Research progress on carotenoid production by Rhodosporidium toruloides.

Carotenoids are natural lipophilic pigments, which have been proven to provide significant health benefits to humans, relying on their capacity to efficiently scavenge singlet oxygen and peroxyl radicals as antioxidants. Strains belonging to the genus Rhodosporidium represent a heterogeneous group known for a number of phenotypic traits including accumulation of carotenoids and lipids and tolerance to heavy metals and oxidative stress. As a representative of these yeasts, Rhodosporidium toruloides naturally produces carotenoids with high antioxidant activity and grows on a wide variety of carbon sources. As a result, R. toruloides is a promising host for the efficient production of more value-added lipophilic compound carotenoids, e.g., torulene and torularhodin. This review provides a comprehensive summary of the research progress on carotenoid biosynthesis in R. toruloides, focusing on the understanding of biosynthetic pathways and the regulation of key enzymes and genes involved in the process. Moreover, the relationship between the accumulation of carotenoids and lipid biosynthesis, as well as the stress from diverse abiotic factors, has also been discussed for the first time. Finally, several feasible strategies have been proposed to promote carotenoid production by R. toruloides. It is possible that R. toruloides may become a critical strain in the production of carotenoids or high-value terpenoids by genetic technologies and optimal fermentation processes. KEY POINTS: • Biosynthetic pathway and its regulation of carotenoids in Rhodosporidium toruloides were concluded • Stimulation of abiotic factors for carotenoid biosynthesis in R. toruloides was summarized • Feasible strategies for increasing carotenoid production by R. toruloides were proposed.

Therapeutic applications of gut microbes in cardiometabolic diseases: current state and perspectives.

Cardiometabolic disease (CMD) encompasses a range of diseases such as hypertension, atherosclerosis, heart failure, obesity, and type 2 diabetes. Recent findings about CMD's interaction with gut microbiota have broadened our understanding of how diet and nutrition drive microbes to influence CMD. However, the translation of basic research into the clinic has not been smooth, and dietary nutrition and probiotic supplementation have yet to show significant evidence of the therapeutic benefits of CMD. In addition, the published reviews do not suggest the core microbiota or metabolite classes that influence CMD, and systematically elucidate the causal relationship between host disease phenotypes-microbiome. The aim of this review is to highlight the complex interaction of the gut microbiota and their metabolites with CMD progression and to further centralize and conceptualize the mechanisms of action between microbial and host disease phenotypes. We also discuss the potential of targeting modulations of gut microbes and metabolites as new targets for prevention and treatment of CMD, including the use of emerging technologies such as fecal microbiota transplantation and nanomedicine. KEY POINTS: • To highlight the complex interaction of the gut microbiota and their metabolites with CMD progression and to further centralize and conceptualize the mechanisms of action between microbial and host disease phenotypes. • We also discuss the potential of targeting modulations of gut microbes and metabolites as new targets for prevention and treatment of CMD, including the use of emerging technologies such as FMT and nanomedicine. • Our study provides insight into identification-specific microbiomes and metabolites involved in CMD, and microbial-host changes and physiological factors as disease phenotypes develop, which will help to map the microbiome individually and capture pathogenic mechanisms as a whole.

Skin microbiome profiling reveals the crucial role of microbial metabolites in anti-photoaging.

BACKGROUND: Skin microbiota is essential for health maintenance. Photoaging is the primary environmental factor that affects skin homeostasis, but whether it influences the skin microbiota remains unclear. OBJECTIVE: The objective of this study is to investigate the relationship between photoaging and skin microbiome. METHODS: A cohort of senior bus drivers was considered as a long-term unilateral ultraviolet (UV) irradiated population. 16S rRNA amplicon sequencing was conducted to assess skin microbial composition variations on different sides of their faces. The microbiome characteristics of the photoaged population were further examined by photoaging guinea pig models, and the correlations between microbial metabolites and aging-related cytokines were analyzed by high-throughput sequencing and reverse transcription polymerase chain reaction. RESULTS: Photoaging decreased the relative abundance of microorganisms including Georgenia and Thermobifida in human skin and downregulated the generation of skin microbe-derived antioxidative metabolites such as ectoin. In animal models, Lactobacillus and Streptobacillus abundance in both the epidermis and dermis dropped after UV irradiation, resulting in low levels of skin antioxidative molecules and leading to elevated expressions of the collagen degradation factors matrix metalloproteinase (MMP)-1 and MMP-2 and inflammatory factors such as interleukin (IL)-1ß and IL-6. CONCLUSIONS: Skin microbial characteristics have an impact in photoaging and the loss of microbe-derived antioxidative metabolites impairs skin cells and accelerates the aging process. Therefore, microbiome-based therapeutics may have potential in delaying skin aging.

Dual-recognition colorimetric platform based on porous Au@Pt nanozymes for highly sensitive washing-free detection of Staphylococcus aureus.

A dual-recognition strategy is reported to construct a one-step washing and highly efficient signal-transduction tag system for high-sensitivity colorimetric detection of Staphylococcus aureus (S. aureus). The porous (gold core)@(platinum shell) nanozymes (Au@PtNEs) as the signal labels show highly efficient peroxidase mimetic activity and are robust. For the sake of simplicity the detection involved the use of a vancomycin-immobilized magnetic bead (MB) and aptamer-functionalized Au@PtNEs for dual-recognition detection in the presence of S. aureus. In addition, we designed a magnetic plate to fit the 96-well microplate to ensure consistent magnetic properties of each well, which can quickly remove unreacted Au@PtNEs and sample matrix while avoiding tedious washing steps. Subsequently, Au@PtNEs catalyze hydrogen peroxide (H2O2) to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) generating a color signal. Finally, the developed Au@PtNEs-based dual-recognition washing-free colorimetric assay displayed a response in the range of S. aureus of 5 × 101-5 × 105 CFU/mL, and the detection limit was 40 CFU/mL within 1.5 h. In addition, S. aureus-fortified samples were analyzed to further evaluate the performance of the proposed method, which yielded average recoveries ranging from 93.66 to 112.44% and coefficients of variation (CVs) within the range 2.72-9.01%. These results furnish a novel horizon for the exploitation of a different mode of recognition and inexpensive enzyme-free assay platforms as an alternative to traditional enzyme-based immunoassays for the detection of other Gram-positive pathogenic bacteria.

Semiquantitative Fingerprinting Based on Pseudotargeted Metabolomics and Deep Learning for the Identification of Listeria monocytogenes and Its Major Serotypes.

The rapid identification of pathogenic microorganism serotypes is still a bottleneck problem to be solved urgently. Compared with proteomics technology, metabolomics technology is directly related to phenotypes and has higher specificity in identifying pathogenic microorganism serotypes. Our study combines pseudotargeted metabolomics with deep learning techniques to obtain a new deep semiquantitative fingerprinting method for Listeria monocytogenes identification at the serotype levels. We prescreened 396 features with orthogonal partial least-squares discrimination analysis (OPLS-DA), and 200 features were selected for deep learning model building. A residual learning framework for L. monocytogenes identification was established. There were 256 convolutional filters in the initial convolution layer, and each hidden layer contained 128 filters. The total depth included seven layers, consisting of an initial convolution layer, a residual layer, and two final fully connected classification layers, with each residual layer containing four convolutional layers. In addition, transfer learning was used to predict new isolates that did not participate in model training to verify the method's feasibility. Finally, we achieved prediction accuracies of L. monocytogenes at the serotype level exceeding 99%. The prediction accuracy of the new strain validation set was greater than 97%, further demonstrating the feasibility of this method. Therefore, this technology will be a powerful tool for the rapid and accurate identification of pathogens.

Washed microbiota transplantation improves renal function in patients with renal dysfunction: a retrospective cohort study.

BACKGROUND: Changes in the gut microbiota composition is a hallmark of chronic kidney disease (CKD), and interventions targeting the gut microbiota present a potent approach for CKD treatment. This study aimed to evaluate the efficacy and safety of washed microbiota transplantation (WMT), a modified faecal microbiota transplantation method, on the renal activity of patients with renal dysfunction. METHODS: A comparative analysis of gut microbiota profiles was conducted in patients with renal dysfunction and healthy controls. Furthermore, the efficacy of WMT on renal parameters in patients with renal dysfunction was evaluated, and the changes in gut microbiota and urinary metabolites after WMT treatment were analysed. RESULTS: Principal coordinate analysis revealed a significant difference in microbial community structure between patients with renal dysfunction and healthy controls (P = 0.01). Patients with renal dysfunction who underwent WMT exhibited significant improvement in serum creatinine, estimated glomerular filtration rate, and blood urea nitrogen (all P < 0.05) compared with those who did not undergo WMT. The incidence of adverse events associated with WMT treatment was low (2.91%). After WMT, the Shannon index of gut microbiota and the abundance of several probiotic bacteria significantly increased in patients with renal dysfunction, aligning their gut microbiome profiles more closely with those of healthy donors (all P < 0.05). Additionally, the urine of patients after WMT demonstrated relatively higher levels of three toxic metabolites, namely hippuric acid, cinnamoylglycine, and indole (all P < 0.05). CONCLUSIONS: WMT is a safe and effective method for improving renal function in patients with renal dysfunction by modulating the gut microbiota and promoting toxic metabolite excretion.

GII.17[P17] and GII.8[P8] noroviruses showed different RdRp activities associated with their epidemic characteristics.

Norovirus is the primary foodborne pathogenic agent causing viral acute gastroenteritis. It possesses broad genetic diversity and the prevalence of different genotypes varies substantially. However, the differences in RNA-dependent RNA polymerase (RdRp) activity among different genotypes of noroviruses remain unclear. In this study, the molecular mechanism of RdRp activity difference between the epidemic strain GII.17[P17] and the non-epidemic strain GII.8[P8] was characterized. By evaluating the evolutionary history of RdRp sequences with Markov Chain Monte Carlo method, the evolution rate of GII.17[P17] variants was higher than that of GII.8[P8] variants (1.22 × 10-3 nucleotide substitutions/site/year to 9.31 × 10-4 nucleotide substitutions/site/year, respectively). The enzyme catalytic reaction demonstrated that the Vmax value of GII.17[P17] RdRp was 2.5 times than that of GII.8[P8] RdRp. And the Km of GII.17[P17] and GII.8[P8] RdRp were 0.01 and 0.15 mmol/L, respectively. Then, GII.8[P8] RdRp fragment mutants (A-F) were designed, among which GII.8[P8]-A/B containing the conserved motif G/F were found to have significant effects on improving RdRp activity. The Km values of GII.8[P8]-A/B reached 0.07 and 0.06 mmol/L, respectively. And their Vmax values were 1.34 times than that of GII.8[P8] RdRp. In summary, our results suggested that RdRp activities were correlated with their epidemic characteristics. These findings will ultimately provide a better understanding in replication mechanism of noroviruses and development of antiviral drugs.

Gut microbiota: Candidates for a novel strategy for ameliorating sleep disorders.

The aim of this review was to evaluate the feasibility of treating sleep disorders using novel gut microbiota intervention strategies. Multiple factors can cause sleep disorders, including an imbalance in the gut microbiota. Studies of the microbiome-gut-brain axis have revealed bidirectional communication between the central nervous system and gut microbes, providing a more comprehensive understanding of mood and behavioral regulatory patterns. Changes in the gut microbiota and its metabolites can stimulate the endocrine, nervous, and immune systems, which regulate the release of neurotransmitters and alter the activity of the central nervous system, ultimately leading to sleep disorders. Here, we review the main factors affecting sleep, discuss possible pathways and molecular mechanisms of the interaction between sleep and the gut microbiota, and compare common gut microbiota intervention strategies aimed at improving sleep physiology.

Brij-58 supplementation enhances menaquinone-7 biosynthesis and secretion in Bacillus natto.

Menaquinone-7 is a form of vitamin K2 that has been shown to have numerous healthy benefits. In this study, several surfactants were investigated to enhance the production of menaquinone-7 in Bacillus natto. Results showed that Brij-58 supplementation influenced the cell membrane via adsorption, and changed the interfacial tension of fermentation broth, while the changes in the state and the composition of the cell membrane enhanced the secretion and biosynthesis of menaquinone-7. The total production and secretion rate of menaquinone-7 increased by 48.0% and 56.2% respectively. During fermentation, the integrity of the cell membrane decreased by 82.9% while the permeability increased by 158% when the maximum secretory rate was reached. Furthermore, Brij-58 supplementation induced the stress response in bacteria, resulting in hyperpolarization of the membrane, and increased membrane ATPase activity. Finally, changes in fatty acid composition increased membrane fluidity by 30.1%. This study provided an effective strategy to enhance menaquinone-7 yield in Bacillus natto and revealed the mechanism of Brij-58 supplementation in menaquinone-7 production. KEY POINTS: • MK-7 yield in Bacillus natto was significantly increased by Brij-58 supplementation. • Brij-58 could be adsorbed on cell surface and change fermentation environment. • Brij-58 supplementation could affect the state and composition of the cell membrane.

Effects of probiotics on hypertension.

Emerging data have suggested that probiotics had good potential in regulating intestinal flora and preventing hypertension. Some studies in human and animal models have demonstrated probiotic intervention could attenuate hypertension, regulate intestinal flora to increase the abundance of beneficial bacteria, and regulate intestinal microbial metabolites such as trimethylamine oxide, short-chain fatty acids, and polyphenols. However, there is still some debate as to whether probiotics exert effective benefits. These recently published reviews did not systematically expound on the heterogeneity between the effect and mechanism of probiotics with different types, doses, and carriers to exert antihypertensive effects, as well as the possible application of probiotics in the prevention and treatment of hypertension in food and clinic. Here we try to systematically review the association between hypertension and intestinal microflora, the effect of probiotics and their metabolites on hypertension, and the recent research progress on the specific mechanism of probiotics on hypertension. In addition, we also summarized the potential application of probiotics in antihypertension. Future challenges include elucidating the functions of metabolites produced by microorganisms and their downstream pathway or molecules, identifying specific strains, not just microbial communities, and developing therapeutic interventions that target hypertension by modulation of gut microbes and metabolites.

Exploration of the molecular mechanisms underlying the antibiotic resistance of Helicobacter pylori: A whole-genome sequencing-based study in Southern China.

BACKGROUND: Although antimicrobial resistance (AMR) in Helicobacter pylori is a global threat to human health and the underlying molecular mechanisms have been explored previously, only a few of them are fully elucidated. MATERIALS AND METHODS: In the present study, we isolated 54 Helicobacter pylori strains from Southern China and assessed their susceptibility to five antibiotics using the agar dilution assay. Whole-genome sequencing was performed to screen the AMR genotypes of the Helicobacter pylori isolates. RESULTS: Our study revealed a high prevalence of resistance to clarithromycin (CLR), levofloxacin (LVX), and metronidazole (MTZ) in the Chinese isolates, 55.56% of which showed multidrug-resistant phenotypes. We screened for the 94 types of previously reported AMR mutations in 12 genes, but only a few of them were related to the AMR phenotype. Furthermore, we discovered four new mutations in the 23S rRNA gene and one mutation in infB related to CLR resistance. Another three mutations in gyrA and one in gyrB were closely correlated with the AMR pattern against LVX. We also demonstrated that the mutations R16C/H in rdxA, V56I in rpsU, and D54A in sodB might contribute to resistance to MTZ, which were previously reported in laboratory experiments but not found in clinical strains. We examined the concordance between the genotype and phenotype of AMR and identified several potential molecular biomarkers for predicting CLR and LVX resistance. CONCLUSIONS: Our study explored the molecular mechanisms underlying the antibiotic resistance of Helicobacter pylori isolates from Southern China. We propose further epidemiologic investigations in China.

Protective effects of peptides on the cell wall structure of yeast under osmotic stress.

Three peptides (LL, LML, and LLL) were used to examine their influences on the osmotic stress tolerance and cell wall properties of brewer's yeast. Results suggested that peptide supplementation improved the osmotic stress tolerance of yeast through enhancing the integrity and stability of the cell wall. Transmission electron micrographs showed that the thickness of yeast cell wall was increased by peptide addition under osmotic stress. Additionally, quantitative analysis of cell wall polysaccharide components in the LL and LLL groups revealed that they had 27.34% and 24.41% higher chitin levels, 25.73% and 22.59% higher mannan levels, and 17.86% and 21.35% higher ß-1,3-glucan levels, respectively, than the control. Furthermore, peptide supplementation could positively modulate the cell wall integrity pathway and up-regulate the expressions of cell wall remodeling-related genes, including FKS1, FKS2, KRE6, MNN9, and CRH1. Thus, these results demonstrated that peptides improved the osmotic stress tolerance of yeast via remodeling the yeast cell wall and reinforcing the structure of the cell wall. KEY POINTS: • Peptide supplementation improved yeast osmotic stress tolerance via cell wall remodeling. • Peptide supplementation enhanced cell wall thickness and stability under osmotic stress. • Peptide supplementation positively modulated the CWI pathway under osmotic stress.

Improved osmotic stress tolerance in brewer's yeast induced by wheat gluten peptides.

The influences of three wheat gluten peptides (WGP-LL, WGP-LML, and WGP-LLL) on the osmotic stress tolerance and membrane lipid component in brewer's yeast were investigated. The results demonstrated that the growth and survival of yeast under osmotic stress were enhanced by WGP supplementation. The addition of WGP upregulated the expressions of OLE1 (encoded the delta-9 fatty acid desaturase) and ERG1 (encoded squalene epoxidase) genes under osmotic stress. At the same time, WGP addition enhanced palmitoleic acid (C16:1) content, unsaturated fatty acids/saturated fatty acids ratio, and the amount of ergosterol in yeast cells under osmotic stress. Furthermore, yeast cells in WGP-LL and WGP-LLL groups were more resistant to osmotic stress. WGP-LL and WGP-LLL addition caused 25.08% and 27.02% increase in membrane fluidity, 22.36% and 29.54% reduction in membrane permeability, 18.38% and 14.26% rise in membrane integrity in yeast cells, respectively. In addition, scanning electron microscopy analysis revealed that the addition of WGP was capable of maintaining yeast cell morphology and reducing cell membrane damage under osmotic stress. Thus, alteration of membrane lipid component by WGP was an effective approach for increasing the growth and survival of yeast cells under osmotic stress. KEY POINTS: •WGP addition enhanced cell growth and survival of yeast under osmotic stress. •WGP addition increased unsaturated fatty acids and ergosterol contents in yeast. •WGP supplementation improved membrane homeostasis in yeast at osmotic stress.

Amplified electrochemical antibiotic aptasensing based on electrochemically deposited AuNPs coordinated with PEI-functionalized Fe-based metal-organic framework.

A facile and versatile competitive electrochemical aptasensor for tobramycin (TOB) detection is described using electrochemical-deposited AuNPs coordinated with PEI-functionalized Fe-based metal-organic framework (AuNPs/P-MOF) as signal-amplification platform and a DNA probe labeled with methylene blue (MB) at the 3'-end (MB-Probe) as a signal producer. First, F-Probe (short complementary DNA strands of both the aptamer and the MB-Probe label with a sulfhydryl group at the 5'-end) was immobilized on the AuNPs/P-MOF modified electrode as detection probes, which competed with TOB in binding to the aptamer. TOB-aptamer binding resulted in F-Probe remaining unhybridized on the electrode surface, so that a significant current response was generated by hybridizing with MB-Probe instead. The developed strategy showed favorable repeatability, with a relative standard deviation (RSD) of 4.3% computed over five independent assays, and high stability, with only 6.8% degradation after 15 days of storage. Under optimal conditions, the proposed aptamer strategy exhibited a linear detection range from 100 pM to 500 nM with a limit of detection (LOD) of 56 pM (S/N = 3). The electrochemical aptasensor demonstrated remarkable selectivity, and its feasibility for accurate and quantitative detection of TOB in milk samples was confirmed (RSD < 4.5%). Due to its simple design, easy operation, and high sensitivity and selectivity, the proposed method could expect to detect other antibiotics by replacing the aptamers. In summary, this study provides a simple and effective new strategy for electrochemical aptasening based on MOF-based sensing interface. Scheme illustration of label-free competitive electrochemical aptamer-based detection of tobramycin based on electrochemically deposited AuNPs coordinated with PEI-functionalized Fe-based metal-organic framework as signal-amplification platform.

Genetic characteristics and virulence of Listeria monocytogenes isolated from fresh vegetables in China.

BACKGROUND: Ready-to-eat (RTE) vegetables have become increasingly popular along with the trend of moving towards a healthy lifestyle. However, RTE vegetables are at a higher risk of containing pathogens, maybe owing to lack of rigorous sanitization procedures. To understand the prevalence and potential risk of Listeria monocytogenes in RTE vegetables, we investigated the contamination level and characteristics of L. monocytogenes isolated from fresh vegetables. RESULTS: Twenty-three (5.49%) of the 419 vegetables samples were positive for L. monocytogenes. Phylogenetic group I.1 (1/2a-3a) and II.2 (1/2b-3b-7) strains were predominant in 30 isolates, which accounted for 33.3 and 50.0%, respectively. Multilocus sequence typing of the 30 isolates grouped them into nine sequence types (STs). The most common STs were ST87 (36.7%) and ST8 (26.7%). Virulence analysis showed that all 30 isolates harbored eight classical virulence genes, 10.0% isolates harbored the llsX gene (ST3 and ST1 strains), and 36.7% carried the ptsA gene and belonged to ST87. Approximately 83.3% isolates carried full-length inlA, whereas five isolates had premature stop codons in inlA, three of which belonged to ST9 and two to ST8. Antibiotic susceptibility showed the isolates were varyingly resistant to 13 antibiotics, 26.7% of the isolates were multi-drug resistant. CONCLUSIONS: The fresh vegetables contain some potential hypervirulent L. monocytogenes (ST1 and ST87) in the Chinese markets. In addition, the high rate of L. monocytogenes isolates was multi-drug resistant. Fresh raw vegetables may be a possible transmission route for L. monocytogenes infection in consumers. Therefore, sanitization of raw fresh vegetables should be strengthened to ensure their microbiological safety when used as RTE vegetables.

Complete genome analysis of a novel phage GW1 lysing Cronobacter.

A newly identified lytic Cronobacter phage, GW1, was isolated from the Pearl River of Guangzhou, China. GW1 had a double-stranded DNA genome of 39,695 nucleotides with an average GC content of 53.18 %. Among the 49 open reading frames (ORFs) identified, genes for rRNA, tRNA, antibiotic resistance, and virulence factors were not found in the phage genome. The morphology, genomic features, and phylogenetic position of GW1 revealed that it represents a new species in the genus T7virus. This novel lytic Cronobacter phage may provide an alternative for phage therapy and biocontrol against Cronobacter.

The resurgence of the norovirus GII.4 variant associated with sporadic gastroenteritis in the post-GII.17 period in South China, 2015 to 2017.

BACKGROUND: Human norovirus is regarded as the leading cause of nonbacterial acute diarrhea in developing and developed countries. Among all genotypes, GII.4 has been the predominant genotype, but in East Asia, it was replaced by the GII.17 in 2014/2015. However, after the prevalence of new GII.17 variant in South China, a sharply increase in the number of norovirus infections associated with sporadic acute diarrhea was detected. In this study, we would investigate the prevalence and genetic diversity of noroviruses in the sporadic acute gastroenteritis cases in the post-GII.17 period in South China. METHODS: Norovirus was screened from 217 patients with sporadic acute gastroenteritis from August 2015 to October 2017 by reverse transcription-polymerase chain reaction. Then, two regions including the partial RNA polymerase and the capsid gene of positive samples were amplified and sequenced. Phylogenetic analyses were performed to determine norovirus genotypes. Complete VP1 sequences of GII.4 strains detected in this study were also amplified and subjected into evolutionary tracing analyses. RESULTS: A total of 43 (19.82%) norovirus samples were confirmed from 217 stool specimens, and it was found that GII.4 resurged as the new predominant variant, accounting for 76.74% (33/43) of positive samples. Only one local strain GZ2015-L550 was clustered with the contemporary GII.P16/GII.4-2012 recombinant variant, and other 32 local strains belonged to the clade with the GII.Pe/GII.4-2012 variant. Other genotypes including GII.17 (n = 4), GII.3 (n = 4), GII.8 (n = 1) and GI. 6 (n = 1) were also detected. Furthermore, all GII.4 strains were phylogenetic analyzed based on their capsid P2 subdomains. Combined with other reported 754 strains, the GII.4-2012 variant could be divided into two clades. Most GII.4 strains collected in 2016 and 2017 in this study (7/8) formed a new cluster A in Clade II with additional 103 contemporaneous strains. In addition, evolutionary tracing of the capsid P2 subdomain of this variant was also analyzed, and one specific amino acid substitutions (N373) was identified for Cluster A. CONCLUSION: In summary, this study confirmed a norovirus infection peak in the post-GII.17 period in South China, which was caused by the resurgence of the GII.4 variant.

High-throughput sequencing analysis of bacterial community composition and quality characteristics in refrigerated pork during storage.

Spoilage bacteria seriously influence meat quality. In this study, the bacterial community, sensory scores, pH, and total volatile basic nitrogen (TVB-N) in refrigerated (4 °C) pork, the most commonly consumed meat in China, were investigated. In a high-throughput sequencing analysis of the V3-V4 region of the 16S rDNA gene, 259 bacterial genera were belonging to 21 phyla were identified. With the passage of time, the bacterial community diversity decreased. After 5 days, Pseudomonas, Acinetobacter and Photobacterium were dominant in refrigerated pork, especially Photobacterium, which rarely associated with meat spoilage. Our results suggest that these taxa contribute to refrigerated pork spoilage. During storage, pH and TVB-N showed similar trends. Additionally, total viable counts (TVC) increased steadily and sensory score decreased. On day 5, TVC, pH, TVB-N and sensory scores changed dramatically, and sensory scores indicating that the shelf life of refrigerated pork was less than 5 days. The predicted metabolic pathways, based o the data of 16S rDNA, indicated an abundant carbohydrate metabolism and amino metabolism in refrigerated pork. This study provides insight into the determinants of shelf life. Furthermore, it provides insight into the process involved in refrigerated pork spoilage.

Reconstituting the History of Cronobacter Evolution Driven by Differentiated CRISPR Activity.

Cronobacter strains harboring the CRISPR-Cas system are important foodborne pathogens causing serious neonatal infections. However, the specific role of the CRISPR-Cas system in bacterial evolution remains relatively unexplored. In this study, we investigated the impact of the CRISPR-Cas system on Cronobacter evolution and obtained 137 new whole-genome Cronobacter sequences by next-generation sequencing technology. Among the strains examined (n = 240), 90.6% (193/213) of prevalent species Cronobacter sakazakii, Cronobactermalonaticus, and Cronobacterdublinensis strains had intact CRISPR-Cas systems. Two rare species, Cronobactercondimenti (n = 2) and Cronobacteruniversalis (n = 6), lacked and preserved the CRISPR-Cas system at a low frequency (1/6), respectively. These results suggest that the presence of one CRISPR-Cas system is important for a Cronobacter species to maintain genome homeostasis for survival. The Cronobacter ancestral strain is likely to have harbored both subtype I-E and I-F CRISPR-Cas systems; during the long evolutionary process, subtype I-E was retained while subtype I-F selectively degenerated in Cronobacter species and was even lost by the major Cronobacter pathovars. Moreover, significantly higher CRISPR activity was observed in the plant-associated species Cdublinensis than in the virulence-related species C. sakazakii and Cmalonaticus Similar spacers of CRISPR arrays were rarely found among species, suggesting intensive change through adaptive acquisition and loss. Differentiated CRISPR activity appears to be the product of environmental selective pressure and might contribute to the bidirectional divergence and speciation of CronobacterIMPORTANCE This study reports the evolutionary history of Cronobacter under the selective pressure of the CRISPR-Cas system. One CRISPR-Cas system in Cronobacter is important for maintaining genome homeostasis, whereas two types of systems may be redundant and not conducive to acquiring beneficial DNA for environmental adaptation and pathogenicity. Differentiated CRISPR activity has contributed to the bidirectional divergence and genetic diversity of Cronobacter This perspective makes a significant contribution to the literature by providing new insights into CRISPR-Cas systems in general, while further expanding the roles of CRISPR beyond conferring adaptive immunity and demonstrating a link to adaptation and species divergence in a genus. Moreover, our study provides new insights into the balance between genome homeostasis and the uptake of beneficial DNA related to CRISPR-based activity in the evolution of Cronobacter.

Proteomics analysis of Listeria monocytogenes ATCC 19115 in response to simultaneous triple stresses.

Listeria monocytogenes can cause listeriosis in humans through consumption of contaminated food. L. monocytogenes can adapt and grow in a vast array of physiochemical stresses in the food production environment. In this study, we performed a proteomics strategy in order to investigate how L. monocytogenes survives with a simultaneous exposure to low pH, high salinity and low temperature. The results showed that the adaptation processes mainly affected the biochemical pathways related to protein synthesis, oxidative stress, cell wall and nucleotide metabolism. Interestingly, enzymes involved in the carbohydrate metabolism of energy, such as glycolysis and pentose phosphate pathway, were derepressed due to the down-regulation of CodY, a global transcriptional repressor. The down-regulation of CodY, together with the up-regulation of carbohydrate metabolism enzymes, likely leads to the accumulation of pyruvate and further to the activation of fatty acid synthesis pathway. Proteomics profiling offered a better understanding of the physiological responses of this pathogen to adapt to harsh environment and would hopefully contribute to improving the food-processing and storage methods.