Combined analysis of transcriptomics and metabolomics provide insights into the antibacterial mechanism of bacteriocin XJS01 against multidrug-resistant Staphylococcus aureus.

Multidrug-resistant (MDR) bacteria are widespread in the environment and pose a serious threat to public health. It has been shown that bacteriocins have a great potential in controlling MDR pathogens, including Staphylococcus aureus. A previously reported Lactobacillus salivarius bacteriocin XJS01 exhibited good antibacterial activity against MDR S. aureus 2612:1606BL1486 (henceforth referred to as S. aureus_26), but its molecular mechanism remains unknown. Herein, we investigated the antibacterial mechanism of XJS01 on S. aureus_26 using an approach combining transcriptomics and metabolomics. The results showed that XJS01 induced significant changes at both transcriptional and metabolic levels in S. aureus_26. In total, 231 differentially expressed genes (DEGs) and 206 differentially abundance metabolites (DAMs) were identified in S. aureus_26 treated with 1 × MIC (minimum inhibition concentration) XJS01 compared with untreated (XJS01-free) cells (control). Functional analysis revealed that these DEGs and DAMs, alone with the related pathways and biological processes, were typically involved in stress response, being primarily related to metal uptake, cell virulence, self-help mechanism, amino acid and energy metabolism, bacterial stress response (e.g., two-component system), and membrane transport (e.g., phosphotransferase system). Overall, this study uncovered the multi-target effects of bacteriocins against MDR S. aureus at the genome-wide transcriptional and metabolic levels. These findings might be useful in the development of bacteriocins for the control of MDR S. aureus and other drug-resistant bacteria.

Proteomic analysis of Staphylococcus aureus exposed to bacteriocin XJS01 and its bio-preservative effect on raw pork loins.

Antibacterial mechanism of bacteriocins against foodborne S. aureus is still to be explored, particularly in proteomics, and a deep and comprehensive study on application of bacteriocins for preservation of raw pork is required. Here, proteomic mechanism of Lactobacillus salivarius bacteriocin XJS01 against foodborne S. aureus 2612:1606BL1486 (S. aureus_26) and its preservation effect on raw pork loins stored at 4 °C for 12 days was investigated. The results showed that 301 differentially abundant proteins (DAPs) were identified between XJS01-treated and -free groups (control group) using Tandem mass tag (TMT) quantitative proteomics technology, which were primarily involved in amino acids and carbohydrate metabolism, cytolysis, defense response, cell apoptosis, cell killing, adhesion, and oxygen utilization of S. aureus_26. Bacterial secretion system (SRP) and cationic antimicrobial peptide resistance may be key pathways to maintain protein secretion and counteract the deleterious effects on S. aureus_26 caused by XJS01. In addition, XJS01 could significantly improve the preservation of raw pork loins by the evaluation results of sensory and antibacterial activity on the meat surface. Overall, this study showed that XJS01 induced a complex organism response in S. aureus, and it could be potential pork preservative.

Antibacterial activity and mechanism of action of bacteriocin LFX01 against Staphylococcus aureus and Escherichia coli and its application on pork model.

Antibacterial activity and mechanism of action of bacteriocins against bacteria that cause pork contamination remain unclear. Here, antibacterial activity of bacteriocin LFX01 against two important indicator strains (i.e., Staphylococcus aureus and Escherichia coli) and its mechanism of action were investigated. The results showed antibacterial activity of LFX01 against growth and biofilm formation of S. aureus_26 (strain 2612:1606BL1486) and E. coli_02 (strain CMCC(B)44102). Additionally, the results demonstrated that LFX01 could decrease cell metabolic activity, disrupt cell membrane permeability and integrity, and trigger leakage of intracellular contents (e.g., K+, ATP, and lactic dehydrogenase). Furthermore, gel retardation showed that LFX01 could bind to the genomic DNA of indicator strains, disrupting DNA structure. These results uncovered mechanism of action of LFX01 against indicator strains from physiological and phenotypic levels. When applied to the surface of fresh pork models, the antibacterial activity of LFX01 against indicator strains was further confirmed. These findings suggested that LFX01 could be a potential pork preservative for controlling foodborne pathogens.

Antibacterial activity and action target of phenyllactic acid against Staphylococcus aureus and its application in skim milk and cheese.

Phenyllactic acid (PLA) has been demonstrated to possess antibacterial activity and capacity to prolong food shelf life. However, studies on the performance of PLA in inhibiting Staphylococcus aureus and its effectiveness when applied to dairy products are largely lacking. Here, antibacterial activity (planktonic and biofilm states) of PLA against S. aureus CICC10145 (S. aureus_45) were investigated. The results showed that PLA inhibited growth of S. aureus_45 and formation of S. aureus_45 biofilm. Next, the antibacterial action target of PLA was uncovered from both physiological and phenotypic perspectives. The results showed that PLA decreased cell metabolic activity and cell viability, damaged cell membrane integrity, triggered leakage of intracellular contents (DNA, proteins, and ATP), and caused oxidative stress damage and morphological deformation of S. aureus_45. In practical application, the antibacterial activity of PLA against S. aureus_45 cells was further confirmed in skim milk and cheese as dairy food models, and the antibacterial effects can be adequately maintained during storage for 21 d, at least at 4°C. These findings suggested that PLA could be a potential candidate for controlling S. aureus outgrowth in dairy foods.

A method for isolating highly purified and active mitochondria from insects.

So far, methods that yield the high purity and activity of the isolated mitochondria from insects have not been reported and determined. Here, we develop methods that combine differential centrifugation and discontinuous Nycodenz density gradient centrifugation to isolate highly purified mitochondria from the thorax muscle of insects, and the methods were widely validated across three orders (Coleoptera, Hymenoptera, and Blattaria) covering four insect species using Western blot and transmission electron microscopy (TEM) analysis. The results showed the removal of the residual contamination with nonmitochondrial components such as nucleus, sarcolemma, cytosol, and endoplasmic reticulum. Furthermore, TEM, mitochondria staining, fluorescence detection, and flow cytometry analyses were employed to assess membrane integrity and activity of the isolated mitochondria. The results showed no loss of mitochondria activity/integrity after isolation. In addition, temporal dynamics in activity of the isolated mitochondria under commonly used laboratory temperature (-20 °C, 4 °C, and 25 °C) were respectively detected using a fluorescence microplate reader. The results showed that it should be avoided to store the isolated mitochondria at room temperature, and the mitochondria can meet the requirements of the most downstream experiments when they were stored at -20 °C. Overall, the study presented a method for isolating highly purified and active mitochondria from insects. This study firstly described a high-speed discontinuous density gradient centrifugation-based method that could be widely applied for mitochondria isolation in insects. The present study also provided an example to assess purity and integrity/activity of the isolated mitochondria.

A novel bacteriocin against Staphylococcus aureus from Lactobacillus paracasei isolated from Yunnan traditional fermented yogurt: Purification, antibacterial characterization, and antibiofilm activity.

Staphylococcus aureus and its biofilm have emerged as a significant threat to the safety of dairy products. In recent years, lactic acid bacteria (LAB) bacteriocins have been widely acknowledged as the potential natural antibacterial substance in food biopreservation due to their excellent antibacterial effects. However, few LAB bacteriocins with antibacterial and antibiofilm activity against S. aureus have been reported in dairy products. In the present study, a novel bacteriocin LSX01 of Lactobacillus paracasei LS-6 isolated from a traditional fermented yogurt produced in Yunnan, China, was purified and characterized extensively. The LSX01 possessed a molecular weight of 967.49 Da and an AA sequence of LDQAGISYT. The minimum inhibitory concentration of LSX01 against S. aureus_45 was 16.90 µg/mL, which was close to or lower than the previously reported bacteriocins. The LSX01 exhibited an extensive antimicrobial spectrum against both gram-positive and gram-negative bacteria. Moreover, LSX01 exhibited excellent tolerance to heat and acid-base treatments, and sensitivity to the proteolytic enzymes, such as pepsin and proteinase K. Furthermore, the treatment of S. aureus_45 planktonic cells with LSX01 significantly reduced their metabolic activity and disrupted the cell membrane integrity. Scan electron microscopy results demonstrated that LSX01 induced cytoplasmic content leakage and cell deformation. Additionally, biofilm formation of S. aureus_45 was also significantly inhibited by LSX01. Overall, the results suggested that the novel LAB bacteriocin LSX01 possessed antibacterial activity and antibiofilm activity against S. aureus and, hence, could have potential for improving safety of dairy products.