HelR is a helicase-like protein that protects RNA polymerase from rifamycin antibiotics.

Rifamycin antibiotics such as rifampin are potent inhibitors of prokaryotic RNA polymerase (RNAP) used to treat tuberculosis and other bacterial infections. Although resistance arises in the clinic principally through mutations in RNAP, many bacteria possess highly specific enzyme-mediated resistance mechanisms that modify and inactivate rifamycins. The expression of these enzymes is controlled by a 19-bp cis-acting rifamycin-associated element (RAE). Guided by the presence of RAE sequences, we identify a helicase-like protein, HelR, in Streptomyces venezuelae that confers broad-spectrum rifamycin resistance. We show that HelR also promotes tolerance to rifamycins, enabling bacterial evasion of the toxic properties of these antibiotics. HelR forms a complex with RNAP and rescues transcription inhibition by displacing rifamycins from RNAP, thereby providing resistance by target protection . Furthermore, HelRs are broadly distributed in Actinobacteria, including several opportunistic Mycobacterial pathogens, offering yet another challenge for developing new rifamycin antibiotics.

Mechanistic plasticity in ApmA enables aminoglycoside promiscuity for resistance.

The efficacy of aminoglycoside antibiotics is waning due to the acquisition of diverse resistance mechanisms by bacteria. Among the most prevalent are aminoglycoside acetyltransferases (AACs) that inactivate the antibiotics through acetyl coenzyme A-mediated modification. Most AACs are members of the GCN5 superfamily of acyltransferases which lack conserved active site residues that participate in catalysis. ApmA is the first reported AAC belonging to the left-handed ß-helix superfamily. These enzymes are characterized by an essential active site histidine that acts as an active site base. Here we show that ApmA confers broad-spectrum aminoglycoside resistance with a molecular mechanism that diverges from other detoxifying left-handed ß-helix superfamily enzymes and canonical GCN5 AACs. We find that the active site histidine plays different functions depending on the acetyl-accepting aminoglycoside substrate. This flexibility in the mechanism of a single enzyme underscores the plasticity of antibiotic resistance elements to co-opt protein catalysts in the evolution of drug detoxification.

Evolution-guided discovery of antibiotics that inhibit peptidoglycan remodelling.

Addressing the ongoing antibiotic crisis requires the discovery of compounds with novel mechanisms of action that are capable of treating drug-resistant infections1. Many antibiotics are sourced from specialized metabolites produced by bacteria, particularly those of the Actinomycetes family2. Although actinomycete extracts have traditionally been screened using activity-based platforms, this approach has become unfavourable owing to the frequent rediscovery of known compounds. Genome sequencing of actinomycetes reveals an untapped reservoir of biosynthetic gene clusters, but prioritization is required to predict which gene clusters may yield promising new chemical matter2. Here we make use of the phylogeny of biosynthetic genes along with the lack of known resistance determinants to predict divergent members of the glycopeptide family of antibiotics that are likely to possess new biological activities. Using these predictions, we uncovered two members of a new functional class of glycopeptide antibiotics-the known glycopeptide antibiotic complestatin and a newly discovered compound we call corbomycin-that have a novel mode of action. We show that by binding to peptidoglycan, complestatin and corbomycin block the action of autolysins-essential peptidoglycan hydrolases that are required for remodelling of the cell wall during growth. Corbomycin and complestatin have low levels of resistance development and are effective in reducing bacterial burden in a mouse model of skin MRSA infection.

Interactions of TonB-dependent transporter FoxA with siderophores and antibiotics that affect binding, uptake, and signal transduction.

The outer membrane of gram-negative bacteria prevents many antibiotics from reaching intracellular targets. However, some antimicrobials can take advantage of iron import transporters to cross this barrier. We showed previously that the thiopeptide antibiotic thiocillin exploits the nocardamine xenosiderophore transporter, FoxA, of the opportunistic pathogen Pseudomonas aeruginosa for uptake. Here, we show that FoxA also transports the xenosiderophore bisucaberin and describe at 2.5 Å resolution the crystal structure of bisucaberin bound to FoxA. Bisucaberin is distinct from other siderophores because it forms a 3:2 rather than 1:1 siderophore-iron complex. Mutations in a single extracellular loop of FoxA differentially affected nocardamine, thiocillin, and bisucaberin binding, uptake, and signal transduction. These results show that in addition to modulating ligand binding, the extracellular loops of siderophore transporters are of fundamental importance for controlling ligand uptake and its regulatory consequences, which have implications for the development of siderophore-antibiotic conjugates to treat difficult infections.

Lessons from assembling a microbial natural product and pre-fractionated extract library in an academic laboratory.

Microbial natural products are specialized metabolites that are sources of many bioactive compounds including antibiotics, antifungals, antiparasitics, anticancer agents, and probes of biology. The assembly of libraries of producers of natural products has traditionally been the province of the pharmaceutical industry. This sector has gathered significant historical collections of bacteria and fungi to identify new drug leads with outstanding outcomes-upwards of 60% of drug scaffolds originate from such libraries. Despite this success, the repeated rediscovery of known compounds and the resultant diminishing chemical novelty contributed to a pivot from this source of bioactive compounds toward more tractable synthetic compounds in the drug industry. The advent of advanced mass spectrometry tools, along with rapid whole genome sequencing and in silico identification of biosynthetic gene clusters that encode the machinery necessary for the synthesis of specialized metabolites, offers the opportunity to revisit microbial natural product libraries with renewed vigor. Assembling a suitable library of microbes and extracts for screening requires the investment of resources and the development of methods that have customarily been the proprietary purview of large pharmaceutical companies. Here, we report a perspective on our efforts to assemble a library of natural product-producing microbes and the establishment of methods to extract and fractionate bioactive compounds using resources available to most academic labs. We validate the library and approach through a series of screens for antimicrobial and cytotoxic agents. This work serves as a blueprint for establishing libraries of microbial natural product producers and bioactive extract fractions suitable for screens of bioactive compounds. ONE-SENTENCE SUMMARY: Natural products are key to discovery of novel antimicrobial agents: Here, we describe our experience and lessons learned in constructing a microbial natural product and pre-fractionated extract library.

Suppression of ß-Lactam Resistance by Aspergillomarasmine A Is Influenced by both the Metallo-ß-Lactamase Target and the Antibiotic Partner.

The rise of Gram-negative pathogens expressing metallo-ß-lactamases (MBLs) is a growing concern, threatening the efficacy of ß-lactam antibiotics, in particular, the carbapenems. There are no inhibitors of MBLs in current clinical use. Aspergillomarasmine A (AMA) is an MBL inhibitor isolated from Aspergillus versicolor with the ability to rescue meropenem activity in MBL-producing bacteria both in vitro and in vivo Here, we systematically explored the pairing of AMA with six ß-lactam antibiotic partners against 19 MBLs from three subclasses (B1, B2, and B3). Cell-based assays performed with Escherichia coli and Klebsiella pneumoniae showed that bacteria producing NDM-1 and VIM-2 of subclass B1 were the most susceptible to AMA inhibition, whereas bacteria producing CphA2 and AIM-1 of subclasses B2 and B3, respectively, were the least sensitive. Intracellular antibiotic accumulation assays and in vitro enzyme assays demonstrated that the efficacy of AMA/ß-lactam combinations did not correlate with outer membrane permeability or drug efflux. We determined that the optimal ß-lactam partners for AMA are the carbapenem antibiotics and that the efficacy of AMA is linked to the Zn2+ affinity of specific MBLs.

A rifamycin inactivating phosphotransferase family shared by environmental and pathogenic bacteria.

Many environmental bacteria are multidrug-resistant and represent a reservoir of ancient antibiotic resistance determinants, which have been linked to genes found in pathogens. Exploring the environmental antibiotic resistome, therefore, reveals the diversity and evolution of antibiotic resistance and also provides insight into the vulnerability of clinically used antibiotics. In this study, we describe the identification of a highly conserved regulatory motif, the rifampin (RIF) -associated element (RAE), which is found upstream of genes encoding RIF-inactivating enzymes from a diverse collection of actinomycetes. Using gene expression assays, we confirmed that the RAE is involved in RIF-responsive regulation. By using the RAE as a probe for new RIF-associated genes in several actinomycete genomes, we identified a heretofore unknown RIF resistance gene, RIF phosphotransferase (rph). The RPH enzyme is a RIF-inactivating phosphotransferase and represents a new protein family in antibiotic resistance. RPH orthologs are widespread and found in RIF-sensitive bacteria, including Bacillus cereus and the pathogen Listeria monocytogenes. Heterologous expression and in vitro enzyme assays with purified RPHs from diverse bacterial genera show that these enzymes are capable of conferring high-level resistance to a variety of clinically used rifamycin antibiotics. This work identifies a new antibiotic resistance protein family and reinforces the fact that the study of resistance in environmental organisms can serve to identify resistance elements with relevance to pathogens.

Total Synthesis and Activity of the Metallo-ß-lactamase Inhibitor Aspergillomarasmine†A.

Resistance to ß-lactam antibiotics is mediated primarily by enzymes that hydrolytically inactivate the drugs by one of two mechanisms: serine nucleophilic attack or metal-dependent activation of a water molecule. Serine ß-lactamases are countered in the clinic by several codrugs that inhibit these enzymes, thereby rescuing antibiotic action. There are no equivalent inhibitors of metallo-ß-lactamases in clinical use, but the fungal secondary metabolite aspergillomarasmine A has recently been identified as a potential candidate for such a codrug. Herein we report the synthesis of aspergillomarasmine A. The synthesis enabled confirmation of the stereochemical configuration of the compound and offers a route for the synthesis of derivatives in the future.

Total Synthesis of Aspergillomarasmine†A and Related Compounds: A Sulfamidate Approach Enables Exploration of Structure-Activity Relationships.

The fungal secondary metabolite aspergillomarasmine A (AMA) has recently been identified as an inhibitor of metallo-ß-lactamases NDM-1 and VIM-2. Described herein is an efficient and practical route to AMA and its related compounds by a sulfamidate approach. In addition, a series of derivatives has been prepared and tested for biological activity in an effort to explore preliminary structure activity relationships. While it was determined that natural LLL isomer of AMA remains the most effective inactivator of NDM-1 enzyme activity both in vitro and in cells, the structure is highly tolerant of the changes in the stereochemistry at positions 3, 6, and 9.

Harnessing the synthetic capabilities of glycopeptide antibiotic tailoring enzymes: characterization of the UK-68,597 biosynthetic cluster.

In this study, a draft genome sequence of Actinoplanes sp. ATCC 53533 was assembled, and an 81-kb biosynthetic cluster for the unusual sulfated glycopeptide UK-68,597 was identified. Glycopeptide antibiotics are important in the treatment of infections caused by Gram-positive bacteria. Glycopeptides contain heptapeptide backbones that are modified by many tailoring enzymes, including glycosyltransferases, sulfotransferases, methyltransferases, and halogenases, generating extensive chemical and functional diversity. Several tailoring enzymes in the cluster were examined in vitro for their ability to modify glycopeptides, resulting in the synthesis of novel molecules. Tailoring enzymes were also expressed in the producer of the glycopeptide aglycone A47934, generating additional chemical diversity. This work characterizes the biosynthetic program of UK-68,597 and demonstrates the capacity to expand glycopeptide chemical diversity by harnessing the unique chemistry of tailoring enzymes.

An unusual class of anthracyclines potentiate Gram-positive antibiotics in intrinsically resistant Gram-negative bacteria.

OBJECTIVES: An orthogonal approach taken towards novel antibacterial drug discovery involves the identification of small molecules that potentiate or enhance the activity of existing antibacterial agents. This study aimed to identify natural-product rifampicin adjuvants in the intrinsically resistant organism Escherichia coli. METHODS: E. coli BW25113 was screened against 1120 actinomycete fermentation extracts in the presence of subinhibitory (2 mg/L) concentrations of rifampicin. The active molecule exhibiting the greatest rifampicin potentiation was isolated using activity-guided methods and identified using mass and NMR spectroscopy. Susceptibility testing and biochemical assays were used to determine the mechanism of antibiotic potentiation. RESULTS: The anthracycline Antibiotic 301A(1) was isolated from the fermentation broth of a strain of Streptomyces (WAC450); the molecule was shown to be highly synergistic with rifampicin (fractional inhibitory concentration index = 0.156) and moderately synergistic with linezolid (FIC index = 0.25) in both E. coli and Acinetobacter baumannii. Activity was associated with inhibition of efflux and the synergistic phenotype was lost when tested against E. coli harbouring mutations within the rpoB gene. Structure-activity relationship studies revealed that other anthracyclines do not synergize with rifampicin and removal of the sugar moiety of Antibiotic 301A(1) abolishes activity. CONCLUSIONS: Screening only a subsection of our natural product library identified a small-molecule antibiotic adjuvant capable of sensitizing Gram-negative bacteria to antibiotics to which they are ordinarily intrinsically resistant. This result demonstrates the great potential of this approach in expanding antibiotic effectiveness in the face of the growing challenge of resistance in Gram-negatives.

Designing analogs of ticlopidine, a wall teichoic acid inhibitor, to avoid formation of its oxidative metabolites.

The thienopyridine antiplatelet agent, ticlopidine and its analog, clopidogrel, have been shown to potentiate the action of ß-lactam antibiotics, reversing the methicillin-resistance phenotype of methicillin-resistant Staphylococcus aureus (MRSA), in vitro. Interestingly, these thienopyridines inhibit the action of TarO, the first enzyme in the synthesis of wall teichoic acid, an important cell wall polymer in Gram-positive bacteria. In the human body, both ticlopidine and clopidogrel undergo a rapid P450-dependent oxidation into their respective antiplatelet-active metabolites, resulting in very low plasma concentrations of intact drug. Herein, a series of analogs of ticlopidine and clopidogrel that would avoid oxidative metabolism were designed, prepared and evaluated as inhibitors of TarO. Specifically, we replaced the P450-labile thiophene ring of ticlopidine and clopidogrel to a more stable phenyl group to generate 2-(2-chlorobenzyl)-1,2,3,4-tetrahydro-isoquinoline) (6) and (2-chloro-phenyl)-(3,4-dihydro-1H-isoquinolin-2-yl)-acetic acid methyl ester (22), respectively. The latter molecules displayed inhibitory activity against TarO and formed the basis of a library of analogs. Most synthesized compounds exhibited comparable efficacy to ticlopidine and clopidogrel. So far, it was introduction of a trifluoromethyl group to compound 6, to generate 2-(2-trifluoromethyl-benzyl)-1,2,3,4-tetrahydro-isoquinoline (13) that exhibited enhanced activity against TarO. Compound 13 represents a novel stable inhibitor of TarO with synergistic impact on ß-lactam antibiotics against MRSA and low potential for P-450 metabolism.

Targeting bacterial nickel transport with aspergillomarasmine A suppresses virulence-associated Ni-dependent enzymes.

Microbial Ni2+ homeostasis underpins the virulence of several clinical pathogens. Ni2+ is an essential cofactor in urease and [NiFe]-hydrogenases involved in colonization and persistence. Many microbes produce metallophores to sequester metals necessary for their metabolism and starve competing neighboring organisms. The fungal metallophore aspergillomarasmine A (AMA) shows narrow specificity for Zn2+, Ni2+, and Co2+. Here, we show that this specificity allows AMA to block the uptake of Ni2+ and attenuate bacterial Ni-dependent enzymes, offering a potential strategy for reducing virulence. Bacterial exposure to AMA perturbs H2 metabolism, ureolysis, struvite crystallization, and biofilm formation and shows efficacy in a Galleria mellonella animal infection model. The inhibition of Ni-dependent enzymes was aided by Zn2+, which complexes with AMA and competes with the native nickelophore for the uptake of Ni2+. Biochemical analyses demonstrated high-affinity binding of AMA-metal complexes to NikA, the periplasmic substrate-binding protein of the Ni2+ uptake system. Structural examination of NikA in complex with Ni-AMA revealed that the coordination geometry of Ni-AMA mimics the native ligand, Ni-(L-His)2, providing a structural basis for binding AMA-metal complexes. Structure-activity relationship studies of AMA identified regions of the molecule that improve NikA affinity and offer potential routes for further developing this compound as an anti-virulence agent.

Glycopeptide sulfation evades resistance.

The incidence of antibiotic resistance among pathogenic microorganisms is increasing at an alarming rate. Resistance against front-line therapeutics such as the glycopeptide antibiotic vancomycin has emerged and has spread to highly virulent pathogens, including Staphylococcus aureus. Glycopeptide antibiotics are natural products from the Actinomycetes that have a characteristic heptapeptide core. The chemical diversity of the class is achieved through glycosylation, halogenation, methylation, and acylation of the core, modifications that are implicated in improved solubility, stability, or activity of the molecule. Sulfation is yet another modification observed infrequently in glycopeptides, but its role is not known. Although glycopeptide sulfotransferases are found in the environmental metagenome and must therefore serve an evolutionary purpose, all previous studies have reported decreased antibiotic activity with sulfation. We report that sulfation of glycopeptides has little effect on the compound's ability to bind its target, the d-Ala-d-Ala peptidoglycan precursors of the bacterial cell wall. However, sulfation does impact glycopeptide dimerization, and importantly, sulfated glycopeptides are significantly less potent inducers of the resistance gene cluster vanHAX in actinomycetes. Our results begin to unravel the mystery of the biological role of glycopeptide sulfation and offer a potential new strategy for the development of new antibiotics that avoid resistance.

The comprehensive antibiotic resistance database.

The field of antibiotic drug discovery and the monitoring of new antibiotic resistance elements have yet to fully exploit the power of the genome revolution. Despite the fact that the first genomes sequenced of free living organisms were those of bacteria, there have been few specialized bioinformatic tools developed to mine the growing amount of genomic data associated with pathogens. In particular, there are few tools to study the genetics and genomics of antibiotic resistance and how it impacts bacterial populations, ecology, and the clinic. We have initiated development of such tools in the form of the Comprehensive Antibiotic Research Database (CARD; http://arpcard.mcmaster.ca). The CARD integrates disparate molecular and sequence data, provides a unique organizing principle in the form of the Antibiotic Resistance Ontology (ARO), and can quickly identify putative antibiotic resistance genes in new unannotated genome sequences. This unique platform provides an informatic tool that bridges antibiotic resistance concerns in health care, agriculture, and the environment.

Synthetic Biology Facilitates Semisynthetic Development of Type V Glycopeptide Antibiotics Targeting Vancomycin-Resistant Enterococcus.

The continued efficacy of glycopeptide antibiotics (GPAs) against Gram-positive bacteria is challenged by the emergence and spread of GPA-resistant pathogens, particularly vancomycin-resistant enterococci (VRE). The growing frequency of GPA resistance propels the need for innovative development of more effective antibiotics. Unlike canonical GPAs like vancomycin, Type V GPAs adopt a distinct mode of action by binding peptidoglycan and blocking the activity of autolysins essential for cell division, rendering them a promising class of antibiotics for further development. In this study, the Type V GPA, rimomycin A, was modified to generate 32 new analogues. Compound 17, derived from rimomycin A through N-terminal acylation and C-terminal amidation, exhibited improved anti-VRE activity and solubility. In a VRE-A neutropenic thigh infection mouse model, compound 17 significantly lowered the bacterial load by 3-4 orders of magnitude. This study sets the stage to develop next-generation GPAs in response to growing VRE infections.

Mechanism and diversity of the erythromycin esterase family of enzymes.

Macrolide antibiotics such as azithromycin and erythromycin are mainstays of modern antibacterial chemotherapy, and like all antibiotics, they are vulnerable to resistance. One mechanism of macrolide resistance is via drug inactivation: enzymatic hydrolysis of the macrolactone ring catalyzed by erythromycin esterases, EreA and EreB. A genomic enzymology approach was taken to gain insight into the catalytic mechanisms and origins of Ere enzymes. Our analysis reveals that erythromycin esterases comprise a separate group in the hydrolase superfamily, which includes homologues of uncharacterized function found on the chromosome of Bacillus cereus, Bcr135 and Bcr136, whose three-dimensional structures have been determined. Biochemical characterization of Bcr136 confirms that it is an esterase that is, however, unable to inactivate macrolides. Using steady-state kinetics, homology-based structure modeling, site-directed mutagenesis, solvent isotope effect studies, pH, and inhibitor profiling performed in various combinations for EreA, EreB, and Bcr136 enzymes, we identified the active site and gained insight into some catalytic features of this novel enzyme superfamily. We rule out the possibility of a Ser/Thr nucleophile and show that one histidine, H46 (EreB numbering), is essential for catalytic function. This residue is proposed to serve as a general base in activation of a water molecule as the reaction nucleophile. Furthermore, we show that EreA, EreB, and Bcr136 are distinct, with only EreA inhibited by chelating agents and hypothesized to contain a noncatalytic metal. Detailed characterization of these esterases allows for a direct comparison of the resistance determinants, EreA and EreB, with their prototype, Bcr136, and for the discussion of their potential connections.

Characterization of a rifampin-inactivating glycosyltransferase from a screen of environmental actinomycetes.

Identifying and understanding the collection of all antibiotic resistance determinants presented in the global microbiota, the antibiotic resistome, provides insight into the evolution of antibiotic resistance and critical information for the development of future antimicrobials. The rifamycins are broad-spectrum antibiotics that target bacterial transcription by inhibition of RNA polymerase. Although mutational alteration of the drug target is the predominant mechanism of resistance to this family of antibiotics in the clinic, a number of diverse inactivation mechanisms have also been reported. In this report, we investigate a subset of environmental rifampin-resistant actinomycete isolates and identify a diverse collection of rifampin inactivation mechanisms. We describe a single isolate, WAC1438, capable of inactivating rifampin by glycosylation. A draft genome sequence of WAC1438 (most closely related to Streptomyces speibonae, according to a 16S rRNA gene comparison) was assembled, and the associated rifampin glycosyltransferase open reading frame, rgt1438, was identified. The role of rgt1438 in rifampin resistance was confirmed by its disruption in the bacterial chromosome, resulting in a loss of antibiotic inactivation and a 4-fold decrease in MIC. Interestingly, examination of the RNA polymerase ß-subunit sequence of WAC1438 suggests that it harbors a resistant target and thus possesses dual mechanisms of rifamycin resistance. Using an in vitro assay with purified enzyme, Rgt1438 could inactivate a variety of rifamycin antibiotics with comparable steady-state kinetics constants. Our results identify rgt1438 as a rifampin resistance determinant from WAC1438 capable of inactivating an assortment of rifamycins, adding a new element to the rifampin resistome.

Inactivation of the lipopeptide antibiotic daptomycin by hydrolytic mechanisms.

The lipopeptide daptomycin is a member of the newest FDA-approved antimicrobial class, exhibiting potency against a broad range of Gram-positive pathogens with only rare incidences of clinical resistance. Environmental bacteria harbor an abundance of resistance determinants orthologous to those in pathogens and thus may serve as an early-warning system for future clinical emergence. A collection of morphologically diverse environmental actinomycetes demonstrating unprecedented frequencies of daptomycin resistance and high levels of resistance by antibiotic inactivation was characterized to elucidate modes of drug inactivation. In vivo studies revealed that hydrolysis plays a key role, resulting in one or both of the following structural modifications: ring hydrolysis resulting in linearization (in 44% of inactivating isolates) or deacylation of the lipid tail (29%). Characterization of the mechanism in actinomycete WAC4713 (a Streptomyces sp. with an MIC of 512 µg/ml) demonstrated a constitutive resistance phenotype and established daptomycin's circularizing ester linkage to be the site of hydrolysis. Characterization of the hydrolase responsible revealed it to be likely a serine protease. These studies suggested that daptomycin is susceptible to general proteolytic hydrolysis, which was further supported by studies using proteases of diverse origin. These findings represent the first comprehensive characterization of daptomycin inactivation in any bacterial class and may not only presage a future mechanism of clinical resistance but also suggest strategies for the development of new lipopeptides.

Small-molecule modulators of Listeria monocytogenes biofilm development.

Listeria monocytogenes is an important food-borne pathogen whose ability to form disinfectant-tolerant biofilms on a variety of surfaces presents a food safety challenge for manufacturers of ready-to-eat products. We developed here a high-throughput biofilm assay for L. monocytogenes and, as a proof of principle, used it to screen an 80-compound protein kinase inhibitor library to identify molecules that perturb biofilm development. The screen yielded molecules toxic to multiple strains of Listeria at micromolar concentrations, as well as molecules that decreased (≤ 50% of vehicle control) or increased (≥ 200%) biofilm formation in a dose-dependent manner without affecting planktonic cell density. Toxic molecules-including the protein kinase C antagonist sphingosine-had antibiofilm activity at sub-MIC concentrations. Structure-activity studies of the biofilm inhibitory compound palmitoyl-d,l-carnitine showed that while Listeria biofilm formation was inhibited with a 50% inhibitory concentration of 5.85 ± 0.24 µM, d,l-carnitine had no effect, whereas palmitic acid had stimulatory effects. Saturated fatty acids between C(9:0) and C(14:0) were Listeria biofilm inhibitors, whereas fatty acids of C(16:0) or longer were stimulators, showing chain length specificity. De novo-synthesized short-chain acyl carnitines were less effective biofilm inhibitors than the palmitoyl forms. These molecules, whose activities against bacteria have not been previously established, are both useful probes of L. monocytogenes biology and promising leads for the further development of antibiofilm strategies.