Establishment of LAMP-CRISPR/Cas12a for rapid detection of Escherichia coli O157:H7 and one-pot detection.

Escherichia coli O157:H7 is a pathogenic serotype of Escherichia coli. Consumption of food contaminated with E. coli O157:H7 could cause a range of diseases. Therefore, it is of great importance to establish rapid and accurate detection methods for E. coli O157:H7 in food. In this study, based on LAMP and combined with the CRISPR/cas12a system, a sensitive and specific rapid detection method for E. coli O157:H7 was established, and One-Pot detection method was also constructed. The sensitivity of this method could stably reach 9.2 × 10° CFU/mL in pure culture, and the whole reaction can be completed within 1 h. In milk, E. coli O157:H7 with an initial contamination of 7.4 × 10° CFU/mL only needed to be cultured for 3 h to be detected. The test results can be judged by the fluorescence curve or by visual observation under a UV lamp, eliminating instrument limitations and One-Pot detection can effectively prevent the problem of false positives. In a word, the LAMP-CRISPR/cas12a system is a highly sensitive and convenient method for detecting E. coli O157:H7.

Transcriptomic analysis reveals that Bacillomycin D-C16 induces multiple pathways of disease resistance in cherry tomato.

BACKGROUND: Bacillomycin D-C16 can induce resistance in cherry tomato against pathogens; however, the underlying molecular mechanism is poorly understood. Here, the effect of Bacillomycin D-C16 on induction of disease resistance in cherry tomato was investigated using a transcriptomic analysis. RESULTS: Transcriptomic analysis revealed a series of obvious enrichment pathways. Bacillomycin D-C16 induced phenylpropanoid biosynthesis pathways and activated the synthesis of defense-related metabolites including phenolic acids and lignin. Moreover, Bacillomycin D-C16 triggered a defense response through both hormone signal transduction and plant-pathogen interactions pathways, and increased the transcription of several transcription factors (e.g., AP2/ERF, WRKY and MYB). These transcription factors might contribute to the further activated the expression of defense-related genes (PR1, PR10 and CHI) and stimulated the accumulation of H2O2. CONCLUSION: Bacillomycin D-C16 can induce resistance in cherry tomato by activating the phenylpropanoid biosynthesis pathway, hormone signal transduction pathway and plant-pathogen interactions pathway, thus activating comprehensive defense reaction against pathogen invasion. These results provided a new insight into the bio-preservation of cherry tomato by the Bacillomycin D-C16.

The global regulator SpoVG regulates Listeria monocytogenes biofilm formation.

Biofilms provide a suitable environment for L. monocytogenes and are the cause of enormous risks in the food industry. SpoVG is a global regulatory factor that plays a vital role in physiological activity of L. monocytogenes. We constructed spoVG mutant strains to investigate the effects of these mutants on L. monocytogenes biofilms. The results show that L. monocytogenes biofilm formation was decreased by 40%. Furthermore, we measured biofilm related phenotypes to study the regulation of SpoVG. The motility capacity of L. monocytogenes was found to decrease after the deletion of spoVG. The cell surface properties changed in the spoVG mutant strains, with an increase in both the cell surface hydrophobicity and the auto-aggregation capacity after spoVG deletion. SpoVG mutant strains were found to be more sensitive to antibiotics, and had a reduced tolerance to inappropriate pH, salt stress and low temperature. The RT-qPCR results showed that SpoVG effectively regulated the expression of genes related to quorum sensing, flagella, virulence and stress factors. These findings suggest that spoVG has potential as a target to decrease biofilm formation and control L. monocytogenes contamination in the food industry.

Biochemical and molecular regulatory mechanism of the pgpH gene on biofilm formation in Listeria monocytogenes.

AIMS: PgpH gene has an important regulatory role on bacterial physiological activity, but studies on its regulation mechanism on biofilm formation of Listeria monocytogenes are lacking. Our aim was to investigate the effect of pgpH gene deletion on biofilm formation in L. monocytogenes. METHODS AND RESULTS: The ΔpgpH deletion strain of L. monocytogenes LMB 33 426 was constructed by homologous recombination. Deletion of the pgpH gene resulted in a significant reduction in biofilm formation. The swimming ability of the ΔpgpH strain on semisolid plates was unchanged compared to the wild-type strain (WT), and the auto-aggregation capacity of L. monocytogenes was decreased. RNA-seq showed that ΔpgpH resulted in the differential expression of 2357 genes compared to WT. pgpH inactivation resulted in the significant downregulation of the cell wall formation-related genes dltC, dltD, walK, and walR and the flagellar assembly related genes fliG and motB. CONCLUSIONS: This study shows that the deletion of pgpH gene regulates biofilm formation and auto-aggregation ability of L. monocytogenes by affecting the expression of flagellar assembly and cell wall related genes. pgpH has a global regulatory effect on biofilm formation in L. monocytogenes.

Effects of the deletion and substitution of thioesterase on bacillomycin D synthesis.

OBJECTIVES: The importance of thioesterase domains on bacillomycin D synthesis and the ability of different thioesterase domains to selectively recognize and catalyze peptide chain hydrolysis and cyclization were studied by deleting and substituting thioesterase domains. RESULTS: No bacillomycin D analogs were found in the thioesterase-deleted strain fmbJ-ΔTE, indicating that the TE domain was essential for bacillomycin D synthesis. Then the thioesterase in bacillomycin D synthetases was replaced by the thioesterase in bacillomycin F, iturin A, mycosubtilin, plipastatin and surfactin synthetases. Except for fmbJ-S-TE, all others were able to synthesize bacillomycin D homologs because a suitable recombination site was selected, which maintained the integrity of NRPSs. In particular, the yield of bacillomycin D in fmbJ-IA-TE, fmbJ-M-TE and fmbJ-P-TE was significantly increased. CONCLUSION: This study expands our understanding of the TE domain in bacillomycin D synthetases and shows that thioesterase has excellent potential in the chemical-enzymatic synthesis of natural products or their analogs.

Establishment of real-time fluorescence and visual LAMP for rapid detection of Escherichia coli O157:H7 and kits construction.

Escherichia coli O157:H7 is a common pathogenic bacterium in food and water that can pose a threat to human health. The aim of this study was to develop loop-mediated isothermal amplification (LAMP) method for the detection of E. coli O157:H7 in food based on the specific gene Ecs_2840 and to construct rapid detection kits based on the established methods. Specifically, we established two methods of real-time fluorescent LAMP (RT-LAMP) and visual LAMP with calcein as an indicator. In pure bacterial culture, the cell sensitivity and genomic sensitivity of the RT-LAMP kit were 8.8 × 100 CFU ml-1 and 4.61 fg µl-1, respectively. The sensitivity of the visual LAMP kit was 2.35 × 100 CFU ml-1 and 4.61 fg µl-1. Both kits had excellent specificity and anti-interference performance. In addition, milk inoculated with 2.26 × 100 CFU ml-1E. coli O157:H7 could be detected within the reaction time after enrichment for 3 h. The results showed that the LAMP kits were rapid, sensitive, and specific for the detection of E. coli O157:H7 in food and had good application prospects in food safety surveillance.

Effect and regulation of fatty acids on bacillomycin D synthesis.

Bacillomycin D is a cyclic antimicrobial lipopeptide that has excellent antifungal effects, but its application is limited due to its low yield. At present, it is not clear whether fatty acids regulate the synthesis of bacillomycin D. Therefore, the effects of nine fatty acids on the yield of bacillomycin D produced by Bacillus amyloliquefaciens fmbJ were studied. The results showed that sodium propionate, propionic acid, and butyric acid could increase the yield of bacillomycin D by 44, 40, and 10%, respectively. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression levels of bacillomycin D synthesis gene, signaling factors and genes related to fatty acid metabolism, so as to explore the mechanism of sodium propionate regulating bacillomycin D synthesis. In conclusion, sodium propionate could accelerate the tricarboxylic acid cycle and promoted spore formation, cell movement, the secretion of extracellular protease and the transcription of bacillomycin D synthesis gene by upregulating the expression of signal factors degU, degQ, sigH, sigM and spo0A and ultimately promoted the synthesis of bacillomycin D. In this study, the mechanism of sodium propionate increasing bacillomycin D production was explored from multiple perspectives, which provided theoretical support for the large-scale production of bacillomycin D and was expected to promote its wide application in food, agriculture and medicine fields.

Acetate and auto-inducing peptide are independent triggers of quorum sensing in Lactobacillus plantarum.

The synthesis of plantaricin in Lactobacillus plantarum is regulated by quorum sensing. However, the nature of the extra-cytoplasmic (EC) sensing domain of the histidine kinase (PlnB1) and the ability to recognize the auto-inducing peptide PlnA1 is not known. We demonstrate the key motif Ile-Ser-Met-Leu of auto-inducing peptide PlnA1 binds to the hydrophobic region Phe-Ala-Ser-Gln-Phe of EC loop 2 of PlnB1 via hydrophobic interactions and hydrogen bonding. Moreover, we identify a new inducer, acetate, that regulates the synthesis of plantaricin by binding to a positively charged region (Arg-Arg-Tyr-Ser-His-Lys) in loop 4 of PlnB1 via electrostatic interaction. The side chain of Phe143 on loop 4 determined the specificity and affinity of PlnB1 to recognize acetate. PlnA1 activates quorum sensing in log phase growth and acetate in stationary phase to maintain the synthesis of plantaricin under conditions of reduced growth. Acetate activation of PlnB was also evident in four types of PlnB present in different Lb. plantarum strains. Finally, we proposed a model to explain the developmental regulation of plantaricin synthesis by PlnA and acetate. These results have potential applications in improving food fermentation and bacteriocin production.

Plantaricin A reverses resistance to ciprofloxacin of multidrug-resistant Staphylococcus aureus by inhibiting efflux pumps.

Overexpression of Staphylococcus aureus efflux pumps is commonly associated with antibiotic resistance, causing conventional antibiotics to be unsuccessful in combating multidrug-resistant bacterial infections. Reducing the activity of the efflux pump is an urgently required to tackle this problem. Here, we found that plantaricin A (PlnA), an antimicrobial peptide derived from Lactobacillus plantarum, had a synergistic effect with ciprofloxacin (CIP), reducing the IC90 of CIP by eight times. Subsequently, changes in membrane permeability, membrane potential, and reactive oxygen species (ROS) were determined; changes that did not explain the synergistic effect were previously observed. Ethidium bromide intake and efflux experiments showed that PlnA inhibited the function of the efflux pump by binding it and altering the structure of MepA, NorA, and LmrS. Then, a series of PlnA mutants were designed to explore the underlying mechanism; they showed that the charge and foaming of PlnA were the predominant factors affecting the structure of NorA. In a skin wound infection model, PlnA significantly reduced the dose of CIP, relieved inflammation, and promoted wound healing, indicating that PlnA and CIP synergy persisted in vivo. Overall, PlnA reduced the use of CIP for combination therapy, and allowing the continued used of CIP to kill MDR S. aureus. Multidrug-resistant Staphylococcus aureus threatens our life as a tenacious pathogen, which causes infections in hospitals, communities and animal husbandry. Various studies have showed that efflux pump inhibitors (EPIs) have been considered potential therapeutic agents for rejuvenating the activity of antibiotics. Unfortunately, small molecule EPIs exhibit several side effects that limit their use for clinical application. The present study showed a new EPI (plantaricin A) produced by Lactobacillus plantarum, which has low cytotoxicity and haemolysis and powerful inhibitory activity on efflux pumps. Therefore, it helps the design of new EPIs and controls the infection of MDR S. aureus.

Plantaricin A, Derived from Lactiplantibacillus plantarum, Reduces the Intrinsic Resistance of Gram-Negative Bacteria to Hydrophobic Antibiotics.

The outer membrane of Gram-negative bacteria is one of the major factors contributing to the development of antibiotic resistance, resulting in a lack of effectiveness of several hydrophobic antibiotics. Plantaricin A (PlnA) intensifies the potency of antibiotics by increasing the permeability of the bacterial outer membrane. Moreover, it has been proven to bind to the lipopolysaccharide of Escherichia coli via electrostatic and hydrophobic interactions and to interfere with the integrity of the bacterial outer membrane. Based on this mechanism, we designed a series of PlnA1 analogs by changing the structure, hydrophobicity, and charge to enhance their membrane-permeabilizing ability. Subsequent analyses revealed that among the PlnA1 analogs, OP4 demonstrated the highest penetrating ability, weaker cytotoxicity, and a higher therapeutic index. In addition, it decelerated the development of antibiotic resistance when the E. coli cells were continuously exposed to sublethal concentrations of erythromycin and ciprofloxacin for 30 generations. Further in vivo studies in mice with sepsis showed that OP4 heightens the potency of erythromycin against E. coli and relieves inflammation. In summary, our results showed that the PlnA1 analogs investigated in the present study, especially OP4, reduce the intrinsic antibiotic resistance of Gram-negative pathogens and expand the antibiotic sensitivity spectrum of hydrophobic antibiotics in Gram-negative bacteria. IMPORTANCE Antibiotic resistance is a global health concern due to indiscriminate use of antibiotics, resistance transfer, and intrinsic resistance of certain Gram-negative bacteria. The asymmetric bacterial outer membrane prevents the entry of hydrophobic antibiotics and renders them ineffective. Consequently, these antibiotics could be employed to treat infections caused by Gram-negative bacteria, after increasing their outer membrane permeability. As PlnA reportedly penetrates outer membranes, we designed a series of PlnA1 analogs and proved that OP4, one of these antimicrobial peptides, effectively augmented the permeability of the bacterial outer membrane. Furthermore, OP4 effectively improved the potency of erythromycin and alleviated inflammatory responses caused by Escherichia coli infection. Likewise, OP4 curtailed antibiotic resistance development in E. coli, thereby prolonging exposure to sublethal antibiotic concentrations. Thus, the combined use of hydrophobic antibiotics and OP4 could be used to treat infections caused by Gram-negative bacteria by decreasing their intrinsic antibiotic resistance.

An endolysin Salmcide-p1 from bacteriophage fmb-p1 against gram-negative bacteria.

AIMS: A novel endolysin Salmcide-p1 was developed as a promising candidate of new preservative and a supplement to effective enzyme preparations against gram-negative bacterial contaminations. METHODS AND RESULTS: Salmcide-p1 was identified by complementing the genomic sequence of a virulent Salmonella phage fmb-p1. Salmcide-p1 of 112 µg ml-1 could quickly kill Salmonella incubated with 100 mmol l-1 EDTA, with no haemolytic activity. Meanwhile, Salmcide-p1 had a high activity of lysing Salmonella cell wall peptidoglycan. At different temperatures (4-75°C), pH (4-11) and NaCl concentration (10-200 mmol l-1 ), the relative activity of Salmcide-p1 was above 60%. At 4°C, the combination of Salmcide-p1 and EDTA-2Na could inhibit the number of Salmonella Typhimurium CMCC 50115 in skim milk to less than 4 log CFU ml-1 by 3 days, and the number of Shigella flexneri CMCC 51571 was lower than 4 log CFU ml-1 by 9 days. CONCLUSIONS: Salmcide-p1 had a wide bactericidal activity against gram-negative bacteria and showed a broader anti-Salmonella spectrum than the phage fmb-p1. The combination strategy of Salmcide-p1 and EDTA-2Na could significantly inhibit the growth of gram-negative bacteria inoculated in skim milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophage endolysin as an antibacterial agent is considered to be a new strategy against bacterial contamination.

A duplex real-time NASBA assay targeting a serotype-specific gene for rapid detection of viable Salmonella Paratyphi C in retail foods of animal origin.

Salmonella enterica serovar Paratyphi C is highly adapted to humans and can cause a typhoid-like disease with high mortality rates. In this study, three serovar-specific genes were identified by comparative genomics for Salmonella Paratyphi C, SPC_0871, SPC_0872, and SPC_0908. Based on the SPC_0908 and xcd genes for testing Salmonella spp., we developed a duplex real-time nucleic acid sequence-based amplification (real-time NASBA) with a molecular beacon approach for the simultaneous detection of viable cells of Salmonella spp. and serotype Paratyphi C. The test selectively and consistently detected 53 Salmonella spp. (representing 31 serotypes) and 18 non-Salmonella strains. Additionally, the method showed high resistance to interference from natural background flora in pork and chicken samples. The sensitivity of the established approach was determined to be 4.89 cfu/25 g in artificially contaminated pork and chicken samples after pre-enrichment. We propose this NASBA-based protocol as a potential detection method for Salmonella spp. and serotype Paratyphi C in foods of animal origin.

Comparison of osteogenesis of bovine bone xenografts between true bone ceramics and decalcified bone matrix.

Xenograft bone scaffolds have certain advantages such as mechanical strength, osteoinductive properties, sufficient source and safety. This study aimed to compare osteogenesis of the two main bovine bone xenografts namely true bone ceramics (TBC) and decalcified bone matrix (DBM), and TBC or DBM combined with bone morphogenetic protein (BMP)-2 (TBC&BMP-2 and DBM&BMP-2). The characteristics of TBC and DBM were investigated by observing the appearance and scanning electron microscopic images, examining mechanical strength, evaluating cytotoxicity and detecting BMP-2 release after being combined with BMP-2 in vitro. The femoral condyle defect and radial defect models were successively established to evaluate the performance of the proposed scaffolds in repairing cortical and cancellous bone defects. General observation, hematoxylin and eosin (HE) staining, mirco-CT scanning, calcein double labeling, X-ray film observation, three-point bending test in vivo were then performed. It indicated that the repair with xenograft bone scaffolds of 8 weeks were needed and the repair results were better than those of 4 weeks whatever the type of defects. To femoral condyle defect, TBC and TBC&BMP-2 were better than DBM and DBM&BMP-2, and TBC&BMP-2 was better than TBC alone; to radial defect, DBM and DBM&BMP-2 were better than TBC and TBC&BMP-2, and DBM&BMP-2 was better than DBM alone. This study has shown that TBC and DBM xenograft scaffolds can be more suitable for the repair of cancellous bone and cortical bone defects for 8 weeks in rats, respectively. We also have exhibited the use of BMP-2 in combination with DBM or TBC provides the possibility to treat bone defects more effectively. We thus believe that we probably need to select the more suitable scaffold according to bone defect types, and both TBC and DBM are promising xenograft materials for bone tissue engineering and regenerative medicine. Graphical abstract.

Thermostability Improvement of L-Asparaginase from Acinetobacter soli via Consensus-Designed Cysteine Residue Substitution.

To extend the application range of L-asparaginase in food pre-processing, the thermostability improvement of the enzyme is essential. Herein, two non-conserved cysteine residues with easily oxidized free sulfhydryl groups, Cys8 and Cys283, of Acinetobacter soli L-asparaginase (AsA) were screened out via consensus design. After saturation mutagenesis and combinatorial mutation, the mutant C8Y/C283Q with highly improved thermostability was obtained with a half-life of 361.6 min at 40 °C, an over 34-fold increase compared with that of the wild-type. Its melting temperature (Tm) value reaches 62.3 °C, which is 7.1 °C higher than that of the wild-type. Molecular dynamics simulation and structure analysis revealed the formation of new hydrogen bonds of Gln283 and the aromatic interaction of Tyr8 formed with adjacent residues, resulting in enhanced thermostability. The improvement in the thermostability of L-asparaginase could efficiently enhance its effect on acrylamide inhibition; the contents of acrylamide in potato chips were efficiently reduced by 86.50% after a mutant C8Y/C283Q treatment, which was significantly higher than the 59.05% reduction after the AsA wild-type treatment. In addition, the investigation of the mechanism behind the enhanced thermostability of AsA could further direct the modification of L-asparaginases for expanding their clinical and industrial applications.

L. johnsonii, L. plantarum, and L. rhamnosus alleviated Enterohaemorrhagic Escherichia coli-induced diarrhoea in mice by regulating gut microbiota.

Enterohaemorrhagic Escherichia coli (EHEC) is a prominent foodborne pathogen that causes infectious intestinal diarrhoea. Lactobacillus is a recognized probiotic that inhibits intestinal pathogens and maintains the balance of the intestinal flora. The purpose of this study was to investigate the regulatory effects of three Lactobacillus strains, L. johnsonii, L. plantarum, and L. rhamnosus, on the intestinal flora of EHEC-infected mice. The initial weight and diarrhoea index of the mice were recorded. After 21 days, the faeces of the mice were subjected to 16S rDNA high-throughput sequencing. The diarrhoea index of mice treated with Lactobacillus improved, their body weight continued to rise, and their liver index gradually decreased. The α diversity analysis showed that the intestinal flora diversity and abundance were lower in mice infected with EHEC than in healthy mice. L. plantarum, L. johnsonii, and L. rhamnosus significantly improved the diversity of the flora species. In terms of flora composition, the three main phyla present were Bacteroidetes, Firmicutes, and Proteobacteria. The abundance of these three phyla was reduced to 93.81% after infection and restored to over 96.30% after treatment. At the genus level, Lactobacillus reduced the abundance of Bacteroides, Helicobacter pylori, and Shigella, while increasing the abundance of butyric acid-producing bacteria and Lactobacillus. Finally, a heat map and non-metric multidimensional scaling analysis showed that the intestinal flora structures in the L. johnsonii, L. plantarum, and L. rhamnosus treatment groups were closest to those of healthy mice. In conclusion, L. johnsonii, L. plantarum, and L. rhamnosus regulated and improved the structure of intestinal flora and relieved diarrhoea caused by EHEC infection.

Maltose effective improving production and regulatory biosynthesis of plantaricin EF in Lactobacillus plantarum 163.

Plantaricin EF, a kind of natural antibacterial substance, has shown inhibitory effect on most pathogen and spoilage microorganisms, which possessed great potential in food preservation. However, the lower production of plantaricin EF has limited its large-scale production and application. In this study, the effect of maltose on plantaricin EF production and its regulation mechanism in Lactobacillus plantarum 163 were investigated. Maltose significantly improved the biomass and plantaricin EF production, which increased by 3.35 and 3.99 times comparing to the control without maltose, respectively. The maximum production of plantaricin E and F in fed-batch fermentation were 10.55 mg/L and 22.94 mg/L, respectively. Besides, qPCR results showed that maltose remarkably improved transcription of plnA, plnB, plnD, plnE, plnF, plnG1 and plnH, and heighten transcription of lamR, lamK, hpk6 and rrp6. These results provided an effective method to enhance plantaricin EF production and revealed a possible regulatory mechanism from transcriptome results that hpk6, rrp6, lamK and lamR were relative to plantaricin EF production. Genes, hpk6 and rrp6, promote transcription of plnG1, whereas lamK and lamR enhance transcription of plnA, plnB and plnD, which increased plantaricin EF production. KEYPOINTS: • Maltose was proved to be effective in promoting the biosynthesis of plantaricin EF. • Maltose promoted the transcription of biosynthesis and secretion genes of plantaricin EF. • Up-regulation of genes lamR, lamK, hpk6 and rrp6 heightened the plantaricin EF production.

Iturin A Induces Resistance and Improves the Quality and Safety of Harvested Cherry Tomato.

The soft rot disease caused by Rhizopus stolonifer is an important disease in cherry tomato fruit. In this study, the effect of iturin A on soft rot of cherry tomato and its influence on the storage quality of cherry tomato fruit were investigated. The results showed that 512 µg/mL of iturin A could effectively inhibit the incidence of soft rot of cherry tomato fruit. It was found that iturin A could induce the activity of resistance-related enzymes including phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), peroxidase (POD), glucanase (GLU), and chitinase (CHI), and active oxygen-related enzymes including ascorbate peroxidases (APX), superoxide dismutases (SOD), catalases (CAT), and glutathione reductase (GR) of cherry tomato fruit. In addition, iturin A treatment could slow down the weight loss of cherry tomato and soften the fruit. These results indicated that iturin A could retard the decay and improve the quality of cherry tomato fruit by both the inhibition growth of R. stolonifera and the inducing the resistance.

Bacillomycin D effectively controls growth of Malassezia globosa by disrupting the cell membrane.

Malassezia globosa is an opportunistic pathogen that causes various skin disorders, which disturbs people's life all the time, and conventional drugs are not completely satisfactory. Bacillomycin D (BD), an antifungal lipopeptide, could inhibit various fungi growth. However, the reports about its effect on M. globosa were not found yet. In this study, we showed that BD and BD-C16 (fatty acid chain had sixteen carbon atoms) completely inhibited growth of M. globosa at concentration of 64 µg/ml in 15 h, which was confirmed with the observation of irregular morphological change of M. globosa treated with BD. Significantly, the study on the working mechanism showed that BD induced cell death by changing cell membrane permeability and thus promoting the release of cellular contents, which may be mediated by the interaction between BD and ergosterol from membrane. Further study showed that BD reduced the overall content of cellular sterol, and interestingly, the expression of some genes involved in membrane and ergosterol synthesis were significantly upregulated, which was likely to be a feedback regulation. Besides, we found that BD had additive and synergistic effects with ketoconazole and amphotericin B, respectively, on inhibition of M. globosa, suggesting that combination use of BD with other commercial drugs could be a promising strategy to relieve skin disorders caused by M. globosa. KEY POINTS: • BD could efficiently inhibit the growth of M. globosa. • BD increases cell membrane permeability and thus promotes the release of cellular contents. • BD has additive or synergistic effect with other antifungal drugs.

Transcriptomic and proteomic profiling response of methicillin-resistant Staphylococcus aureus (MRSA) to a novel bacteriocin, plantaricin GZ1-27 and its inhibition of biofilm formation.

Methicillin-resistant Staphylococcus aureus (MRSA) has become a worrisome superbug, due to its wide distribution and multidrug resistance. To characterize effects of a newly identified plantaricin GZ1-27 on MRSA, transcriptomic and proteomic profiling of MRSA strain ATCC43300 was performed in response to sub-MIC (16 µg/mL) plantaricin GZ1-27 stress. In total, 1090 differentially expressed genes (padj < 0.05) and 418 differentially expressed proteins (fold change > 1.2, p < 0.05) were identified. Centralized protein expression clusters were predicted in biological functions (biofilm formation, DNA replication and repair, and heat-shock) and metabolic pathways (purine metabolism, amino acid metabolism, and biosynthesis of secondary metabolites). Moreover, a capacity of inhibition MRSA biofilm formation and killing biofilm cells were verified using crystal violet staining, scanning electron microscopy, and confocal laser-scanning microscopy. These findings yielded comprehensive new data regarding responses induced by plantaricin and could inform evidence-based methods to mitigate MRSA biofilm formation.

Bacillomycin D inhibits growth of Rhizopus stolonifer and induces defense-related mechanism in cherry tomato.

The inhibitory effect of Bacillomycin D, a cyclic lipopeptide, on Rhizopus stolonifer colonization of cherry tomato was studied, and its possible mechanism of action was explored. Bacillomycin D showed a direct inhibitory effect on R. stolonifer spore germination and mycelial growth in vitro. It conferred both a direct inhibitory effect on R. stolonifer growth in cherry tomato in vivo and induced host resistance in cherry tomato. Moreover, Bacillomycin D treatment significantly increased the activities of plant defense-related enzymes, including chitinase (CHI), ß-1,3-glucanase (GLU), phenylalanine ammonia-lyase (PAL), and peroxidase (POD). Real-time PCR (RT-PCR) showed that defense-related genes involved in the salicylic acid defense signaling pathway and genes encoding pathogenesis-related proteins were up-regulated in Bacillomycin D treatment. Furthermore, Bacillomycin D-C16 resulted in direct inhibition and a remarkable induced resistance to R. stolonifer which was higher than as induced by Bacillomycin D-C14. Together, the data indicated that Bacillomycin D can control the growth of R. stolonifer through both the direct inhibition of the fungus and the activation of defense-related genes and enzymes in cherry tomato.