Antibacterial Activity of Hexanol Vapor In Vitro and on the Surface of Vegetables.

Hexanol is a volatile alcohol and a major component of plant essential oils (EOs). However, the antibacterial activity of hexanol vapor has not been well studied. This study aimed to evaluate the antibacterial activity of hexanol. In this study, seven food-related bacteria were exposed to 1-, 2- or 3-hexanol vapor on agar media to evaluate their growth. Additionally, the total viable counts in three vegetables when exposed to 1-hexanol vapor were measured. The results showed that 1-hexanol exhibited antibacterial effects against Gram-negative bacteria but did not affect Gram-positive bacteria. However, compounds 2- and 3-hexanol did not show antimicrobial activity against any bacteria. For the vegetables, exposure to 1-hexanol vapor decreased the total viable bacterial counts in cabbage and carrot and inhibited bacterial growth in eggplants. In cabbage, 1-hexanol vapor at concentrations over 50 ppm decreased the total viable count within 72 h, and 25 ppm of vapor showed bacteriostatic activity for 168 h. However, 1-hexanol vapor also caused discoloration in cabbage. In summary, 1-hexanol has the potential to act as an antibacterial agent, but further studies are required for practical use. Moreover, the study results may help determine the antimicrobial activity of various EOs in the future.

Identification of changes in the microflora composition of Japanese horse mackerel (Trachurus japonicus) during storage to identify specific spoilageorganisms.

Japanese horse mackerel (Trachurus japonicus) is an important marine resource, and its loss and waste should be reduced. This study aimed to identify the changes in the microflora composition during storage and specific spoilage organisms (SSOs) in Japanese horse mackerel, for spoilage prevention. They were stored at either 20 °C or 4 °C aerobically, and the bacterial viable counts, concentration of total volatile basic nitrogen (TVB-N), and microflora composition for each group were analyzed. Samples stored at 20 °C for 48 h showed similar viable counts to those stored at 4 °C for 168 h; however, the TVB-N concentrations increased at 20 °C, but not at 4 °C. 16S rRNA metagenome analysis showed that Shewanella became dominant genus in the microflora regardless of the storage temperature. However, dominant amplicon sequence variants (ASVs), which are a more detailed classification level than the genus, differed depending on the storage temperatures; therefore, dominant ASVs at 20 °C were assumed to be potential SSOs. Shewanella sp. Strain NFH-SH190041, which was genetically closely related to the dominant ASVs at 20 °C, was isolated, and its spoilage ability was verified. The strain NFH-SH190041 may be considered a novel SSO of Japanese horse mackerel because its 16S rRNA sequence is clearly different from those of known species.

Comparative Evaluation of Selective Media for the Detection of Bacillus cereus.

The commercially available 3 types of selective media in Japan were compared for the detection of Bacillus cereus. When assessed inclusivity using 25 B. cereus strains, MYP agar, NGKG agar, and chromogenic X-BC agar demonstrated excellent inclusivity. For exclusivity study using 50 non-B. cereus strains, MYP, NGKG, and X-BC allowed to grow 11, 7, and 3 strains, respectively. Of the grown bacteria on each strains tested, only 2 strains of B. thuringiensis formed typical B. cereus colonies on all selective media tested. The NGKG and X-BC were compared with MYP as a reference using artificially contaminated food (fried rice, plain rice, fried noodle, and potato salad ), since MYP is recommended in ISO 7932: 2004. The both correlation coefficients between NGKG and MYP, and X-BC and MYP were 0.999. Therefore, we demonstrated that NGKG and X-BC can be adapted to ISO 7932: 2004 method for selected food as well as MYP.

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.

Evaluation of the Novel Dry Sheet Culture Method for the Enumeration of Enterobacteriaceae.

MC-Media PadTM EB (MMP-EB) , a novel sheet culture method for the enumeration of Enterobacteriaceae, has been evaluated. When both inclusivity and exclusivity of MMP-EB were assessed using 104 microbes including 51 Enterobacteriaceae strains, all tested Enterobacteriaceae strains grew and formed obvious red-colored colonies and all tested non-Enterobacteriaceae strains were shown different appearance from Enterobacteriaceae strains. For the comparison study of the method, MMP-EB was compared with violet red bile glucose agar (VRBG) according to ISO 21528-2:2017 and PetrifilmTM Enterobacteriaceae Count Plate (Petrifilm EB) method using 100 naturally contaminated food samples. The correlation coefficients between MMP-EB and VRBG, and MMP-EB and Petrifilm EB were 0.940 and 0.972, respectively. Furthermore, there were no statistically significant difference between MMP-EB and both reference methods. Our results demonstrated that MMP-EB was a suitable alternative method for the enumeration of Enterobacteriaceae in food samples.

Complete Genome Sequence of Lactobacillus curvatus NFH-Km12, Isolated from the Japanese Traditional Fish Fermented Food Kabura-zushi.

Kabura-zushi is a traditional Japanese fermented food made from yellowtail, rice, salt, and koji. In this study, the complete genomic sequence of Lactobacillus curvatus NFH-Km12, isolated from this unique food, is reported. NFH-Km12 has a 1.9-Mbp chromosome and contains 5 plasmids.

Comparison of Chromogenic Selective Media for the Detection of Cronobacter spp. (Enterobacter sakazakii).

　The four types of chromogenic selective media that are commercially available in Japan were compared for establishing a Japanese standard method for detecting Cronobacter spp. based on ISO/TS 22964:2006. When assessed using 9 standard Cronobacter spp. strains and 29 non-Cronobacter strains, Enterobacter sakazakii isolation agar, ChromocultTM Enterobacter sakazakii agar, CHROMagarTM E. sakazakii, and XM-sakazakii agar demonstrated excellent inclusivity and exclusivity. Using the ISO/TS 22964:2006 method, the recovered numbers of 38 Cronobacter spp. strains, including 29 C. sakazakii isolates obtained from each medium, were equivalent, indicating that there was no significant difference (p > 0.05) among the four types of chromogenic selective media. Thus, we demonstrated that these four chromogenic selective media are suitable alternatives when using the standard method for detecting Cronobacter spp. in Japan, based on the ISO/TS 22964:2006.

Increase in hydrophobicity of Bacillus subtilis spores by heat, hydrostatic pressure, and pressurized carbon dioxide treatments.

The effects of heat treatment (HT), hydrostatic pressure treatment (HPT), and pressurized carbon dioxide treatment (CT) on surface hydrophobicity of B. subtilis 168 spores were investigated. The spore surface hydrophobicity was measured by determining the ratio of hydrophobic spores (RHS) that were partitioned into the n-hexadecane phase from the aqueous spore suspension. The RHS after HT generally increased in a temperature-dependent manner and reached approximately 10% at temperatures above 60°C. The effects of pressurization by HPT and accompanying temperature on increased RHS were complex. The highest RHS after HPT was approximately 17%. Following CT, RHS reached approximately 80% at 5 MPa at 80°C for 30 min. An increased treatment temperature enhanced RHS by CT. The increase in RHS by CT led to the formation of spore clumps and adhesion of spores to hydrophobic surfaces. Acidification of spore suspension to pH 3.2, expected pH during CT, by HCl also increased the adhesion of spores at the similar degree with CT. The spore surface zeta potential distribution was not changed by CT. Furthermore, spores with increased RHS after CT had germination-like phenomena including loss of their refractility and enhanced staining by 4',6-diamidino-2-phenylindole. Physiological germination that was induced by the addition of l-alanine also increased the RHS. From these results, it is clear that CT under heating considerably increases RHS. CT under heating considerably increases RHS. This increase in RHS may be due to acidification or germination-like phenomena during CT.

Evaluation of the Quantitative Dry Culture Method (Sanitakun(TM) SA) for the Enumeration of Staphylococcus aureus in Artificially Contaminated Food Samples.

Sanita-kun(TM) SA for Staphylococcus aureus (SkSA), a novel dry sheet quantitative culture system, was evaluated. When the inclusivity and exclusivity of SkSA were assessed using 121 microorganisms including 47 S. aureus strains, the tested S. aureus strains formed blue-colored colonies on the SkSA and all the other microbes failed to grow. The SkSA was then compared with Baird-Parker agar (BP) according to ISO 6888-1, Mannitol salt agar with egg yolk (MSEY), and 3M Petrifilm(TM) STX (3M-STX) in 100 artificially contami nated food samples. The correlation coefficients between SkSA and BP, SkSA and MSEY, and SkSA and 3M-STX were 0.971, 0.989 and 0.996, respectively. Our results demonstrated that SkSA is a suitable alternative for the enumeration of S. aureus in foods.

Evaluation of a Novel Dry Sheet Culture Method for Rapid Enumeration of Total Aerobic Count in Foods.

A novel dry sheet culture method (Sanita-kun ACplus; SkACp) for rapid enumeration of total viable count has been developed. This rehydrated plate system comprises an adhesive sheet, nonwoven fabric coated with nutrients, and two types of water absorption polymers. In addition, SkACp facilitates methods for both rapid count (rapid mode: 24-h incubation) and accurate enumeration (standard mode: 48-h incubation) because it not only contains conventional 2,3,5-triphenyltetrazolium chloride but also contains two kinds of new tetrazolium salts for rapid and accurate enumeration of total aerobic count. When SkACp was assessed with 91 microorganisms, 87 strains (95.6%), excluding lactic acid and psychrotrophic bacteria, formed red-colored colonies within 24 h, whereas all microorganisms tested formed colonies within 48 h. The SkACp method, with both 24 and 48 h of incubation, was compared with plate count agar (PCA) and 3M Petrifilm AC (PAC) by using 107 naturally contaminated foods. For all foods tested (n = 107), the linear correlation coefficients of 48-h counts on SkACp compared with PCA and PAC were 0.98 and 0.75, respectively, while the 24-h counts on SkACp compared with PCA and PAC were 0.77 and 0.96, respectively. For foods tested, excluding yogurt and lactic beverages ( n = 101), the linear correlation coefficients of 48-h counts on SkACp compared with PCA and PAC were 0.98 and 0.96, respectively, while the 24-h counts on SkACp compared with PCA and PAC were 0.96 and 0.95, respectively. These results demonstrated that SkACp (48 h) is a useful alternative for the enumeration of the total aerobic count for all foods, whereas SkACp (24 h) was also an effective method for rapid enumeration in foods, excluding yogurt and lactic beverages.

Development of a Novel Chromogenic Medium for Improved Campylobacter Detection from Poultry Samples.

The presence of expanded-spectrum ß-lactamase (ESBL)-producing Escherichia coli is a common problem in the isolation of Campylobacter from poultry samples using conventional cefoperazone-based selective media. A novel chromogenic medium (CM-HT), based on modified charcoal cefoperazone deoxycholate agar (mCCDA), has been developed as a solution for improved Campylobacter detection from poultry samples. Although the basic components of CM-HT are the same as mCCDA, CM-HT uses both granular charcoal and sodium cefoxitin to enhance viewability and inhibit ESBL-producing bacteria. All tested Campylobacter jejuni (n = 31) and Campylobacter coli (n = 6) strains grew and formed purple-colored colonies on CM-HT. In contrast, the growth of all other tested microorganisms, including ESBL-producing E. coli strains, was suppressed by this medium. Additionally, 84 poultry samples were examined for the presence of Campylobacter using the ISO 10272-1 method (enrichment with Bolton broth) and the NIHSJ-02 method (enrichment with Preston broth) with mCCDA and CM-HT media for the isolation. The numbers of samples from which Camplylobacter was detected on CM-HT using Preston and Bolton broth were 22 and 18, whereas the numbers on mCCDA were 22 and 13, respectively. Only Campylobacter was detected on CM-HT using both enrichment broths; however, there were 5 and 19 samples from which ESBL-producing E. coli was detected on mCCDA using Preston and Bolton broth, respectively. Thus, there was a significant difference between CM-HT and mCCDA in selectivity for ESBL-producing E. coli regardless of which enrichment broth was used. The results obtained demonstrated that CM-HT is a possible solution for the improved isolation of Campylobacter from poultry samples.

Awa1p on the cell surface of sake yeast inhibits biofilm formation and the co-aggregation between sake yeasts and Lactobacillus plantarum ML11-11.

We examined mixed-species biofilm formation between Lactobacillus plantarum ML11-11 and both foaming and non-foaming mutant strains of Saccharomyces cerevisiae sake yeasts. Wild-type strains showed significantly lower levels of biofilm formation compared with the non-foaming mutants. Awa1p, a protein involved in foam formation during sake brewing, is a glycosylphosphatidylinositol (GPI)-anchored protein and is associated with the cell wall of sake yeasts. The AWA1 gene of the non-foaming mutant strain Kyokai no. 701 (K701) has lost the C-terminal sequence that includes the GPI anchor signal. Mixed-species biofilm formation and co-aggregation of wild-type strain Kyokai no. 7 (K7) were significantly lower than K701 UT-1 (K701 ura3/ura3 trp1/trp1), while the levels of strain K701 UT-1 carrying the AWA1 on a plasmid were comparable to those of K7. The levels of biofilm formation and co-aggregation of the strain K701 UT-1 harboring AWA1 with a deleted GPI anchor signal were similar to those of K701 UT-1. These results clearly demonstrate that Awa1p present on the surface of sake yeast strain K7 inhibits adhesion between yeast cells and L. plantarum ML11-11, consequently impeding mixed-species biofilm formation.

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.

Evaluation of a novel dry sheet culture method (Sanita-kun(R)) for rapid enumeration of yeasts and molds in foods.

Sanita-kun(R) Yeasts and Molds (SkYM), a novel dry sheet culture method for rapid enumeration of fungi, has been developed. This re-hydrated plate consists of a unique adhesive sheet, non-woven fabric coated with nutrients, antibiotic, water absorption polymer and uniquely synthesized 2-(2-methoxyphenyl)-3-(4-nitrophenyl)-5-phenyl-tetrazolium chloride for rapid enumeration of yeasts and molds. When SkYM was assessed using 37 microbes including 33 fungal strains, 29 fungal strains (87.9%) were formed red colored colonies within 48h whereas all yeasts and molds tested formed colonies within 72 h. All tested bacteria failed to grow. The SkYM method, with both 48 and 72 h of incubation, was compared with Dichloran Rose-Bengal Chloramphenicol Agar (DRBC; 5 days) according to ISO 21527-1, and with 3M Petrifilm YM (PYM; 5 days) and Nissui Compact Dry YM (CDYM; 5 days) commercially available dry culture methods using 100 naturally contaminated foods. The linear correlation coefficients of SkYM (48h) with DRBC, PYM and CDYM were 0.921, 0.929 and 0.947, respectively, whereas the linear correlation coefficients between SkYM (72 h) and DRBC, SkYM (72h) and PYM, SkYM (72h) and CDYM were 0.948, 0.877 and 0.911, respectively. These results demonstrated that SkYM was a useful alternative for rapid enumeration of yeasts and molds in foods.

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.

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.

Manganese Ion Increases LAB-yeast Mixed-species Biofilm Formation.

Remarkable LAB-yeast mixed-species biofilm was formed by lactic acid bacteria (LAB) Lactobacillus plantarum ML11-11 isolated from Fukuyama pot vinegar and Saccharomyces cerevisiae. This mixed-species biofilm formation increased in proportion to the YPD medium concentration but decreased in proportion to the MRS medium concentration. The effect of MRS components on mixed-species biofilm formation was investigated in a YPD medium environment, and it was clarified that beef extract (one of the MRS medium components) decreased mixed-species biofilm formation. On the other hand, manganese sulfate (another component in MRS) remarkably increased both LAB single- and LAB-yeast mixed-species biofilm formation. LAB single- and mixed-species biofilm formation were increased in proportion to the manganese sulfate concentration up to 1 mM and 100 µM, respectively. The growth of L. plantarum ML11-11 was increased significantly by the addition of 10 µM manganese sulfate and was resistant to higher concentration of up to 100 mM, but growth of S. cerevisiae was sensitive to manganese ion above 100 µM. These results suggested that mixed-species biofilm formation could be controlled artificially by controlling the manganese ion level.

Cronobacter spp. in commercially available dried food in Japan.

A total of 140 samples of dried food sold in Japan were surveyed and tested for the presence of viable bacteria, distribution of coliform bacteria, and contamination with Cronobacter spp. The samples were purchased from retail stores in Tokyo and Kanagawa Prefecture. Out of the 140 samples tested, viable bacteria were found in 135 samples and coliform bacteria were found in 23 samples. Qualitative and quantitative testing revealed the presence of Cronobacter spp. in 35 (25.0%) and 11 samples (7.9%), respectively. The most commonly found Cronobacter species were C. sakazakii, with the next most common, in order, being C. muytjensii and C. turicensis. The actual numbers of Cronobacter species in the tested dried foods were low, but the widespread contamination particularly in dried herbs and vegetables was confirmed.

Antimicrobial activity of the carnivorous plant Dionaea muscipula against food-related pathogenic and putrefactive bacteria.

Solvent extracts from the carnivorous plant Dionaea muscipula (Venus flytrap) were prepared using eight different organic solvents, and examined for antibacterial activity against food-related pathogenic and putrefactive bacteria. All solvent extracts showed higher antibacterial activity against gram positive bacteria than against gram negative bacteria. The TLC-bioautography analysis of the extracts revealed that a yellow spot was detected at Rf value of 0.85, which showed strong antibacterial activity. The UV, MS, and NMR analyses revealed that the antibacterial compound was plumbagin.

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.