Identification of microbial communities and multi-species biofilms contamination in seafood processing environments with different hygiene conditions.

Biofilms formed by spoilage and pathogenic bacteria increase microbial persistence, causing an adverse influence on the quality of seafood. The mono-species biofilms are widely reported, however, the contamination of multi-species biofilms and their matrix in food environments are still not fully understood. Here, we assessed the contamination of multi-species biofilms in three seafood processing environments with different hygiene levels by detecting bacterial number and three biofilm matrix components (carbohydrates, extracellular DNA (eDNA), and proteins). Samples comprising seven food matrix surfaces and eight food processing equipment surfaces were collected from two seafood processing plants (XY and XC) and one seafood market (CC). The results showed that the bacterial counts ranged from 1.89 to 4.91 CFU/cm2 and 5.68 to 9.15 BCE/cm2 in these surfaces by cultivation and real-time PCR, respectively. Six biofilm hotspots were identified, including four in CC and two in XY. Among the three processing environments, the amplicon sequence variants (ASVs) of Proteobacteria, Bacteroidetes, and Actinobacteria decreased with improved processing hygiene, while Firmicutes showed a decrease in the four most abundant phyla. The most prevalent bacteria belonged to genera Psychrobacter, Acinetobacter, and Pseudomonas, demonstrating the significant differences and alteration in bacterial community composition during different environments. From the biofilm hotspots, 15 isolates with strong biofilm forming ability were identified, including 7 Pseudomonas, 7 Acinetobacter, and 1 Psychrobacter. The Pseudomonas isolates exhibited the highest production of EPS components and three strong motilities, whose characteristics were positively correlated. Thus, this study verified the presence of multi-species biofilms in seafood processing environments, offering preliminary insights into the diversity of microbial communities during processing. It highlights potential contamination sources and emphasizes the importance of understanding biofilms composition to control biofilms formation in seafood processing environments.

Fabrication and characterization of sodium alginate/blueberry anthocyanins/hinokitiol loaded ZIF-8 nanoparticles composite films with antibacterial activity for monitoring pork freshness.

A smart film was developed to detect the freshness of pork by incorporating blueberry anthocyanins (BAs) and hinokitiol (HIN) loaded zeolite-imidazolium framework (HIN@ZIF-8) with into a sodium alginate matrix, and its microstructure and physicochemical properties were studied. The SA matrix was doped with BAs and HIN@ZIF-8 nanoparticles (SA-BAs/HIN@ZIF-8) to increase its tensile strength and reduce its water vapor permeability. HIN@ZIF-8 has low cytotoxicity, and SA-BAs/HIN@ZIF-8 membranes have long-lasting antimicrobial and highly sensitive color development properties against Escherichia coli and Staphylococcus aureus. The results of pork preservation experiments showed that SA-BA/HIN@ZIF-8 could extend the shelf life of pork to 6 days at 4 ℃. E-nose evaluation experiments showed that SA-BAs/HIN@ZIF-8 could inhibit compounds that cause unpleasant and irritating odours. Therefore, SA-BAs/HIN@ZIF-8 was considered to be an effective method to improve the freshness of pork, and the results showed that it has a promising application in food preservation.

Effect of chewing ability on in vivo oral digestive characteristics and in vitro gastrointestinal starch hydrolysis of three different types of cooked rice.

Chewing ability has a strong effect on food digestion. However, little is known about the relationship between the food mastication degree and the subsequent gastric emptying. This study was to explore the effects of individual chewing ability (strong and weak) on the in vivo oral processing characteristics and in vitro dynamic gastrointestinal starch hydrolysis of three types of rice (japonica rice, indica rice and waxy rice). Results showed that the swallowable bolus in the weak chewing group had larger holes and a looser microstructure with more small rice particles, while the strong chewing ones obtained a bolus with higher saliva content (up to 28%) and starch hydrolysis degree (up to 13.55%). Moreover, the gastric retention and starch hydrolysis of the strong chewing ability group were higher in the artificial gastric dynamic system (AGDS). The indica rice particles with the higher degree of fragmentation contacted enzymes easier and hydrolyzed quicker, thus emptying through the stomach faster (81.76%). However, the oral chewing properties of rice mainly influenced the starch digestion in the stomach and the initial stage of the small intestine (∼5 min). This study suggested that the chewing ability and rice variety can influence the bolus properties, which in turn affected the gastric emptying and the degree of starch hydrolysis during digestion.

Isolation, antimicrobial resistance and virulence characterization of Salmonella spp. from fresh foods in retail markets in Hangzhou, China.

Salmonella can cause severe foodborne diseases. This study investigated the prevalence of Salmonella spp. in fresh foods in Hangzhou market and their harborage of antibiotic resistance and virulence genes, antibiotic susceptibility, and pathogenicity. A total of 500 samples (pork, n = 140; chicken, n = 128; vegetable, n = 232) were collected over a one-year period. Salmonella was found in 4.2% (21) of samples with the detection rate in pork, chicken and vegetables as 4.3% (6), 6.3% (8), and 3% (7), respectively. One Salmonella strain was recovered from each positive sample. The isolates were identified as six serotypes, of which S. Enteritidis (n = 7) and S. Typhimurium (n = 6) were the most predominant serotypes. The majority of isolates showed resistance to tetracycline (85.7%) and/or ciprofloxacin (71.4%). Tetracycline resistance genes showed the highest prevalence (90.5%). The occurrence of resistance genes for ß-lactams (blaTEM-1, 66.7%; and blaSHV, 9.5%) and aminoglycosides (aadA1, 47.6%; Aac(3)-Ia, 19%) was higher than sulfonamides (sul1, 42.9%) and quinolones (parC, 38.1%). The virulence gene fimA was detected in 57.1% of isolates. Gene co-occurrence analysis implied that resistance genes were associated with virulence genes. Furthermore, selected S. Typhimurium isolates (n = 4) carrying different resistance and virulence genes up-regulated the secretions of cytokines IL-6 and IL-8 by Caco-2 cells in different degrees, suggesting that virulence genes may play a role in inflammatory transcription. In in vivo virulence test, microbiological counts in mouse feces and tissues showed that all included S. Typhimurium were able to infect mice, with one strain showing significantly higher virulence than others. In conclusion, this study indicates Salmonella contamination in fresh foods in Hangzhou market poses a risk to public health and it should be closely monitored to prevent and control foodborne diseases.

Characterization and fitness cost analysis of two plasmids carrying different subtypes of blaNDM in aquaculture farming.

In recent years, the blaNDM gene, which mediate resistance to carbapenems, has disseminated all over the world, and has also been detected in animals. Understanding the dissemination and accumulation of antibiotic resistance genes (ARGs) in a human-impacted environment is essential to solve the food safety problems caused by antibiotics. In this study, two strains of carbapenem bacteria carrying blaNDM were screened from 244 strains isolated from two T. sinensis farms in Zhejiang province, China. After their plasmids were isolated and sequenced, their structure and gene environment were analyzed and the mechanism of blaNDM gene transfer was explored. The study measured the fitness cost of plasmids carrying different blaNDM subtypes by four biological characteristics experiments. The results showed that the fitness cost of IncC plasmid carrying blaNDM-1 was higher than that of IncX3 plasmid carrying blaNDM-5. Furthermore, the real-time PCR showed that the decrease of transcription level of fitness-related genes lead to the different fitness cost of plasmids carrying different blaNDM subtypes. Fitness of many blaNDM-harboring plasmids enhanced the further dissemination of this gene and increase the risk of blaNDM gene spreading in aquatic environment, and thus further investigation of carbapenem-resistant bacterias among food animals are in urgent need.

Encapsulation of Salmonella phage SL01 in alginate/carrageenan microcapsules as a delivery system and its application in vitro.

Phages can be used successfully to treat pathogenic bacteria including zoonotic pathogens that colonize the intestines of animals and humans. However, low pH and digestive enzyme activity under harsh gastric conditions affect phage viability, thereby reducing their effectiveness. In this study, alginate (ALG)/κ-carrageenan (CG) microcapsules were developed to encapsulate and release phage under simulated gastrointestinal conditions. The effects of ALG and CG concentrations on the encapsulation and loading efficiency of microcapsules, as well as the release behavior and antibacterial effects of microcapsules in simulating human intestinal pH and temperature, were investigated. Based on various indicators, when the concentration of ALG and CG were 2.0 and 0.3%, respectively, the obtained microcapsules have high encapsulation efficiency, strong protection, and high release efficiency in simulated intestinal fluid. This effect is attributed to the formation of a more tightly packed biopolymer network within the composite microcapsules based on the measurements of their microstructure properties. Bead-encapsulation is a promising, reliable, and cost-effective method for the functional delivery of phage targeting intestinal bacteria.

The diversity in antimicrobial resistance of MDR Enterobacteriaceae among Chinese broiler and laying farms and two mcr-1 positive plasmids revealed their resistance-transmission risk.

This research aimed to investigate the microbial composition and diversity of antimicrobial resistance genes (ARGs) found in Chinese broiler and layer family poultry farms. We focused on the differences in resistance phenotypes and genotypes of multidrug-resistant Enterobacteriaceae (MDRE) isolated from the two farming environments and the existence and transmissibility of colistin resistance gene mcr-1. Metagenomic analysis showed that Firmicutes and Bacteroides were the dominant bacteria in broiler and layer farms. Many aminoglycoside and tetracycline resistance genes were accumulated in these environments, and their absolute abundance was higher in broiler than in layer farms. A total of 526 MDRE were isolated with a similar distribution in both farms. The results of the K-B test showed that the resistance rate to seven antimicrobials including polymyxin B and meropenem in broiler poultry farms was significantly higher than that in layer poultry farms (P ≤ 0.05). PCR screening results revealed that the detection rates of mcr-1, aph(3')Ia, aadA2, bla oxa-1 , bla CTX-M , fosB, qnrD, sul1, tetA, and catA1 in broiler source MDRE were significantly higher than those in layers (P ≤0.05). A chimeric plasmid p20432-mcr which carried the novel integron In1866 was isolated from broiler source MDRE. The high frequency of conjugation (10-1 to 10-3) and a wide range of hosts made p20432-mcr likely to play an essential role in the high detection rate of mcr-1, aph(3')-Ia, and aadA2 in broiler farms. These findings will help optimize disinfection and improve antimicrobial-resistant bacteria surveillance programs in poultry farms, especially broilers.

In vitro Digestion Characteristics of Hydrolyzed Infant Formula and Its Effects on the Growth and Development in Mice.

Infant formula, an important food for babies, is convenient and nutritious, and hydrolyzed formulas have attracted much attention due to their non-allergicity. However, it is uncertain whether hydrolyzed formulars cause obesity and other side effects in infants. Herein, three infant formulas, standard (sIF), partially hydrolyzed (pHIF), and extensively hydrolyzed (eHIF), were analyzed in an in vitro gastrointestinal digestion model. With increasing degree of hydrolysis, the protein moleculars, and allergenicity of the proteins decreased and the long-chain polyunsaturated fatty acid content increased. Moreover, the digestion model solutions quickly digested the small fat globules and proteins in the hydrolyzed formula, allowing it to become electrostatically stable sooner. The eHIF-fed mice presented larger body sizes, and exhibited excellent exploratory and spatial memory abilities in the maze test. Based on villus height and crypt depth histological characterizations and amplicon sequencing, eHIF promoted mouse small intestine development and changed the gut microbiota composition, eventually favoring weight gain. The mouse spleen index showed that long-term infant formula consumption might be detrimental to immune system development, and the weight-bearing swimming test showed that eHIF could cause severe physical strength decline. Therefore, long-term consumption of infant formula, especially eHIF, may have both positive and negative effects on mouse growth and development, and our results might shed light on feeding formula to infants.

Targeting early proximal-rod component substrate FlgB to FlhB for flagellar-type III secretion in Salmonella.

The Salmonella flagellar secretion apparatus is a member of the type III secretion (T3S) family of export systems in bacteria. After completion of the flagellar motor structure, the hook-basal body (HBB), the flagellar T3S system undergoes a switch from early to late substrate secretion, which results in the expression and assembly of the external, filament propeller-like structure. In order to characterize early substrate secretion-signals in the flagellar T3S system, the FlgB, and FlgC components of the flagellar rod, which acts as the drive-shaft within the HBB, were subject to deletion mutagenesis to identify regions of these proteins that were important for secretion. The ß-lactamase protein lacking its Sec-dependent secretion signal (Bla) was fused to the C-terminus of FlgB and FlgC and used as a reporter to select for and quantify the secretion of FlgB and FlgC into the periplasm. Secretion of Bla into the periplasm confers resistance to ampicillin. In-frame deletions of amino acids 9 through 18 and amino acids 39 through 58 of FlgB decreased FlgB secretion levels while deleting amino acid 6 through 14 diminished FlgC secretion levels. Further PCR-directed mutagenesis indicated that amino acid F45 of FlgB was critical for secretion. Single amino acid mutagenesis revealed that all amino acid substitutions at F45 of FlgB position impaired rod assembly, which was due to a defect of FlgB secretion. An equivalent F49 position in FlgC was essential for assembly but not for secretion. This study also revealed that a hydrophobic patch in the cleaved C-terminal domain of FlhB is critical for recognition of FlgB at F45.

Characterization and comparative genomics analysis of RepA_N multi-resistance plasmids carrying optrA from Enterococcus faecalis.

Introduction: This research aimed to investigate the antibiotic resistance of Enterococcus faecalis from swine farms in Zhejiang Province and the prevalence and transmission mechanism of oxazolidone resistance gene optrA. Method: A total of 226 Enterococcus faecalis were isolated and their resistance to 14 antibiotics was detected by broth microdilution. The resistance genes were detected by PCR. Results: The antibiotic resistance rate of 226 isolates to nearly 57% (8/14) of commonly used antibiotics was higher than 50%. The resistance rate of tiamulin was highest (98.23%), that of tilmicosin, erythromycin, tetracycline and florfenicol was higher than 80%, and that of oxazolidone antibiotic linezolid was 38.49%. The overall antibiotics resistance in Hangzhou, Quzhou and Jinhua was more serious than that in the coastal cities of Ningbo and Wenzhou. The result of PCR showed that optrA was the main oxazolidinone and phenicols resistance gene, with a detection rate of 71.68%, and optrA often coexisted with fexA in the isolates. Through multi-locus sequence typing, conjugation transfer, and replicon typing experiments, it was found that the horizontal transmission mediated by RepA_N plasmid was the main mechanism of optrA resistance gene transmission in E. faecalis from Zhejiang Province. Two conjugative multi-resistance plasmids carrying optrA, RepA_N plasmid pHZ318-optrA from Hangzhou and Rep3 plasmid from Ningbo, were sequenced and analyzed. pHZ318-optrA contain two multidrug resistance regions (MDR), which contributed to the MDR profile of the strains. optrA and fexA resistance genes coexisted in IS1216E-fexA-optrA-ferr-erm(A)-IS1216E complex transposon, and there was a partial sequence of Tn554 transposon downstream. However, pNB304-optrA only contain optrA, fexA and an insertion sequence ISVlu1. The presence of mobile genetic elements at the boundaries can possibly facilitate transfer among Enterococcus through inter-replicon gene transfer. Discussion: This study can provide theoretical basis for ensuring the quality and safety of food of animal origin, and provide scientific guidance for slowing down the development of multi-antibiotic resistant Enterococcus.

Characterization and Comparative Genomics Analysis of lncFII Multi-Resistance Plasmids Carrying bla CTX - M and Type1 Integrons From Escherichia coli.

This research aimed to investigate the presence and transferability of the extended-spectrum ß-lactamase resistance genes to identify the genetic context of multi-drug resistant (MDR) loci in two Escherichia coli plasmids from livestock and poultry breeding environment. MICs were determined by broth microdilution. A total of 137 E. coli resistant to extended-spectrum ß-lactam antibiotics were screened for the presence of the ESBL genes by PCR. Only two E. coli out of 206 strains produced carbapenemases, including strain 11011 that produced enzyme A, and strain 417957 that produced enzyme B. The genes were bla KPC and bla NDM , respectively. The plasmids containing bla CTX - M were conjugatable, and the plasmids containing carbapenem resistance gene were not conjugatable. Six extended-spectrum ß-lactamase resistance genes were detected in this research, including bla TEM, bla CTX - M, bla SHV, bla OAX - 1, bla KPC, and bla NDM , and the detection rates were 94.89% (130/137), 92.7% (127/137), 24.81% (34/137), 20.43% (28/137), 0.72% (1/137), and 0.72% (1/137), respectively. Two conjugative lncFII multi-resistance plasmids carrying bla CTX - M, p11011-fosA and p417957-CTXM, were sequenced and analyzed. Both conjugative plasmids were larger than 100 kb and contained three accessory modules, including MDR region. The MDR region of the two plasmids contained many antibiotic resistance genes, including bla CTX - M, mph (A), dfrA17, aadA5, sul1, etc. After transfer, both the transconjugants displayed elevated MICs of the respective antimicrobial agents. A large number of resistance genes clusters in specific regions may contribute to the MDR profile of the strains. The presence of mobile genetic elements at the boundaries can possibly facilitate transfer among Enterobacteriaceae through inter-replicon gene transfer. Our study provides beta-lactam resistance profile of bacteria, reveals the prevalence of ß-lactamase resistance genes in livestock and poultry breeding environment in Zhejiang Province, and enriches the research on IncFII plasmids containing bla CTX - M.

CRISPR-Cas systems are present predominantly on chromosome and its relationship with MEGs in Vibrio species.

Bacteria have developed diverse strategies to counteract virus predation, one of which is the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR associated (Cas) proteins immune defense system. In this study, the structure and function of the CRISPR-Cas system in 120 Vibrio strains were analyzed by bioinformatics methods, as well as the correlation between CRISPR and mobile genetic elements (MEGs). Only 61 Vibrio strains contained one or more CRISPR structures, and finally 102 CRISPRs were identified. The typical repeat size was 28 bp, and the total length of CRISPRs is nearly 60 bp, which was the most stable length of CRISPR in Vibrio strains. The types of CRISPR-Cas present in 61 strains were I-C, I-E, I-F, II-B, III-B, III-D and the rare type IV systems. Through principal component analysis, we found that Cas gene was most closely related to CRISPR. In addition, phages and plasmids were also highly correlated, showing negative correlation with CRISPR-Cas system. CRISPR-Cas predominantly present on chromosome within Vibrio while rarely in plasmids. Comparing the structural characteristics of plasmids containing CRISPR and without CRISPR, we found plasmid pMBL287 with CRISPR contained a bacteriophage f237, with more MGES, suggesting the diversity was greater. In addition, the same mobile genetic elements IS256 and ISL3 were found in the upstream and downstream of CRISPR. This study provides the prevalence, diversity and phylogenetic distribution of CRISPR-Cas in Vibrio, revealing which type of CRISPR-Cas system is predominant, and the factors affecting its function, as well as its relationship with mobile genetic elements.

Fitness cost and compensation mechanism of sulfonamide resistance genes (sul1, sul2, and sul3) in Escherichia coli.

The fitness cost of antibiotic resistance is a crucial factor to determine the evolutionary and transmission success of resistant bacteria. Exploring the fitness cost and compensation mechanism of antibiotic resistance genes (ARGs) in bacteria may effectively reduce the transmission of drug-resistant genes in the environment. Engineered bacteria with the same genetic background that carry sulfonamide resistance gene were generated to explore the fitness cost of sulfonamide resistance gene in Escherichia coli. There were significant differences in the protein expression of the two-component system pathway (fliZ, fliA, fliC and lrhA), folate biosynthesis pathway (sul1, sul2 and sul3), ABC transporter system (ugpC, rbsA and gsiA), and outer membrane pore protein OmpD through the comparative analysis of differential proteins compared to sensitive bacteria. Thus, we could speculate the possible fitness compensation mechanism. Finally, quantitative Real-time PCR (qRT-PCR) was used to verify the functions of some differential proteins at the transcriptional level. The fitness cost and compensatory evolution of antibiotic resistance are an essential part of bacterial evolution.

The Clustered Regularly Interspaced Short Palindromic Repeats-Associated System and Its Relationship With Mobile Genetic Elements in Klebsiella.

Microorganisms have developed many strategies in the process of long-term defense against external attacks, one of which is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) bacterial immunological system. In this study, the whole genome of 300 strains of Klebsiella was collected, the CRISPR-Cas system in the strains was statistically analyzed, and the types and structures of CRISPR system in Klebsiella were explored, as well as the correlation between CRISPR and mobile genetic elements (MGEs). Through principal component analysis (PCA), we found that Cas gene, plasmids, integron, IS1, IS609, and enzymes of DNA metabolism were closely related to CRISPR-Cas. Compared the structural characteristics of plasmids, the DinG family helicases, Cas6, Csf2, and IS5 were observed near the CRISPR loci in plasmid, which is also confirmed by the results of PCA that they may be important factors affecting the plasmid with CRISPR.

Milk phospholipid supplementation mediates colonization resistance of mice against Salmonella infection in association with modification of gut microbiota.

Gut microbiota-mediated colonization resistance against enteropathogens is known to be greatly influenced by bioactive food compounds. This work aims to investigate the effects of milk phospholipid (MP) supplementation on the colonization resistance of mice to Salmonella enterica serovar Typhimurium (S. Typhimurium) infection, with the focus mainly on the change of gut microbiota. Comparative microbiota analysis based on 16S rRNA gene sequence data of mice under different MP supplementation situations allowed us to identify specific microbiota characteristics associated with the varying degree of susceptibility to S. Typhimurium infection. We found that a moderate dietary intake of MPs (0.05 wt%) significantly increased the relative abundance of Bacteroides spp. (p < 0.05) and the propionate level (p < 0.05) in the mouse colon and enhanced colonization resistance against S. Typhimurium infection, when compared with the un-supplemented S. Typhimurium-infected mice, whereas excessive MP supplementation (0.25 wt%) did not significantly change the level of Bacteroides spp. (p > 0.05) and propionate (p > 0.05) and even enhanced the susceptibility and severity of S. Typhimurium infection. Furthermore, the inhibitory effects of Bacteroides spp. and propionate on S. Typhimurium intestinal colonization were verified in an ex vivo S. Typhimurium-infected 3D colonoid culture system. Our results showed that the supplementation of nutraceuticals may not always be the more the better, particularly under specific pathological conditions, and identification of specific gut microbiota characteristics may have the potential to become an indicator of appropriate supplementation in specific cases.

After In Vitro Digestion, Jackfruit Flake Affords Protection against Acrylamide-Induced Oxidative Damage.

Some studies have demonstrated that acrylamide (AA) was correlated with oxidative stress, resulting in physical damage. The jackfruit flake was an immature pulp that contained a high level of antioxidant activity. This study aimed to assess the defensive efficacy of jackfruit flake in AA-induced oxidative stress before and after simulated gastrointestinal digestion. Our results indicate that the total polyphenol content of Jackfruit flake digest (Digestive products of jackfruit flake after gastrointestinal, JFG) was diminished; however, JFG had raised the relative antioxidant capacity compared to Jackfruit flake extract (JFE). Additionally, the results of High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) implied that a proportion of compounds were degraded/converted into other unknown and/or undetected metabolites. Further, by high content analysis (HCA) techniques, JFG markedly reduced cytotoxicity and excessive production of reactive oxygen species (ROS) in cells, thereby alleviating mitochondrial disorders. In this study, it may be converted active compounds after digestion that had preferable protective effects against AA-induced oxidative damage.

Analysis of CRISPR/Cas system of Proteus and the factors affected the functional mechanism.

BACKGROUND: The Proteus is one of the most common human and animal pathogens. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR/Cas) are inheritable genetic elements found in a variety of archaea and bacteria in the evolution, providing immune function against foreign invasion. OBJECTIVES: To analyze the characteristics and functions of the CRISPR/Cas system in Proteus genomes, as well as the internal and external factors affecting the system. METHODS: CRISPR loci were identified and divided into groups based on the repeat sequence in 96 Proteus strains by identification. Compared the RNA secondary structure and minimum free energy of CRISPR loci through bioinformatics, the evolution of cas genes, and the effects of related elements were also discussed. RESULTS: 85 CRISPR loci were identified and divided into six groups based on the sequence of repeats, and the more stable the secondary structure of RNA, the smaller the minimum free energy, the fewer base mutations in the repeat, the more stable the CRISPR and the more complete the evolution of the system. In addition, Cas1 gene can be a symbol to distinguish species to some extent. Of all the influencing factors, CRISPR/Cas had the greatest impact on plasmids. CONCLUSIONS: This study examined the diversity of CRISPR/Cas system in Proteus and found statistically significant positive/negative correlations between variety factors (the RNA stability, free energy, etc.) and the CRISPR locus, which played a vital role in regulating the CRISPR/Cas system.

Comparative analysis of KPC-2-encoding chimera plasmids with multi-replicon IncR:IncpA1763-KPC:IncN1 or IncFIIpHN7A8:IncpA1763-KPC:IncN1.

BACKGROUND: IncR, IncFII, IncpA1763-KPC, and IncN1 plasmids have been increasingly found among Enterobacteriaceae species, but plasmids with hybrid structures derived from the above-mentioned incompatibility groups have not yet been described. METHODS: Plasmids p721005-KPC, p504051-KPC, and pA3295-KPC were fully sequenced and compared with previously sequenced related plasmids pHN84KPC (IncR), pKPHS2 (IncFIIK), pKOX_NDM1 (IncFIIY), pHN7A8 (IncFIIpHN7A8), and R46 (IncN1). RESULTS: The backbone of p721005-KPC/p504051-KPC was a hybrid of the entire 10-kb IncR-type backbone from pHN84KPC, the entire 64.3-kb IncFIIK-type maintenance, and conjugal transfer regions from pKPHS2, a 15.5-kb IncFIIY-type maintenance region from pKOX_NDM1 and a 5.6-kb IncpA1763-KPC-type backbone region from pA1763-KPC, and it contained a primary IncR replicon and two auxiliary IncpA1763-KPC and IncN1 replicons. The backbone of pA3295-KPC was a hybrid of a 7.2-kb IncFIIpHN7A8-type backbone region from pHN7A8, the almost entire 33.3-kb IncN1-type maintenance and conjugal transfer regions highly similar to R46, a 26.2-kb IncFIIK-type maintenance regions from pKPHS2, the above 15.5-kb IncFIIY-type maintenance region, and the above 5.6-kb IncpA1763-KPC-type backbone region, and it contained a primary Inc-FIIpHN7A8 replicon and two auxiliary IncpA1763-KPC and IncN1 replicons. Each of p721005-KPC, p504051-KPC, and pA3295-KPC acquired a wealth of accessory modules, carrying a range of intact and residue mobile elements (such as insertion sequences, unit transposons, and integrons) and resistance markers (such as bla KPC, tetA, dfrA, and qnr). CONCLUSION: In each of p721005-KPC, p504051-KPC, and pA3295-KPC, multiple replicons in coordination with maintenance and conjugation regions of various origins would maintain a broad host range and a stable replication at a steady-state plasmid copy number.

Synergistic Effects of The Enhancements to Mitochondrial ROS, p53 Activation and Apoptosis Generated by Aspartame and Potassium Sorbate in HepG2 Cells.

The safety of food additives has been widely concerned. Using single additives in the provisions of scope is safe, but the combination of additives, may induce additive, synergy, antagonism and other joint effects. This study investigated the cytotoxicity of aspartame (AT) together with potassium sorbate (PS). Thiazolyl Blue Tetrazolium Bromide (MTT) assay indicated that AT and PS had IC50 values of 0.48 g/L and 1.25 g/L at 24 h, respectively. High content analysis (HCA) showed that both AT and PS had a negative effect on mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and DNA damage while the joint group behaved more obviously. The biochemical assays revealed typical cell morphological changes and the activation of cytochrome c and caspase-3 verified apoptosis induced by AT together with PS. With dissipation of MMP and increase of cell membrane permeability (CMP), it indicated AT together with PS-induced apoptosis was mediated by mitochondrial pathway. Meanwhile, p53 were involved in DNA damage, and the ratio of Bax/Bcl-2 was increased. Moreover, excessive ROS induced by AT together with PS is a key initiating factor for apoptosis. All these results proved that p53 was involved in apoptosis via mitochondria-mediated pathway and the process was regulated by ROS.

Inactivation of Vibrio parahaemolyticus by Aqueous Ozone.

Vibrio parahaemolyticus contamination causes serious foodborne illness and has become a global health problem. As a disinfectant, aqueous ozone can effectively kill a number of bacteria, viruses, parasites, and other microorganisms. In this study, three factors, namely, the aqueous ozone concentration, the exposure time, and the bacterial density, were analyzed by response surface methodology, and the aqueous ozone concentration was the most influential factor in the sterilization ratio. Under low aqueous ozone concentrations (less than 0.125 mg/l), the bacterial cell membranes remained intact, and the ozone was detoxified by intracellular antioxidant enzymes (e.g., superoxide dismutase and catalase). Under high aqueous ozone concentrations (more than 1 mg/l), cell membranes were damaged by the degree of peripheral electronegativity at the cell surface and the concentration of lactate dehydrogenase released into the extracellular space, and the ultrastructures of the cells were confirmed by transmission electron microscopy. Aqueous ozone penetrated the cells through leaking membranes, inactivated the enzymes, inhibited almost all the genes, and degraded the genetic materials of gDNA and total RNA, which eventually led to cell death.