Background The ability of plant-pathogenic bacteria to develop antimicrobial resistance against crop protection products represents a significant challenge. An alternative to conventional crop protecting products could be random peptide mixtures (RPMs), which potentially target the phospholipid-containing cell membrane. The randomized arrangement of the peptides minimizes the risk of bacterial resistance developing against the RPMs. However, not all plant-pathogenic bacteria exhibited growth inhibition after RPM treatment. Our prior studies revealed correlations between bacterial growth inhibition and changes in the fatty acid pattern following treatment. However, additional data on the intact phospholipid composition are essential to further understand and improve novel RPMs. Results Accordingly, we developed an analytical setup for in-depth bacterial lipid membrane characterization based on two complementary methods in conjunction with chemometric data evaluation to study the impact of RPM treatment on phospholipid class and species level. An efficient phospholipid class quantitation using hydrophilic interaction liquid chromatography (HILIC)-based lipid class separation with uniform charged aerosol detection (CAD) revealed distinct differences in the class composition of six plant-pathogenic bacteria. Moreover, branched-chain fatty acid (BCFA)-comprising phospholipid profiling via liquid chromatography-tandem mass spectrometry (LC-MS/MS) provided additional lipid species information to classify the investigated bacteria based on the number of bound BCFA. The combination of these techniques served for a comprehensive characterization of the bacterial membrane adaptation to the RPM treatment, which showed some correlations with the inhibitory effects of the RPMs. Significance In this proof-of-concept study, HILIC-CAD phospholipid quantitation and BCFA-comprising phospholipid profiling were introduced as complementary techniques for in-depth characterization of bacterial cell membranes as well as membrane adaptations at both phospholipid class and species level. Our developed analytical setup may facilitate future studies targeting in-depth characterization of bacterial lipid membranes.

AbstractAgricultural yields are often limited by damage caused by pathogenic microorganisms, including plant‐pathogenic bacteria. The chemical control options to cope with bacterial diseases in agriculture are limited, predominantly relying on copper‐based products. These compounds, however, possess limited efficacy. Therefore, there is an urgent need to develop novel technologies to manage bacterial plant diseases and reduce food loss. In this study, a new antimicrobial agent was developed using a doping method that entraps small bioactive organic molecules inside copper as the metal matrix. The food preservative agent lauroyl arginate ethyl ester (ethyl lauroyl arginate; LAE) was chosen as the doped organic compound. The new composites were termed LAE@[Cu]. Bactericidal assays against Acidovorax citrulli, a severe plant pathogen, revealed that LAE and copper in the composites possess a synergistic interaction as compared with each component individually. LAE@[Cu] composites were further characterised in terms of chemical properties and in planta assays demonstrated their potential for further development as crop protection agents.

Plant-pathogenic bacteria are one of the major constraints on agricultural yield. In order to selectively treat these bacteria, it is essential to understand the molecular structure of their cell membrane. Previous studies have focused on analyzing hydrolyzed fatty acids (FA) due to the complexity of bacterial membrane lipids. These studies have highlighted the occurrence of branched-chain fatty acids (BCFA) alongside normal-chain fatty acids (NCFA) in many bacteria. As several FA are bound in the intact phospholipids of the bacterial membrane, the presence of isomeric FA complicates lipid analysis. Furthermore, commercially available reference standards do not fully cover potential lipid isomers. To address this issue, we have developed a reversed-phase high-performance liquid chromatography (RP-HPLC) method with tandem mass spectrometry (MS/MS) to analyze the phospholipids of various plant-pathogenic bacteria with a focus on BCFA containing phospholipids. The study revealed the separation of three isomeric phosphatidylethanolamines (PE) depending on the number of bound BCFA to NCFA. The validation of the retention order was based on available reference standards in combination with the analysis of hydrolyzed fatty acids through gas chromatography with mass spectrometry (GC/MS) after fractionation. Additionally, the transferability of the retention order to other major lipid classes, such as phosphatidylglycerols (PG) and cardiolipins (CL), was thoroughly examined. Using the information regarding the retention behavior, the phospholipid profile of six plant-pathogenic bacteria was structurally elucidated. Furthermore, the developed LC-MS/MS method was used to classify the plant-pathogenic bacteria based on the number of bound BCFA in the phospholipidome.

Resistance of plant-pathogenic bacteria to classic antibiotics has prompted the search for suitable alternative antimicrobial substances. One promising strategy could be the use of purposely synthesized random peptide mixtures (RPMs). Six plant-pathogenic bacteria were cultivated and treated with two RPMs previously found to show antimicrobial activity mainly by bacterial membrane disruption. Here, we show that bacteria treated with RPMs showed partly remarkable changes in the fatty acid pattern while those unaffected did not. Quantitative changes could be verified by compound specific isotope analysis of δ 13 C values (‰). This technique was employed due to the characteristic feature of stronger bonds between heavier isotopes in (bio)chemical reactions. As a proof of concept, the increase in abundance of a fatty acid group after RPM treatment was accompanied with a decrease in the 13 C content and vice versa. We propose that our findings will help designing and synthesizing more selective antimicrobial peptides.

LebensmittelchemieVolume 76, Issue S1 p. S1-151-S1-151 Article Fettsäurebestimmung verschiedener Bakterienstämme vor und nach Behandlung mit antibakteriellen zufälligen Peptidmischungen (RPMs) Nina Wiedmaier-Czerny, Nina Wiedmaier-Czerny Institut für Lebensmittelchemie (170b), Universität Hohenheim, Garbenstraße 28, 70599 StuttgartSearch for more papers by this authorSchroth Dorothee, Schroth Dorothee Institut für Lebensmittelchemie (170b), Universität Hohenheim, Garbenstraße 28, 70599 StuttgartSearch for more papers by this authorStephanie Krauß, Stephanie Krauß Institut für Lebensmittelchemie (170b), Universität Hohenheim, Garbenstraße 28, 70599 StuttgartSearch for more papers by this authorShiri Topman-Rakover, Shiri Topman-Rakover Institut für Lebensmittelchemie (170b), Universität Hohenheim, Garbenstraße 28, 70599 Stuttgart Institute of Biochemistry, Food Science and Nutrition, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, Rehovot, 76100 IsraelSearch for more papers by this authorAya Brill, Aya Brill Institute of Biochemistry, Food Science and Nutrition, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, Rehovot, 76100 Israel Department of Plant Pathology and Microbiology, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, Rehovot, 76100 IsraelSearch for more papers by this authorSaul Burdman, Saul Burdman Department of Plant Pathology and Microbiology, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, Rehovot, 76100 IsraelSearch for more papers by this authorZvi Hayouka, Zvi Hayouka Institute of Biochemistry, Food Science and Nutrition, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, Rehovot, 76100 IsraelSearch for more papers by this authorWalter Vetter, Walter Vetter Institut für Lebensmittelchemie (170b), Universität Hohenheim, Garbenstraße 28, 70599 StuttgartSearch for more papers by this author Nina Wiedmaier-Czerny, Nina Wiedmaier-Czerny Institut für Lebensmittelchemie (170b), Universität Hohenheim, Garbenstraße 28, 70599 StuttgartSearch for more papers by this authorSchroth Dorothee, Schroth Dorothee Institut für Lebensmittelchemie (170b), Universität Hohenheim, Garbenstraße 28, 70599 StuttgartSearch for more papers by this authorStephanie Krauß, Stephanie Krauß Institut für Lebensmittelchemie (170b), Universität Hohenheim, Garbenstraße 28, 70599 StuttgartSearch for more papers by this authorShiri Topman-Rakover, Shiri Topman-Rakover Institut für Lebensmittelchemie (170b), Universität Hohenheim, Garbenstraße 28, 70599 Stuttgart Institute of Biochemistry, Food Science and Nutrition, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, Rehovot, 76100 IsraelSearch for more papers by this authorAya Brill, Aya Brill Institute of Biochemistry, Food Science and Nutrition, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, Rehovot, 76100 Israel Department of Plant Pathology and Microbiology, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, Rehovot, 76100 IsraelSearch for more papers by this authorSaul Burdman, Saul Burdman Department of Plant Pathology and Microbiology, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, Rehovot, 76100 IsraelSearch for more papers by this authorZvi Hayouka, Zvi Hayouka Institute of Biochemistry, Food Science and Nutrition, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, Rehovot, 76100 IsraelSearch for more papers by this authorWalter Vetter, Walter Vetter Institut für Lebensmittelchemie (170b), Universität Hohenheim, Garbenstraße 28, 70599 StuttgartSearch for more papers by this author First published: 01 March 2022 https://doi.org/10.1002/lemi.202258018AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume76, IssueS1Supplement: Abstracts der Vorträge der Regionalverbände und die der Posterflashtalks der AG JLCMarch 2022Pages S1-151-S1-151 RelatedInformation

Antibiotic resistance is one of the greatest crises in human medicine. Increased incidents of antibiotic resistance are linked to clinical overuse and overreliance on antibiotics. Among the ESKAPE pathogens, Acinetobacter baumannii, especially carbapenem-resistant isolates, has emerged as a significant threat in the context of blood, urinary tract, lung, and wound infections. Therefore, new approaches that limit the emergence of antibiotic resistant A. baumannii are urgently needed. Recently, we have shown that random peptide mixtures (RPMs) are an attractive alternative class of drugs to antibiotics with strong safety and pharmacokinetic profiles. RPMs are antimicrobial peptide mixtures produced by incorporating two amino acids at each coupling step, rendering them extremely diverse but still defined in their overall composition, chain length, and stereochemistry. The extreme diversity of RPMs may prevent bacteria from evolving resistance rapidly. Here, we demonstrated that RPMs rapidly and efficiently kill different strains of A. baumannii, inhibit biofilm formation, and disrupt mature biofilms. Importantly, RPMs attenuated bacterial burden in mouse models of acute pneumonia and soft tissue infection and significantly reduced mouse mortality during sepsis. Collectively, our results demonstrate RPMs have the potential to be used as powerful therapeutics against antibiotic-resistant A. baumannii.

Bacteria show distinct and characteristic fatty acid (FA) patterns which can be modified by environmental conditions. In this study, we cultivated six plant-pathogenic bacteria of agricultural concern and performed a detailed analysis of the fatty acid composition. The study covered four strains of the gram-negative Xanthomonas campestris pathovar (pv) campestris (Xcc), Xanthomonas perforans (Xp), Acidovorax citrulli (Ac) and Pseudomonas syringae pv. tomato (Pst), and two strains of the gram-positive Clavibacter michiganensis subsp. michiganensis (Cmm) and Streptomyces scabies (Ssc). After cultivation, freeze-dried bacteria samples were transesterified and analysed by gas chromatography with mass spectrometry in full scan and selected ion monitoring (SIM) modes. Altogether, 44 different FAs were detected in the six strains with individual contributions of 0.01-43.8% to the total FAs. The variety in the six strains ranged between 12 and 31 individual FAs. The FA composition of Xcc, Xp, Cmm and Ssc were dominated by iso- and anteiso-fatty acids (especially i15:0, a15:0, i16:0), which is typical for most bacteria. In contrast to this, Ac and Pst showed only saturated and monounsaturated FAs. Four of the six bacteria showed similar FA patterns as reported before in the literature. Differences were observed in the case of Cmm where many more FAs were detected in the present study. In addition, to the best of our knowledge, the FA pattern of Xp was presented for the first time.