Characterization and complete genome analysis of Bacillus velezensis CB6 revealed ATP synthase subunit α against foodborne pathogens.

Given the serious threat of foodborne multidrug-resistant bacteria to animals and humans, finding an effective antibacterial compound has always been an important topic for scientists. Here, from the soil of Changbaishan, we have identified a bacterium that can inhibit the growth of Staphylococcus aureus. Nr genome database analysis and phylogenetic analysis showed that strain CB6 belongs to Bacillus velezensis. We found that the crude extract of strain CB6 has broad-spectrum antibacterial activity against foodborne pathogens. In addition, we showed that the crude extract loses antibacterial activity after treatment with papain. Next, strain CB6 was purified using ammonium sulfate precipitation, a Sephadex G-75 gel filtration column and high-performance liquid chromatography system (HPLC). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis indicated that the antibacterial compound was the protein ATP synthase subunit α (ATP-1), with a molecular weight of 55.397 KDa. Moreover, we reported the complete genome sequence of strain CB6, which is composed of a unique circular 3,963,507 bp chromosome with 3749 coding genes and a G + C content of 46.53%. The genome contained 12 gene clusters with antibacterial functions, which constituted over 20.947% of the complete genome. Of note, the amino acid sequence encoding the ATP-1 protein in the strain CB6 genome was identified. In addition to these findings, we speculate that the ATP-1 protein may provide energy for secondary metabolites, which in turn will improve the antibacterial activity of the secondary metabolites. All the above important features make the ATP-1 as a potential candidate for the development of new antibacterial drugs and food preservatives in the future.

An effective live attenuated vaccine against Aeromonas veronii infection in the loach (Misgurnus anguillicaudatus).

Aeromonas veronii is a major pathogenic bacterium in humans and animals. When it causes outbreaks, there are enormous economic losses to the aquaculture industry. An effective live attenuated vaccine strain, ΔhisJ, was obtained in our previous studies by gene knockout in Aeromonas veronii TH0426 using the suicide vector pRE112. Here, we evaluated whether the live attenuated vaccine ΔhisJ was suitable for prevention of Aeromonas veronii infection by injection and immersion in loaches. Compared with that of the TH0426 wild-type strain, the virulence of the live vaccine was significantly weakened. Vaccine safety assessment results also indicated that 1 × 107 CFU/mL live vaccine was safe and did not induce clinical symptoms or obvious pathological changes. Additionally, after challenging loaches with Aeromonas veronii TH0426, the relative percent survival of the IN3 injection group was 65.66%, and that of the IM group was 50.78%. Our data show that the live attenuated vaccine ΔhisJ can improve the immune protection rate of loaches. Furthermore, increased enzyme activity parameters (SOD, LZM, ACP, and AKP) in the skin mucus, increased enzyme activity parameters (SOD, LZM, ACP, AKP, and GPx) in the serum, increased specific IgM antibodies and cytokine IL-1ß contents in the serum, and increased cytokine (IL-15, pIgR, IL-1ß, and TNF-α) expression in the liver and spleen were observed. These data are the first to indicate that the live attenuated vaccine ΔhisJ is suitable for the development of a safe and effective vaccine against Aeromonas veronii infection in loach aquaculture.

Analysis of an IncR plasmid carried by carbapenem-resistant Klebsiella pneumoniae: A survey of swine Klebsiella pneumoniae in Jilin Province.

OBJECTIVES: This study was conducted in Jilin Province to investigate the mechanism involved in the antibiotic resistance and pathogenicity of Klebsiella pneumoniae. METHODS: Lung samples were collected from large-scale pig farms in Jilin Province. Antimicrobial susceptibility and mouse lethality assays were carried out. K. pneumoniae isolate JP20, with high virulence and antibiotic resistance, was chosen for whole-genome sequencing. The complete sequence of its genome was annotated, and the virulence and antibiotic resistance mechanism were analysed. RESULTS: A total of 32 K. pneumoniae strains were isolated and tested for antibiotic resistance and pathogenicity. Among them, the JP20 strain showed high levels of resistance to all tested antimicrobial agents and strong pathogenicity in mice (lethal dose of 1.35 × 1011 CFU/mL). Sequencing of the multidrug-resistant and highly virulent K. pneumoniae JP20 strain revealed that the antibiotic resistance genes were mainly carried by an IncR plasmid. We speculate that extended-spectrum ß-lactamases and loss of outer membrane porin OmpK36 play an important role in carbapenem antibiotic resistance. This plasmid contains a mosaic structure consisting of a large number of mobile elements. CONCLUSION: Through genome-wide analysis, we found that an lncR plasmid carried by the JP20 strain may have evolved in pig farms, possibly leading to multidrug resistance in the JP20 strain. It is speculated that the antibiotic resistance of K. pneumoniae in pig farms is mainly mediated by mobile elements (insertion sequences, transposons, and plasmids). These data provide a basis for monitoring the antibiotic resistance of K. pneumoniae and lay a foundation for an improved understanding of the genomic characteristics and antibiotic resistance mechanism of K. pneumoniae.

Transcriptomic Changes and satP Gene Function Analysis in Pasteurella multocida with Different Levels of Resistance to Enrofloxacin.

Pasteurella multocida (Pm) is one of the major pathogens of bovine respiratory disease (BRD), which can develop drug resistance to many of the commonly used antibiotics. Our earlier research group found that with clinical use of enrofloxacin, Pm was more likely to develop drug resistance to enrofloxacin. In order to better understand the resistance mechanism of Pm to enrofloxacin, we isolated PmS and PmR strains with the same PFGE typing in vitro, and artificially induced PmR to obtain the highly resistant phenotype, PmHR. Then transcriptome sequencing of clinically isolated sensitive strains, resistant and highly drug-resistant strains, treated with enrofloxacin at sub-inhibitory concentrations, were performed. The satP gene, of which the expression changed significantly with the increase in drug resistance, was screened. In order to further confirm the function of this gene, we constructed a satP deletion (ΔPm) strain using suicide vector plasmid pRE112, and constructed the C-Pm strain using pBBR1-MCS, and further analyzed the function of the satP gene. Through a continuously induced resistance test, it was found that the resistance rate of ΔPm was obviously lower than that of Pm in vitro. MDK99, agar diffusion and mutation frequency experiments showed significantly lower tolerance of ΔPm than the wild-type strains. The pathogenicity of ΔPm and Pm was measured by an acute pathogenicity test in mice, and it was found that the pathogenicity of ΔPm was reduced by about 400 times. Therefore, this study found that the satP gene was related to the tolerance and pathogenicity of Pm, and may be used as a target of enrofloxacin synergistic effect.

Evaluation of structural, functional, and anti-oxidant potential of differentially extracted polysaccharides from potatoes peels.

Potato peel was used for the extraction of three types of polysaccharides (PW, PAL, and PAC) using water, alkaline, and acid treatments, respectively. The structure of the PP polysaccharides was examined by means of Fourier transform infrared spectroscopy (FT-IR) analysis and Gas chromatography-mass spectrometry (GC-MS). The results suggest that the extracted polysaccharides constituted essentially three functional groups: CO, CH, and OH. The polysaccharides were comprised of low proportions of proteins, 17-23% uronic acids, and approximately 70% carbohydrates. PAL, PW, and PAC with molecular weights of 2.25 × 103, 2.18 × 103, and 1.92 × 103 kDa, respectively, were composed of rhamnose, xylose, mannose, arabinose, glucose, and galactose. Functional properties (solubility, oil holding capacity, foaming, and emulsion properties) of these polysaccharides were evaluated. Among three, PAL showed the highest fat-binding capacity which was 7.50 g/g with the solubility of 95.06%. The three polysaccharides possessed appreciable in vitro anti-oxidant (scavenging DPPH and ABTS+ radicals, chelating ferrous ions, and reducing power) potential. PAL exhibited the strongest reducing power, scavenging activity on DPPH radicals and chelating capability on ferrous ions. PP polysaccharides can be used as promising natural antioxidants in food, pharmaceutical, and cosmetic preparations.

The rheological properties of differentially extracted polysaccharides from potatoes peels.

The effects of concentration, temperature, pH, and salt ion concentration on the apparent viscosity of potato peel (PP) polysaccharide solutions were studied. The results showed that the PP polysaccharides exhibited the characteristics of "non-Newtonian fluid". The apparent viscosity of the PP polysaccharides decreased under acidic, alkaline conditions, and with the increasing temperature. At the increasing concentration of PAC with the addition of four ion concentrations, the apparent viscosity was decreased gradually. The apparent viscosity of PAL increased with four ion concentrations, while that of PW increased initially with the rising concentration of Na+, Mg2+, and K+ and then decreased. Addition of Ca2+ lowered the apparent viscosity initially which was followed by further increase. The results of dynamic rheological experiment showed that the loss modulus G″ of PP polysaccharide was higher than the storage modulus G' at lower frequencies, and vice versa at higher frequencies. With the increasing concentration of PP polysaccharides, the values of the intersection of G' and G″ were lowered. The results showed that the rheological properties of PP polysaccharide solutions were affected by concentration, temperature, pH, and salt ions.

In vitro Prebiotic Effects of Bamboo Shoots and Potato Peel Extracts on the Proliferation of Lactic Acid Bacteria Under Simulated GIT Conditions.

The present study explored the possible prebiotic application of potato peel and bamboo shoot extracts for the proliferation of lactic acid bacteria (LAB) from diverse niches and their tolerance ability to simulated gastrointestinal tract (GIT) conditions was also examined. Initially, the complete 16S rDNA sequencing of selected isolates revealed them as Lactobacillus paracasei (6), Staphylococcus simulans (2), and Streptococcus thermophilus (1). Higher cell densities and rapid pH change were obtained from cultured media supplemented with BS (2%) and PP (2%) as a carbon source. Their higher tolerance and the lowest reducing sugar abilities were obtained for BS at pH 2.5 and 9.0, while at pH 3.5 and 8.0 for PP. The isolates were screened for additional functional and technological properties to harvest the most appropriate starter. The selected isolates harbored promising functional properties such as amylase presence, cell surface hydrophobicity, autoaggregation, proteolytic and lipolytic activity, antifungal action, as well as exopolysaccharide production. On the basis of these attributes, microencapsulated strain K3 was found resistant to gastrointestinal conditions after 2 h, resulting in significantly (p ≤ 0.05) improved survival compared to non-capsulated strain. The current approach presents an interesting economical strategy to modulate LAB through supplementation of plant-derived carbon sources as well as to enhance their survival under GIT.