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819300.9974Sinorhizobium meliloti Functions Required for Resistance to Antimicrobial NCR Peptides and Bacteroid Differentiation. Legumes of the Medicago genus have a symbiotic relationship with the bacterium Sinorhizobium meliloti and develop root nodules housing large numbers of intracellular symbionts. Members of the nodule-specific cysteine-rich peptide (NCR) family induce the endosymbionts into a terminal differentiated state. Individual cationic NCRs are antimicrobial peptides that have the capacity to kill the symbiont, but the nodule cell environment prevents killing. Moreover, the bacterial broad-specificity peptide uptake transporter BacA and exopolysaccharides contribute to protect the endosymbionts against the toxic activity of NCRs. Here, we show that other S. meliloti functions participate in the protection of the endosymbionts; these include an additional broad-specificity peptide uptake transporter encoded by the yejABEF genes and lipopolysaccharide modifications mediated by lpsB and lpxXL, as well as rpoH1, encoding a stress sigma factor. Strains with mutations in these genes show a strain-specific increased sensitivity profile against a panel of NCRs and form nodules in which bacteroid differentiation is affected. The lpsB mutant nodule bacteria do not differentiate, the lpxXL and rpoH1 mutants form some seemingly fully differentiated bacteroids, although most of the nodule bacteria are undifferentiated, while the yejABEF mutants form hypertrophied but nitrogen-fixing bacteroids. The nodule bacteria of all the mutants have a strongly enhanced membrane permeability, which is dependent on the transport of NCRs to the endosymbionts. Our results suggest that S. meliloti relies on a suite of functions, including peptide transporters, the bacterial envelope structures, and stress response regulators, to resist the aggressive assault of NCR peptides in the nodule cells. IMPORTANCE The nitrogen-fixing symbiosis of legumes with rhizobium bacteria has a predominant ecological role in the nitrogen cycle and has the potential to provide the nitrogen required for plant growth in agriculture. The host plants allow the rhizobia to colonize specific symbiotic organs, the nodules, in large numbers in order to produce sufficient reduced nitrogen for the plants' needs. Some legumes, including Medicago spp., produce massively antimicrobial peptides to keep this large bacterial population in check. These peptides, known as NCRs, have the potential to kill the rhizobia, but in nodules, they rather inhibit the division of the bacteria, which maintain a high nitrogen-fixing activity. In this study, we show that the tempering of the antimicrobial activity of the NCR peptides in the Medicago symbiont Sinorhizobium meliloti is multifactorial and requires the YejABEF peptide transporter, the lipopolysaccharide outer membrane, and the stress response regulator RpoH1.202134311575
908510.9974Mode of action and resistance studies unveil new roles for tropodithietic acid as an anticancer agent and the γ-glutamyl cycle as a proton sink. While we have come to appreciate the architectural complexity of microbially synthesized secondary metabolites, far less attention has been paid to linking their structural features with possible modes of action. This is certainly the case with tropodithietic acid (TDA), a broad-spectrum antibiotic generated by marine bacteria that engage in dynamic symbioses with microscopic algae. TDA promotes algal health by killing unwanted marine pathogens; however, its mode of action (MoA) and significance for the survival of an algal-bacterial miniecosystem remains unknown. Using cytological profiling, we herein determine the MoA of TDA and surprisingly find that it acts by a mechanism similar to polyether antibiotics, which are structurally highly divergent. We show that like polyether drugs, TDA collapses the proton motive force by a proton antiport mechanism, in which extracellular protons are exchanged for cytoplasmic cations. The α-carboxy-tropone substructure is ideal for this purpose as the proton can be carried on the carboxyl group, whereas the basicity of the tropylium ion facilitates cation export. Based on similarities to polyether anticancer agents we have further examined TDA's cytotoxicity and find it to exhibit potent, broad-spectrum anticancer activities. These results highlight the power of MoA-profiling technologies in repurposing old drugs for new targets. In addition, we identify an operon that confers TDA resistance to the producing marine bacteria. Bioinformatic and biochemical analyses of these genes lead to a previously unknown metabolic link between TDA/acid resistance and the γ-glutamyl cycle. The implications of this resistance mechanism in the context of the algal-bacterial symbiosis are discussed.201626802120
601620.9973Investigating human-derived lactic acid bacteria for alcohol resistance. BACKGROUND: Excessive alcohol consumption has been consistently linked to serious adverse health effects, particularly affecting the liver. One natural defense against the detrimental impacts of alcohol is provided by alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH), which detoxify harmful alcohol metabolites. Recent studies have shown that certain probiotic strains, notably Lactobacillus spp., possess alcohol resistance and can produce these critical enzymes. Incorporating these probiotics into alcoholic beverages represents a pioneering approach that can potentially mitigate the negative health effects of alcohol while meeting evolving consumer preferences for functional and health-centric products. RESULTS: Five lactic acid bacteria (LAB) isolates were identified: Lactobacillus paracasei Alc1, Lacticaseibacillus rhamnosus AA, Pediococcus acidilactici Alc3, Lactobacillus paracasei Alc4, and Pediococcus acidilactici Alc5. Assessment of their alcohol tolerance, safety, adhesion ability, and immunomodulatory effects identified L. rhamnosus AA as the most promising alcohol-tolerant probiotic strain. This strain also showed high production of ADH and ALDH. Whole genome sequencing analysis revealed that the L. rhamnosus AA genome contained both the adh (encoding for ADH) and the adhE (encoding for ALDH) genes. CONCLUSIONS: L. rhamnosus AA, a novel probiotic candidate, showed notable alcohol resistance and the capability to produce enzymes essential for alcohol metabolism. This strain is a highly promising candidate for integration into commercial alcoholic beverages upon completion of comprehensive safety and functionality evaluations.202438659044
909630.9973Enhanced antibacterial activity of surface re-engineered lysozyme against Gram-negative bacteria without accumulated resistance. In this study, we show a way to improve the antibacterial activity of lysozyme by incorporating guanidino functional groups onto its surface (Lyz-Gua), which could treat pathogenic bacteria without accumulated resistance and shows advantages over commercial antibiotics.202235876097
910240.9972An Organogold Compound as Potential Antimicrobial Agent against Drug-Resistant Bacteria: Initial Mechanistic Insights. The rise of antimicrobial resistance has necessitated novel strategies to efficiently combat pathogenic bacteria. Metal-based compounds have been proven as a possible alternative to classical organic drugs. Here, we have assessed the antibacterial activity of seven gold complexes of different families. One compound, a cyclometalated Au(III) C^N complex, showed activity against Gram-positive bacteria, including multi-drug resistant clinical strains. The mechanism of action of this compound was studied in Bacillus subtilis. Overall, the studies point towards a complex mode of antibacterial action, which does not include induction of oxidative stress or cell membrane damage. A number of genes related to metal transport and homeostasis were upregulated upon short treatment of the cells with gold compound. Toxicity tests conducted on precision-cut mouse tissue slices ex vivo revealed that the organogold compound is poorly toxic to mouse liver and kidney tissues, and may thus, be treated as an antibacterial drug candidate.202134181818
14150.9972Plant growth-promoting activities and genomic analysis of the stress-resistant Bacillus megaterium STB1, a bacterium of agricultural and biotechnological interest. In this work, the stress-resistant Bacillus megaterium STB1 is characterized and its ability to promote plant growth under normal and stress conditions is demonstrated. The genomic sequence of this bacterium, and a detailed analysis of the genes involved in facilitating its stress resistance and plant growth-promoting activities is also reported. The B. megaterium STB1 genome is rich in genetic elements involved in multiple stress resistance, xenobiotic degradation, pathogen antagonistic activities, and other traits related to soil and rhizosphere colonization. Moreover, genes participating in the biosynthesis of auxins and cytokinins, the modulation of polyamines, GABA, brassinosteroids and ethylene levels were also found. Ultimately, this study brings new insights into the role of B. megaterium as a plant growth-promoting bacterium and opens new opportunities for the development of novel strategies for agriculture and biotechnology.202031886139
909460.9972Pathogen-Specific Polymeric Antimicrobials with Significant Membrane Disruption and Enhanced Photodynamic Damage To Inhibit Highly Opportunistic Bacteria. Highly pathogenic Gram-negative bacteria and their drug resistance are a severe public health threat with high mortality. Gram-negative bacteria are hard to kill due to the complex cell envelopes with low permeability and extra defense mechanisms. It is challenging to treat them with current strategies, mainly including antibiotics, peptides, polymers, and some hybrid materials, which still face the issue of drug resistance, limited antibacterial selectivity, and severe side effects. Together with precise bacteria targeting, synergistic therapeutic modalities, including physical membrane damage and photodynamic eradication, are promising to combat Gram-negative bacteria. Herein, pathogen-specific polymeric antimicrobials were formulated from amphiphilic block copolymers, poly(butyl methacrylate)- b-poly(2-(dimethylamino) ethyl methacrylate- co-eosin)- b-ubiquicidin, PBMA- b-P(DMAEMA- co-EoS)-UBI, in which pathogen-targeting peptide ubiquicidin (UBI) was tethered in the hydrophilic chain terminal, and Eosin-Y was copolymerized in the hydrophilic block. The micelles could selectively adhere to bacteria instead of mammalian cells, inserting into the bacteria membrane to induce physical membrane damage and out-diffusion of intracellular milieu. Furthermore, significant in situ generation of reactive oxygen species was observed upon light irradiation, achieving further photodynamic eradication. Broad-spectrum bacterial inhibition was demonstrated for the polymeric antimicrobials, especially highly opportunistic Gram-negative bacteria, such as Pseudomona aeruginosa ( P. aeruginosa) based on the synergy of physical destruction and photodynamic therapy, without detectable resistance. In vivo P. aeruginosa-infected knife injury model and burn model both proved good potency of bacteria eradication and promoted wound healing, which was comparable with commercial antibiotics, yet no risk of drug resistance. It is promising to hurdle the infection and resistance suffered from highly opportunistic bacteria.201930632740
831470.9972Interactions between Bacteria and Bile Salts in the Gastrointestinal and Hepatobiliary Tracts. Bile salts and bacteria have intricate relationships. The composition of the intestinal pool of bile salts is shaped by bacterial metabolism. In turn, bile salts play a role in intestinal homeostasis by controlling the size and the composition of the intestinal microbiota. As a consequence, alteration of the microbiome-bile salt homeostasis can play a role in hepatic and gastrointestinal pathological conditions. Intestinal bacteria use bile salts as environmental signals and in certain cases as nutrients and electron acceptors. However, bile salts are antibacterial compounds that disrupt bacterial membranes, denature proteins, chelate iron and calcium, cause oxidative damage to DNA, and control the expression of eukaryotic genes involved in host defense and immunity. Bacterial species adapted to the mammalian gut are able to endure the antibacterial activities of bile salts by multiple physiological adjustments that include remodeling of the cell envelope and activation of efflux systems and stress responses. Resistance to bile salts permits that certain bile-resistant pathogens can colonize the hepatobiliary tract, and an outstanding example is the chronic infection of the gall bladder by Salmonella enterica. A better understanding of the interactions between bacteria and bile salts may inspire novel therapeutic strategies for gastrointestinal and hepatobiliary diseases that involve microbiome alteration, as well as novel schemes against bacterial infections.201729043249
950780.9971Bacteriocins: Classification, synthesis, mechanism of action and resistance development in food spoilage causing bacteria. Huge demand of safe and natural preservatives has opened new area for intensive research on bacteriocins to unravel the novel range of antimicrobial compounds that could efficiently fight off the food-borne pathogens. Since food safety has become an increasingly important international concern, the application of bacteriocins from lactic acid bacteria that target food spoilage/pathogenic bacteria without major adverse effects has received great attention. Different modes of actions of these bacteriocins have been suggested and identified, like pore-forming, inhibition of cell-wall/nucleic acid/protein synthesis. However, development of resistance in the food spoilage and pathogenic bacteria against these bacteriocins is a rising concern. Emergence and spread of mutant strains resistant to bacteriocins is hampering food safety. It has spurred an interest to understand the bacteriocin resistance phenomenon displayed by the food pathogens, which will be helpful in mitigating the resistance problem. Therefore, present review is focused on the different resistance mechanisms adopted by food pathogens to overcome bacteriocin.201930610901
914790.9971A Critical Review on the Potential of Inactivated Bacteria in Counteracting Human Pathogens. Bacterial infections are a major global public health challenge, especially with increasing antibiotic resistance. Postbiotics, bioactive compounds produced by probiotics, have been proposed as a novel strategy to inhibit the growth of pathogenic bacteria and address antibiotic resistance. Similar to probiotics and certain food ingredients, postbiotics can also modulate beneficial microbial communities and ultimately contribute to host health. Postbiotics derived from probiotics may affect the physical and chemical conditions of the intestinal environment, and by enhancing the host's immune system, directly interfere with the metabolic pathways and signaling of pathogenic bacteria. Postbiotics inhibit biofilm formation, reduce the expression of antibiotic resistance genes, and enhance the efficacy of antibiotic therapies. They are effective against resistant bacteria such as Escherichia coli and Clostridium difficile and reduce the risk of dental infections caused by Streptococcus mutans. Some postbiotics, such as lactic acid and antimicrobial peptides derived from Lactobacillus and Bifidobacterium genus, help the immune system dealing resistant bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, and Helicobacter pylori. The review investigates the mechanisms of action and applications of postbiotics in the control of pathogenic bacteria and their role as a complement to existing treatments.202540394322
9013100.9971Two antimicrobial genes from Aegilops tauschii Cosson identified by the Bacillus subtilis expression system. Antimicrobial genes play an important role as a primary defense mechanism in all multicellular organisms. We chose Bacillus subtilis as a target pathogen indicator and transferred the Aegilops tauschii Cosson cDNA library into B. subtilis cells. Expression of the candidate antimicrobial gene can inhibit B. subtilis cell growth. Using this strategy, we screened six genes that have an internal effect on the indicator bacteria. Then, the secreted proteins were extracted and tested; two genes, AtR100 and AtR472, were found to have strong external antimicrobial activities with broad-spectrum resistance against Xanthomonas oryzae pv. oryzicola, Clavibacter fangii, and Botrytis cinerea. Additionally, thermal stability tests indicated that the antimicrobial activities of both proteins were thermostable. Furthermore, these two proteins exhibited no significant hemolytic activities. To test the feasibility of application at the industrial level, liquid fermentation and spray drying of these two proteins were conducted. Powder dilutions were shown to have significant inhibitory effects on B. cinerea. Fluorescence microscopy and flow cytometry results showed that the purified protein impaired and targeted the cell membranes. This study revealed that these two antimicrobial peptides could potentially be used for replacing antibiotics, which would provide the chance to reduce the emergence of drug resistance.202032770019
9093110.9971Antibacterial activity of positively charged carbon quantum dots without detectable resistance for wound healing with mixed bacteria infection. Widespread bacterial infection and the spread of antibiotic resistance exhibit increasing threat to the public and thus require new antibacterial strategies. Carbon quantum dots (CQDs) have been extensively investigated to play fluorescent, catalytic roles and even potential biomedical functions containing sterilization. However, synthetic understanding of the interaction of CQDs and bacteria, the exhibition of antibacterial ability, and the risk of resistance evolution remain lacking. Herein, a simple one-pot method was fabricated to prepare positively charged CQDs (PC-CQDs) as a broad-spectrum antibacterial agent. PC-CQDs possessed effective antibacterial activity against all tested Gram-positive, Gram-negative, and drug-resistant bacteria. Investigation of the antibacterial mechanism of PC-CQDs indicated that small-sized PC-CQDs functionalized with -NH(2) and -NH induced strong adherence behavior on the bacterial cell membrane. Moreover, the entry of PC-CQDs caused conformational changes in the genes and generation of reactive oxygen species in the bacteria. Safety evaluation illustrated that PC-CQDs did not trigger detectable drug resistance or hemolysis. Furthermore, PC-CQDs effectively promoted the antibacterial treatment of mixed Staphylococcus aureus and Escherichia coli infected wound in rats with low in vivo toxicity. These results suggested that PC-CQDs are a potential antibacterial candidate for real wound healing applications in complex bacterial infections and even resistant bacteria-caused infections.202133812599
9508120.9971Nisin and class IIa bacteriocin resistance among Listeria and other foodborne pathogens and spoilage bacteria. Food safety has been an important issue globally due to increasing foodborne diseases and change in food habits. To inactivate foodborne pathogens, various novel technologies such as biopreservation systems have been studied. Bacteriocins are ribosomally synthesized peptides or proteins with antimicrobial activity produced by different groups of bacteria, but the bacteriocins produced by many lactic acid bacteria offer potential applications in food preservation. The use of bacteriocins in the food industry can help reduce the addition of chemical preservatives as well as the intensity of heat treatments, resulting in foods that are more naturally preserved. However, the development of highly tolerant and/or resistant strains may decrease the efficiency of bacteriocins as biopreservatives. Several mechanisms of bacteriocin resistance development have been proposed among various foodborne pathogens. The acquiring of resistance to bacteriocins can significantly affect physiological activity profile of bacteria, alter cell-envelope lipid composition, and also modify the antibiotic susceptibility/resistance profile of bacteria. This article presents a brief review on the scientific research about the various possible mechanisms involved in the development of resistance to nisin and Class IIa bacteriocins among the foodborne pathogens.201121417775
154130.9970Classification of acetic acid bacteria and their acid resistant mechanism. Acetic acid bacteria (AAB) are obligate aerobic Gram-negative bacteria that are commonly used in vinegar fermentation because of their strong capacity for ethanol oxidation and acetic acid synthesis as well as their acid resistance. However, low biomass and low production rate due to acid stress are still major challenges that must be overcome in industrial processes. Although acid resistance in AAB is important to the production of high acidity vinegar, the acid resistance mechanisms of AAB have yet to be fully elucidated. In this study, we discuss the classification of AAB species and their metabolic processes and review potential acid resistance factors and acid resistance mechanisms in various strains. In addition, we analyze the quorum sensing systems of Komagataeibacter and Gluconacetobacter to provide new ideas for investigation of acid resistance mechanisms in AAB in the form of signaling pathways. The results presented herein will serve as an important reference for selective breeding of high acid resistance AAB and optimization of acetic acid fermentation processes.202133595734
8159140.9970Quaternary Ammonium Salts: Insights into Synthesis and New Directions in Antibacterial Applications. The overuse of antibiotics has led to the emergence of a large number of antibiotic-resistant genes in bacteria, and increasing evidence indicates that a fungicide with an antibacterial mechanism different from that of antibiotics is needed. Quaternary ammonium salts (QASs) are a biparental substance with good antibacterial properties that kills bacteria through simple electrostatic adsorption and insertion into cell membranes/altering of cell membrane permeability. Therefore, the probability of bacteria developing drug resistance is greatly reduced. In this review, we focus on the synthesis and application of single-chain QASs, double-chain QASs, heterocyclic QASs, and gemini QASs (GQASs). Some possible structure-function relationships of QASs are also summarized. As such, we hope this review will provide insight for researchers to explore more applications of QASs in the field of antimicrobials with the aim of developing systems for clinical applications.202336748912
6042150.9970Limosilactobacillus fermentum ING8, a Potential Multifunctional Non-Starter Strain with Relevant Technological Properties and Antimicrobial Activity. Lactic acid bacteria (LAB) have gained particular attention among different exopolysaccharide-producing microorganisms due to their safety status and effects on human health and food production. Exopolysaccharide-producing LAB play a crucial role in different ways, such as improving texture, mouthfeel, controlling viscosity, and for low-calorie food production. In this study, we isolated a multifunctional strain with good exopolysaccharide production properties. Limosilactobacillus fermentum ING8 was isolated from an Indian traditional fermented milk (Dahi) and evaluated for its safety, enzymatic activity, NaCl resistance and temperature tolerance, milk coagulation, and storage stability. Finally, the complete genome of this strain was sequenced and subjected to safety in silico evaluation and genomic analysis. The results revealed that L. fermentum ING8 possesses relevant technological properties, such as exopolysaccharide production, antimicrobial activity, and galactose utilization. Besides, this strain showed very high stability to storage conditions at refrigeration temperature. In addition, the genomic analysis did not evidence any possible deleterious elements, such as acquired antibiotic resistance genes, virulence genes, or hemolysis-related genes. However, all structural genes related to the galactose operon and EPS production were detected. Therefore, L. fermentum ING8 can be considered a promising multifunctional bacterium to be proposed as non-starter in different types of dairy productions.202235267336
9155160.9970Polyphenols and their nanoformulations as potential antibiofilm agents against multidrug-resistant pathogens. The emergence of multidrug-resistant (MDR) pathogens is a major problem in the therapeutic management of infectious diseases. Among the bacterial resistance mechanisms is the development of an enveloped protein and polysaccharide-hydrated matrix called a biofilm. Polyphenolics have demonstrated beneficial antibacterial effects. Phenolic compounds mediate their antibiofilm effects via disruption of the bacterial membrane, deprivation of substrate, protein binding, binding to adhesion complex, viral fusion blockage and interactions with eukaryotic DNA. However, these compounds have limitations of chemical instability, low bioavailability, poor water solubility and short half-lives. Nanoformulations offer a promising solution to overcome these challenges by enhancing their antibacterial potential. This review summarizes the antibiofilm role of polyphenolics, their underlying mechanisms and their potential role as resistance-modifying agents.202438305223
8813170.9970Enhancing Escherichia coli abiotic stress resistance through ornithine lipid formation. Escherichia coli is a common host for biotechnology and synthetic biology applications. During growth and fermentation, the microbes are often exposed to stress conditions, such as variations in pH or solvent concentrations. Bacterial membranes play a key role in response to abiotic stresses. Ornithine lipids (OLs) are a group of membrane lipids whose presence and synthesis have been related to stress resistance in bacteria. We wondered if this stress resistance could be transferred to bacteria not encoding the capacity to form OLs in their genome, such as E. coli. In this study, we engineered different E. coli strains to produce unmodified OLs and hydroxylated OLs by expressing the synthetic operon olsFC. Our results showed that OL formation improved pH resistance and increased biomass under phosphate limitation. Transcriptome analysis revealed that OL-forming strains differentially expressed stress- and membrane-related genes. OL-producing strains also showed better growth in the presence of the ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP), suggesting reduced proton leakiness in OL-producing strains. Furthermore, our engineered strains showed improved heterologous violacein production at phosphate limitation and also at low pH. Overall, this study demonstrates the potential of engineering the E. coli membrane composition for constructing robust hosts with an increased abiotic stress resistance for biotechnology and synthetic biology applications. KEY POINTS: • Ornithine lipid production in E. coli increases biomass yield under phosphate limitation. • Engineered strains show an enhanced production phenotype under low pH stress. • Transcriptome analysis and CCCP experiments revealed reduced proton leakage.202438587638
9153180.9970Mycoplasma Contamination of Cell Cultures: Vesicular Traffic in Bacteria and Control over Infectious Agents. Cell cultures are subject to contamination either with cells of other cultures or with microorganisms, including fungi, viruses, and bacteria. Mycoplasma contamination of cell cultures is of particular importance. Since cell cultures are used for the production of vaccines and physiologically active compounds, designing a system for controlling contaminants becomes topical for fundamental science and biotechnological production. The discovery of extracellular membrane vesicles in mycoplasmas makes it necessary to take into consideration the bacterial vesicular traffic in systems designed for controlling infectious agents. The extracellular vesicles of bacteria mediate the traffic of proteins and genes, participate in cell-to-cell interactions, as well as in the pathogenesis and development of resistance to antibiotics. The present review discusses the features of mycoplasmas, their extracellular vesicles, and the interaction between contaminants and eukaryotic cells. Furthermore, it provides an analysis of the problems associated with modern methods of diagnosis and eradication of mycoplasma contamination from cell cultures and prospects for their solution.201425349713
8862190.9970Vibrio anguillarum Is Genetically and Phenotypically Unaffected by Long-Term Continuous Exposure to the Antibacterial Compound Tropodithietic Acid. Minimizing the use of antibiotics in the food production chain is essential for limiting the development and spread of antibiotic-resistant bacteria. One alternative intervention strategy is the use of probiotic bacteria, and bacteria of the marine Roseobacter clade are capable of antagonizing fish-pathogenic vibrios in fish larvae and live feed cultures for fish larvae. The antibacterial compound tropodithietic acid (TDA), an antiporter that disrupts the proton motive force, is key in the antibacterial activity of several roseobacters. Introducing probiotics on a larger scale requires understanding of any potential side effects of long-term exposure of the pathogen to the probionts or any compounds they produce. Here we exposed the fish pathogen Vibrio anguillarum to TDA for several hundred generations in an adaptive evolution experiment. No tolerance or resistance arose during the 90 days of exposure, and whole-genome sequencing of TDA-exposed lineages and clones revealed few mutational changes, compared to lineages grown without TDA. Amino acid-changing mutations were found in two to six different genes per clone; however, no mutations appeared unique to the TDA-exposed lineages or clones. None of the virulence genes of V. anguillarum was affected, and infectivity assays using fish cell lines indicated that the TDA-exposed lineages and clones were less invasive than the wild-type strain. Thus, long-term TDA exposure does not appear to result in TDA resistance and the physiology of V. anguillarum appears unaffected, supporting the application of TDA-producing roseobacters as probiotics in aquaculture. IMPORTANCE: It is important to limit the use of antibiotics in our food production, to reduce the risk of bacteria developing antibiotic resistance. We showed previously that marine bacteria of the Roseobacter clade can prevent or reduce bacterial diseases in fish larvae, acting as probiotics. Roseobacters produce the antimicrobial compound tropodithietic acid (TDA), and we were concerned regarding whether long-term exposure to this compound could induce resistance or affect the disease-causing ability of the fish pathogen. Therefore, we exposed the fish pathogen Vibrio anguillarum to increasing TDA concentrations over 3 months. We did not see the development of any resistance to TDA, and subsequent infection assays revealed that none of the TDA-exposed clones had increased virulence toward fish cells. Hence, this study supports the use of roseobacters as a non-risk-based disease control measure in aquaculture.201627235441