Prevalence of Staphylococcus argenteus among food handlers, kitchen utensils, and food samples in Japan.

Staphylococcus argenteus has received increased attention from an aspect of food safety since several food poisoning outbreaks caused by the bacterium were reported in Japan. However, S. argenteus prevalence among food handlers and utensils has not yet been investigated. In this study, we investigated S. argenteus prevalence among a collection of coagulase-positive staphylococci (CPS) that were isolated during food sanitary inspections in Japan. Out of a total of 191 CPS isolates, 14 were identified as S. argenteus. One was isolated from shelled shrimp, nine were isolated from food handlers' hand swabs, and four were isolated from kitchen utensils. Whole-genome sequencing revealed that transmission of S. argenteus from human hands to utensils was possible. Though all 14 isolates were negative for the pvl and tst-1 genes, 6 harbored the seb gene. Only 21.4% of S. argenteus isolates were resistant to antibiotics, while 62.1% of the S. aureus isolates from the same sources were confirmed to be resistant. To the best of our knowledge, this is the first report to demonstrate possible transmission of S. argenteus from food handlers to utensils in food-processing environments.

Improving carotenoid production in recombinant yeast, Saccharomyces cerevisiae, using ultrasound-irradiated two-phase extractive fermentation.

Carotenoids are hydrophobic compounds that exhibit excellent bioactivity and can be produced by recombinant S. cerevisiae. Irradiating microorganisms with ultrasonic waves increase the productivity of various useful chemicals. Ultrasonic waves are also used to extract useful chemicals that accumulate in microbial cells. In this study, we aimed to improve the carotenoid production efficiency of a recombinant S. cerevisiae using an ultrasonic-irradiation based two-phase extractive fermentation process. When isopropyl myristate was used as the extraction solvent, a total of 264 mg/L of carotenoid was produced when batches were subjected to ultrasonic-irradiation at 10 W, which was a 1.3-fold increase when compared to the control. Transcriptome analysis suggested that one of the reasons for this improvement was an increase in the number of living cells. In fact, after 96 h of fermentation, the number of living cells increased by 1.4-fold upon irradiation with ultrasonic waves. Consequently, we succeeded in improving the carotenoid production in a recombinant S. cerevisiae strain using a ultrasonic-irradiated two-phase extractive fermentation and isopropyl myristate as the solvent. This fermentation strategy has the potential to be widely applied during the production of hydrophobic chemicals in recombinant yeast, and future research is expected to further develop this process.

Isolation and characterization of Staphylococcus argenteus strains from retail foods and slaughterhouses in Japan.

Staphylococcus argenteus has been recently established as a novel species of Staphylococcus aureus complex. It is known to cause various human diseases, such as skin and soft-tissue infections, sepsis, and staphylococcal food poisoning, although the source of infection has not been clearly described. In food poisoning cases, the source of bacterial contamination in food is unknown. This study examined the prevalence of S. argenteus among retail fresh food and poultry slaughterhouses in Japan. Among 642 food samples examined, successful isolation of S. argenteus was achieved in 21 of 151 (13.9%) chicken samples. No isolations from pork, beef, fish, or vegetables in retail markets were confirmed. Multiple-locus sequence typing revealed that the 21 isolates were classified into four sequence types (ST) that were divided into 14 subtypes using spa-typing. All food isolates were susceptible to methicillin and did not show positivity for the Panton-Valentine leukocidin gene. When bacteria were isolated from two poultry slaughterhouses in the same region, 14 S. argenteus strains were successfully isolated from only one slaughterhouse. Thirteen of 14 strains were isolated from a poultry carcass and slaughterhouse environments during a certain sampling period and were all classified as ST5961 with identical spa-type. Also, the number of single nucleotide variants (SNVs) detected on the core genomes of the same 13 strains were between 0 and 17, suggesting a long-term inhabitation of an S. argenteus strain inside the facility. Furthermore, one isolate from chicken meat was also genetically linked with the same lineage of slaughterhouse isolates, with ≤15 SNVs being detected. Additionally, one slaughterhouse isolate from chiller water and three chicken isolates were classified into the same cluster by phylogenetic analysis, although the number of pairwise SNVs ranged from 62 to 128. To the best of the authors' knowledge, this is the first study that demonstrated S. argenteus in a food processing facility and the possible bacterial contamination on food during food processing.

Improvement of 2,3-butanediol tolerance in Saccharomyces cerevisiae by using a novel mutagenesis strategy.

Although the yeast Saccharomyces cerevisiae has been used to produce various bio-based chemicals, including solvents and organic acids, most of these products inhibit yeast growth at high concentrations. In general, it is difficult to rationally improve stress tolerance in yeast by modifying specific genes, because many of the genes involved in stress response remain unidentified. Previous studies have reported that various forms of stress tolerance in yeast were improved by introducing random mutations, such as DNA point mutations and DNA structural mutations. In this study, we developed a novel mutagenesis strategy that allows for the simultaneous performance of these two types of mutagenesis to construct a yeast variant with high 2,3-butanediol (2,3-BDO) tolerance. The mutations were simultaneously introduced into S. cerevisiae YPH499, accompanied by a stepwise increase in the concentration of 2,3-BDO. The resulting mutant YPH499/pol3δ/BD_392 showed 4.9-fold higher cell concentrations than the parental strain after 96 h cultivation in medium containing 175 g/L 2,3-BDO. Afterwards, we carried out transcriptome analysis to characterize the 2,3-BDO-tolerant strain. Gene ontology enrichment analysis with RNA sequence data revealed an increase in expression levels of genes related to amino acid metabolic processes. Therefore, we hypothesize that the yeast acquired high 2,3-BDO tolerance by amino acid function. Our research provides a novel mutagenesis strategy that achieves efficient modification of the genome for improving tolerance to various types of stressors.

Construction of lactic acid-tolerant Saccharomyces cerevisiae by using CRISPR-Cas-mediated genome evolution for efficient D-lactic acid production.

Lactic acid (LA) is chemically synthesized or fermentatively produced using glucose as substrate, mainly using lactic acid bacteria. Polylactic acid is used as a biodegradable bioplastic for packaging materials, medical materials, and filaments for 3D printers. In this study, we aimed to construct a LA-tolerant yeast to reduce the neutralization cost in LA production. The pHLA2-51 strain was obtained through a previously developed genome evolution strategy, and transcriptome analysis revealed the gene expression profile of the mutant yeast. Furthermore, the expression of the genes associated with glycolysis and the LA synthesis pathway in the LA-tolerant yeast was comprehensively and randomly modified to construct a D-LA-producing, LA-tolerant yeast. In detail, DNA fragments expressing thirteen genes, HXT7, HXK2, PGI1, PFK1, PFK2, FBA1, TPI1, TDH3, PGK1, GPM1, ENO2, and PYK2, and D-lactate dehydrogenase (D-LDH) from Leuconostoc mesenteroides were randomly integrated into the genomic DNA in the LA-tolerant yeast. The resultant engineered yeast produced about 33.9 g/L of D-LA from 100 g/L glucose without neutralizing agents in a non-neutralized condition and 52.2 g/L of D-LA from 100 g/L glucose with 20 g/L CaCO3 in a semi-neutralized condition. Our research provides valuable insights into non-neutralized fermentative production of LA. KEY POINTS: • Lactic acid (LA) tolerance of yeast was improved by genome evolution. • The transcription levels of 751 genes were changed under LA stress. • Rapid LA production with semi-neutralization was achieved by modifying glycolysis. • A versatile yeast strain construction method based on the CRISPR system was proposed.

A specific combination of dual index adaptors decreases the sensitivity of amplicon sequencing with the Illumina platform.

Amplicon sequencing is a powerful approach in microbiome studies as it detects live organisms with high sensitivity. This approach determines the composition of sequence variants of marker genes using high-throughput DNA sequencers. The use of dual index adaptors is the fundamental technique for pooling DNA libraries for Illumina sequencers and is believed not to affect the results. However, here, we observed a decrease of sequence quality in samples containing a specific combination of indexes, namely N704 and S507 in Nextera kits, in multiple runs on the Illumina MiSeq sequencer operated in different facilities. This decrease was also observed when sequencing randomly fragmented DNA of Escherichia coli and was not observed when either individual adaptor was used. Each end of the DNA library with this index combination contains a complementary sequence motif, which potentially inhibits proper cluster generation and/or subsequent sequencing. Community analysis of the 16S and 18S rRNA amplicons using QIIME2 revealed significant decreases in α-diversity in the samples containing the N704/S507 index combination, resulting from loss of low-abundance sequence variants during denoising. Our data underscore the importance of quality validation of sequence reads in developing dual index techniques and suggest cautious interpretation of microbiome data containing low-quality sequence reads.

Role of gut microbiota in sex- and diet-dependent metabolic disorders that lead to early mortality of androgen receptor-deficient male mice.

The gut microbiota is involved in metabolic disorders induced by androgen deficiency after sexual maturation in males (late-onset hypogonadism). However, its role in the energy metabolism of congenital androgen deficiency (e.g., androgen-insensitive syndrome) remains elusive. Here, we examined the link between the gut microbiota and metabolic disease symptoms in androgen receptor knockout (ARKO) mouse by administering high-fat diet (HFD) and/or antibiotics. HFD-fed male, but not standard diet-fed male or HFD-fed female, ARKO mice exhibited increased feed efficiency, obesity with increased visceral adipocyte mass and hypertrophy, hepatic steatosis, glucose intolerance, insulin resistance, and loss of thigh muscle. In contrast, subcutaneous fat mass accumulated in ARKO mice irrespective of the diet and sex. Notably, all HFD-dependent metabolic disorders observed in ARKO males were abolished after antibiotics administration. The ratios of fecal weight-to-food weight and cecum weight-to-body weight were specifically reduced by ARKO in HFD-fed males. 16S rRNA sequencing of fecal microbiota from HFD-fed male mice revealed differences in microbiota composition between control and ARKO mice. Several genera or species (e.g., Turicibacter and Lactobacillus reuteri, respectively) were enriched in ARKO mice, and antibiotics treatment spoiled the changes. Furthermore, the life span of HFD-fed ARKO males was shorter than that of control mice, indicating that androgen deficiency causes metabolic dysfunctions leading to early death. These findings also suggest that AR signaling plays a role in the prevention of metabolic dysfunctions, presumably by influencing the gut microbiome, and improve our understanding of health consequences in subjects with hypogonadism and androgen insensitivity.

Chronic kidney disease after 5/6 nephrectomy disturbs the intestinal microbiota and alters intestinal motility.

Organ-organ crosstalk is involved in homeostasis. Gastrointestinal symptoms are common in patients with renal failure. The aim of this study was to elucidate the relationship between gastrointestinal motility and gastrointestinal symptoms in chronic kidney disease. We performed studies in C57BL/6 mice with chronic kidney disease after 5/6 nephrectomy. Gastrointestinal motility was evaluated by assessing the ex vivo responses of ileum and distal colon strips to electrical field stimulation. Feces were collected from mice, and the composition of the gut microbiota was analyzed using 16S ribosomal RNA sequencing. Mice with chronic kidney disease after 5/6 nephrectomy showed a decreased amount of stool, and this constipation was correlated with a suppressed contraction response in ileum motility and decreased relaxation response in distal colon motility. Spermine, one of the uremic toxins, inhibited the contraction response in ileum motility, but four types of uremic toxins showed no effect on the relaxation response in distal colon motility. The 5/6 nephrectomy procedure disturbed the balance of the gut microbiota in the mice. The motility dysregulation and constipation were resolved by antibiotic treatments. The expression levels of interleukin 6, tumor necrosis factor-α, and iNOS in 5/6 nephrectomy mice were increased in the distal colon but not in the ileum. In addition, macrophage infiltration in 5/6 nephrectomy mice was increased in the distal colon but not in the ileum. We found that 5/6 nephrectomy altered gastrointestinal motility and caused constipation by changing the gut microbiota and causing colonic inflammation. These findings indicate that renal failure was remarkably associated with gastrointestinal dysregulation.

Value-adding conversion and volume reduction of sewage sludge by anaerobic co-digestion with crude glycerol.

In this study, conversion of sewage sludge to biogas by anaerobic co-digestion with crude glycerol was examined. When 0.126g/L crude glycerol was added to the reactor, only methane was produced. Upon addition of 5.04g/L crude glycerol, hydrogen production occurred, and a significant amount of 1,3-propanediol (1,3-PDO) was generated in the liquid phase. On day 6, the dry weight was largely composed of organic acids (48%) and 1,3-PDO (17%), which are water-soluble. Degradation of 1,3-PDO was very slow, which is advantageous for recovery. Crude glycerol, which contains alkaline substances, promoted organic matter degradation by microorganisms, which possibly affected biogas and 1,3-PDO production. Addition of 0.630-2.52g/L glycerol initially led to hydrogen production, followed by methane production a few days later, which stabilized within 1week. In conclusion, adjustment of the crude glycerol concentration allows controllable conversion to value-added products for co-digestion.

Estimation of the adhesive force distribution for the flagellar adhesion of Escherichia coli on a glass surface.

The effects of the presence or absence of microbial flagella and the microbial motility on the colloidal behaviors of microbial cells were quantitatively evaluated. The microbial cell attachment and detachment processes on a glass surface were observed directly using a parallel-plate flow chamber. Wild-type, flagellar paralyzed, and nonflagellated Escherichia coli strains were used as model microbial cells. In the cell attachment tests, the microbial adhesion rate in a 160mM NaCl solution was approximately 10 times higher than that in a 10mM solution, for all E. coli strains. The colloidal behavior of the microbial cells agreed well with the predictions of the DLVO theory. In addition, the microbial flagella and motility did not significantly affect the cell attachment, regardless of the existence of a potential barrier between the cell and the glass substratum. In the cell detachment tests, the cumulative number of microbial cells detached from the glass substratum with increasing flow rate was fit well with the Weibull distribution function. The list of strains arranged in order of increasing median drag force required to remove them was nonflagellated strain, flagellar paralyzed strain, and wild-type strain. These results indicated that the flagella and the flagellar motility inhibited the cell detachment from the glass substratum. Furthermore, a large external force would likely be required to inhibit the microbial adhesion in the early stage of the biofilm formation.

Measurement of microbial adhesive forces with a parallel plate flow chamber.

HYPOTHESIS: It was predicted that the colloidal behaviors of archaea and bacteria with disparate surface structure were different. In this study, the effects of the physicochemical properties of microbial cell surfaces on colloidal behavior were analyzed with Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, thermodynamics, and powder technology. EXPERIMENTS: Cell attachment and detachment from model substrates were directly observed using a parallel plate flow chamber. Gram-negative Escherichia coli and archaeal Methanosarcina barkeri were used as model microbial cells, and positively and negatively charged glass slides were used as model substrates. FINDINGS: Microbial adhesion on both substrates agreed well with predictions calculated from DLVO theory, using experimental parameters. The total number of cells detached from the substrates as a function of flow rate was fit with the Weibull distribution function. In addition, the drag force required for detachment, which was estimated from the hydrodynamic forces, had a wide distribution; however, the forces became smaller with increasing ionic strength because of reduced electrostatic interactions between the cells and the substrate. M. barkeri could not be detached from positively charged substrates because it would entail a negative change in the interfacial energy of interaction. Thus adhesion was thermodynamically favored in this case.

Exposure of the yeast Saccharomyces cerevisiae to functionalized polystyrene latex nanoparticles: influence of surface charge on toxicity.

Novel nanoparticles with unique physicochemical characteristics are being developed with increasing frequency, leading to higher probability of nanoparticle release and environmental accumulation. Therefore, it is important to assess the potential environmental and biological adverse effects of nanoparticles. In this study, we investigated the toxicity and behavior of surface-functionalized nanoparticles toward yeast (Saccharomyces cerevisiae). The colony count method and confocal microscopy were used to examine the cytotoxicity of manufactured polystyrene latex (PSL) nanoparticles with various functional groups (amine, carboxyl, sulfate, and nonmodified). S. cerevisiae were exposed to PSL nanoparticles (40 mg/L) dispersed in 5-154 mM NaCl solutions for 1 h. Negatively charged nanoparticles had little or no toxic effect. Interestingly, nanoparticles with positively charged amine groups (p-Amine) were not toxic in 154 mM NaCl, but highly toxic in 5 mM NaCl. Confocal microscopy indicated that in 154 mM NaCl, the p-Amine nanoparticles were internalized by endocytosis, whereas in 5 mM NaCl they covered the dead cell surfaces. This demonstrates that nanoparticle-induced cell death might to be related to their adhesion to cells rather than their internalization. Together, these findings identify important factors in determining nanoparticle toxicity that might affect their impact on the environment and human health.

Changes in dominant fermentation type during anaerobic digestion of high-loading glycerol with slight glucose content.

High-loading glycerol containing slight amounts of five different monosaccharides was inoculated with seed sludge obtained from a methane fermentation reactor. The use of different monosaccharides as fermentation promoters resulted in changes in fermentation types; in particular, glucose induced the formation of 1,3-propanediol. After 9 days incubation with glucose, glycerol levels had fallen by 81%, while molar yields of organic acids and 1,3-propanediol (per mole of glycerol degraded) were 0.22 and 0.39, respectively. Other monosaccharides enhanced methane production after 14 days of incubation in the following order: galactose, galacturonic acid, mannose and arabinose. Hydrogen was generated (together with a negligible amount of methane) only in the presence of glucose. When glucose was introduced to a methane-producing reactor (promoted by galacturonic acid), hydrogen production began 5 days later and displaced the methane production after 12 days. These results suggest that glucose catalyzes glycerol degradation, resulting in the production of hydrogen.

Novel anaerobic digestion induced by bacterial components for value-added byproducts from high-loading glycerol.

A novel bioprocessing system was developed and tested that involved anaerobic fermentation to degrade high-loading glycerol by a fermentation promoter, and which could be used for the production of important resources. In the absence of a promoter, there was no anaerobic digestion when glycerol (4.0-6.0%, v/v) was added to the reactor. By contrast, glycerol was readily decomposed when sewage sludge, acting as a fermentation promoter, was added to the anaerobic reactor. Fermentation resulted in the generation of hydrogen, 1,3-propanediol (1,3-PDO) and various organic acids. In 7 days, glycerol decomposition reached 88%; hydrogen production was 3.1mg/kg-glycerol (0.0004 g/day/L), and 1,3-PDO yield reached 0.35 kg/kg-glycerol (0.05 g/day/L). Further experiments confirmed that the bacteria Escherichia coli and particularly Schizosaccharomyces pombe (found within sewage sludge) and especially glucose (found within bacterial components (i.e., cell walls)) acted as efficient promoters of fermentation.

Efficient, high-speed methane fermentation for sewage sludge using subcritical water hydrolysis as pretreatment.

A novel biomass-energy process for the production of methane from sewage sludge using a subcritical water (sub-CW) hydrolysis reaction as pretreatment is proposed. The main substances of sewage sludge hydrolyzed by sub-CW at 513 K for 10 min were acetic acid, formic acid, pyroglutamic acid, alanine, and glycine. Fermentation experiments were conducted in an anaerobic-sludge reactor for two different samples: real sewage sludge and a model solution containing components typically produced by the sub-CW pretreatment of sewage sludge. In the experiment for the sub-CW pretreatment of sewage sludge, methane generation was twice that for non-pretreatment after 3 days of incubation. In the model experiment, the methane conversion was about 40% with the application of mixture of organic acids and amino acids after 5 days of incubation. Furthermore, the methane conversion was about 60% for 2 days when only organic acids, such as acetic acid and formic acid, were applied. Because acetic acid is the key intermediate and main precursor of the methanogenesis step, fermentation experiments were conducted in an anaerobic-sludge reactor with high concentrations of acetic acid (0.01-0.1M). Nearly 100% of acetic acid was converted to methane and carbon dioxide in 1-3 days.

Xyloglucan breakdown during cotton fiber development.

Cotton (Gossypium herbaceum L.) fibers elongated almost linearly up to about 20 days post anthesis. The molecular mass of xyloglucans in fiber cell walls decreased gradually during the elongation stage. When enzymatically active (native) cell wall preparations of fibers were autolyzed, the molecular mass of xyloglucans decreased. The decrease was most prominent in wall preparations obtained from the rapidly elongating fibers. The xyloglucan-degrading activity was recovered from the fiber cell walls with 3 mol/L NaCl, and the activity was high at the stages in which fibers elongated vigorously. These results suggest the possible involvement of xyloglucan metabolism in the regulation of cotton fiber elongation.

Changes in the sugar composition and molecular mass distribution of matrix polysaccharides during cotton fiber development.

Cotton (Gossypium herbaceum L.) fiber development consists of a fiber elongation stage (up to 20 d post-anthesis) and a subsequent cell wall thickening stage. Cell wall analysis revealed that the extractable matrix (pectic and hemicellulosic) polysaccharides accounted for 30-50% of total sugar content in the fiber elongation stage but less than 3% in the cell wall thickening stage. By contrast, cellulose increased dramatically after the fiber elongation ceased. The amounts of extractable xyloglucans and arabinose- and galactose-containing polymers per seed increased in the early fiber elongation stage and decreased thereafter. The amounts of extractable acidic polymers and non-cellulosic beta-glucans (mainly composed of beta-1,3-glucans) increased in parallel with fiber elongation and then decreased. The molecular masses of extractable non-cellulosic beta-glucans, and arabinose- and galactose-containing polymers decreased during both fiber elongation and cell wall thickening stages. The molecular mass of extractable xyloglucans also decreased during the fiber elongation stage, but this decrease ceased during the cell wall thickening stage. Conversely, the molecular size of acidic polymers in the extractable pectic fraction increased during both stages. Thus, not only the amounts but also the molecular size of the extractable matrix polysaccharides showed substantial changes during cotton fiber development.