigmoid colon cancer, having a massive invasion to the urinary bladder. He received SOX therapy following a pelvic exenteration operation. After the third course of SOX therapy, he presented with general fatigue and repeated seizures, and blood examination showed a high level of serum ammonium. He was diagnosed as hyperammonemic encephalopathy. The second patients was a 60-year-old woman with ascending colon cancer and liver metastasis having portal vein tumor thrombosis, who was given a palliative resection of ascending colon, and then underwent modified FOLFOX6 therapy. At the second course, she fell into a deep coma, and blood examination revealed a high level of serum ammonium. In both patients, treatment with infusion of branched-chain amino acid solutions resolved the symptoms of encephalopathy. Acute neurotoxicity caused by hyperammonemic encephalopathy during chemotherapy for colorectal cancer is rare and not well recognized, but it is a clinically important complication. We should pay more attention to hyperammonemic encephalopathy of patients receiving chemotherapy for colorectal cancer.

Metachronous Liver Metastasis from Alpha-Fetoprotein-Producing Gastric Cancer Successfully Treated with Capecitabine/Oxaliplatin Combination Chemotherapy.

A consensus regarding the treatment of recurrent alpha-fetoprotein-producing gastric carcinoma due to its rarity is lacking. We herein describe a case of such a carcinoma that was associated with metachronous liver metastasis. A 73-year-old man was referred for the surgical treatment of a type 2 gastric tumor that extended from the greater curvature of the gastric corpus to the pylorus. As no remote metastases were detected, the patient underwent open total gastrectomy with lymphadenectomy and Roux-en-Y reconstruction. Histopathological examination revealed regional lymph node metastasis and the invasion of the muscularis propria by a moderately differentiated adenocarcinoma. Immunostaining of the primary tumor was positive for alpha-fetoprotein and negative for human epidermal growth factor receptor 2. Serum alpha-fetoprotein levels decreased to within normal limits after eight courses of S-1 monotherapy; however, levels started to increase, and a hypovascular nodule in segment 5/6 of the liver was detected 3.5 years later. Serum alpha-fetoprotein levels returned to the normal range, and the tumor was undetectable after four courses of capecitabine and oxaliplatin therapy. No recurrence was detected at 1.5-year follow-up. This case demonstrates that combined capecitabine and oxaliplatin therapy can successfully treat metachronous liver metastasis from alpha-fetoprotein-producing gastric carcinoma.

Lower Eyelid Reconstruction Using a Myotarsocutaneous Flap while Considering the Superior and Inferior Palpebral Sulci.

Various reconstructive procedures have been reported for full-thickness defects of the lower eyelid after tumor excision or trauma. In eyelid reconstruction, not only functionality but also cosmetic results are important; furthermore, making scars inconspicuous is challenging. The purpose of this study is to make the scars less noticeable in lower eyelid reconstruction. We reconstructed the anterior lamella using a myotarsocutaneous flap and the posterior lamella of the donor site using a palatal mucosal graft in a 61-year-old man with basal cell carcinoma of the lower eyelid. In designing the myotarsocutaneous flap, we matched the upper edge of the flap with the superior palpebral sulcus, and the lower edge with the inferior palpebral sulcus. The flap length was the same as the width of the defect. The lateral side of the postoperative scar was hidden by the excess skin of the upper eyelid, while the caudal side of the scar and the trapdoor deformity was covered with a tear trough. After movement, the flap was not located outside the lateral canthus, and good cosmetic results were obtained. This report describes our surgical procedure.

Novel multicellular prokaryote discovered next to an underground stream.

A diversity of prokaryotes currently exhibit multicellularity with different generation mechanisms in a variety of contexts of ecology on Earth. In the present study, we report a new type of multicellular bacterium, HS-3, isolated from an underground stream. HS-3 self-organizes its filamentous cells into a layer-structured colony with the properties of a nematic liquid crystal. After maturation, the colony starts to form a semi-closed sphere accommodating clusters of coccobacillus daughter cells and selectively releases them upon contact with water. This is the first report that shows that a liquid-crystal status of cells can support the prokaryotic multicellular behavior. Importantly, the observed behavior of HS-3 suggests that the recurrent intermittent exposure of colonies to water flow in the cave might have been the ecological context that cultivated the evolutionary transition from unicellular to multicellular life. This is the new extant model that underpins theories regarding a role of ecological context in the emergence of multicellularity.

Inhibition of Streptococcus mutans biofilm formation by Streptococcus salivarius FruA.

The oral microbial flora consists of many beneficial species of bacteria that are associated with a healthy condition and control the progression of oral disease. Cooperative interactions between oral streptococci and the pathogens play important roles in the development of dental biofilms in the oral cavity. To determine the roles of oral streptococci in multispecies biofilm development and the effects of the streptococci in biofilm formation, the active substances inhibiting Streptococcus mutans biofilm formation were purified from Streptococcus salivarius ATCC 9759 and HT9R culture supernatants using ion exchange and gel filtration chromatography. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis was performed, and the results were compared to databases. The S. salivarius HT9R genome sequence was determined and used to indentify candidate proteins for inhibition. The candidates inhibiting biofilms were identified as S. salivarius fructosyltransferase (FTF) and exo-beta-d-fructosidase (FruA). The activity of the inhibitors was elevated in the presence of sucrose, and the inhibitory effects were dependent on the sucrose concentration in the biofilm formation assay medium. Purified and commercial FruA from Aspergillus niger (31.6% identity and 59.6% similarity to the amino acid sequence of FruA from S. salivarius HT9R) completely inhibited S. mutans GS-5 biofilm formation on saliva-coated polystyrene and hydroxyapatite surfaces. Inhibition was induced by decreasing polysaccharide production, which is dependent on sucrose digestion rather than fructan digestion. The data indicate that S. salivarius produces large quantities of FruA and that FruA alone may play an important role in multispecies microbial interactions for sucrose-dependent biofilm formation in the oral cavity.

The importance of inter-species cell-cell co-aggregation between Lactobacillus plantarum ML11-11 and Saccharomyces cerevisiae BY4741 in mixed-species biofilm formation.

Cells of Lactobacillus plantarum ML11-11, an isolate from Fukuyama pot vinegar, and the yeast Saccharomyces cerevisiae formed significant mixed-species biofilms with concurrent inter-species co-aggregation. The co-aggregation did not occur with heated or proteinase K-treated ML11-11 cells, or in the presence of D-mannose, suggesting that surface proteins of ML11-11 and mannose-containing surface substance(s) of yeast were the predominant contributing factors. Sugar fatty acid ester inhibited mixed-species biofilm formation, but did not inhibit co-aggregation, suggesting that the cell-cell adhesion and cell-polystylene adhesion are controlled by different mechanisms. Microscopic observation and microflora analysis revealed that inter-species co-aggregation plays an important role in the formation of the mixed-species biofilm.

Roles of multidrug efflux pumps on the biofilm formation of Escherichia coli K-12.

We genetically analyzed the roles of multidrug efflux pumps on the biofilm formation of Escherichia coli K-12 BW25113. We used 22 mutants missing various genes related to the multidrug efflux pumps and found that the biofilm formation of emrD, emrE, emrK, acrD, acre and mdtE-deleted mutants was extremely decreased. These results indicate that some multidrug efflux pumps significantly contribute to the biofilm formation of E. coli.

Curry Leaf Triggers Cell Death of P. gingivalis with Membrane Blebbing.

Periodontal disease has become a serious public health problem, as indicated by accumulating evidence that periodontal disease is not only a major cause of tooth loss but is also associated with various systemic diseases. The present study assessed the anti-bacterial activities of three herbal products (curry leaf, clove, and cinnamon) against Porphyomonas gingivalis, a keystone pathogen for periodontal diseases. The curry leaf extract (CLE) showed the strongest growth inhibitory activity among them, and the activity was maintained even after extensive heat treatment. Of note, while clove and cinnamon extracts at sub-minimum inhibitory concentrations (sub-MICs) significantly enhanced the biofilm formation of P. gingivalis, CLE at sub-MIC did not have any effect on the biofilm formation. The MIC of CLE against P. gingivalis was higher than those against a wide range of other oral bacterial species. P. gingivalis cells were completely killed within 30 min after treatment with CLE. Spatiotemporal analysis using high-speed atomic force microscopy revealed that CLE immediately triggered aberrant membrane vesicle formation on the bacterial surface. Bacterial membrane potential assay revealed that CLE induced depolarization of the bacterial membrane. Taken together, these findings suggest the mechanism behind early bactericidal activity of CLE and its therapeutic applicability in patients with periodontal diseases.

Two-way Revascularization to Manage Celiac Artery Stenosis during Pancreaticoduodenectomy: A Case Report.

Celiac artery (CA) occlusion, or stenosis, is not uncommon, and most cases are asymptomatic. If the CA is occluded, collateral circulation from the superior mesenteric artery (SMA) is maintained through the pancreaticoduodenal arcade. However, the pancreaticoduodenal arcade is removed if pancreaticoduodenectomy (PD) is performed, which results in ischemia of the liver, stomach, and residual pancreas. Thus, these patients require CA revascularization, which can include antegrade endovascular reconstruction and retrograde reconstruction using vascular anastomosis from the SMA system to the CA system. Both methods carry risks of restenosis or anastomotic thrombosis. We report a technique that involves a combination of both revascularization methods in an 89-year-old man who underwent PD for lower bile duct cancer. Preoperative endovascular stent placement in the CA preserved antegrade blood flow to the liver, and intraoperative vascular anastomosis of the jejunal artery and right gastroepiploic artery achieved retrograde blood flow. Although we confirmed both stent and anastomosis patency and blood circulation in our case, obstruction of 1 of these revascularization pathways would not likely lead to ischemia of the liver. Thus, our 2-way revascularization technique for managing celiac artery stenosis during PD may reduce the risk of organ ischemia.

Mixed-species biofilm formation by direct cell-cell contact between brewing yeasts and lactic acid bacteria.

Mixed-species biofilm was remarkably formed in a static co-culture of Lactobacillus plantarum ML11-11 and Saccharomyces cerevisiae Y11-43 isolated from brewing samples of Fukuyama pot vinegar. Mixed-species biofilm is probably formed by direct cell-cell contact between ML11-11 and S. cerevisiae including Y11-43 and laboratory yeast strains. Scanning electron microscopic observation suggested that the mixed-species biofilm had a thick, bi-layer structure.