Viral Capsid Trafficking along Treadmilling Tubulin Filaments in Bacteria.

Cargo trafficking along microtubules is exploited by eukaryotic viruses, but no such examples have been reported in bacteria. Several large Pseudomonas phages assemble a dynamic, tubulin-based (PhuZ) spindle that centers replicating phage DNA sequestered within a nucleus-like structure. Here, we show that capsids assemble on the membrane and then move rapidly along PhuZ filaments toward the phage nucleus for DNA packaging. The spindle rotates the phage nucleus, distributing capsids around its surface. PhuZ filaments treadmill toward the nucleus at a constant rate similar to the rate of capsid movement and the linear velocity of nucleus rotation. Capsids become trapped along mutant static PhuZ filaments that are defective in GTP hydrolysis. Our results suggest a transport and distribution mechanism in which capsids attached to the sides of filaments are trafficked to the nucleus by PhuZ polymerization at the poles, demonstrating that the phage cytoskeleton evolved cargo-trafficking capabilities in bacteria.

Architecture and self-assembly of the jumbo bacteriophage nuclear shell.

Bacteria encode myriad defences that target the genomes of infecting bacteriophage, including restriction-modification and CRISPR-Cas systems1. In response, one family of large bacteriophages uses a nucleus-like compartment to protect its replicating genomes by excluding host defence factors2-4. However, the principal composition and structure of this compartment remain unknown. Here we find that the bacteriophage nuclear shell assembles primarily from one protein, which we name chimallin (ChmA). Combining cryo-electron tomography of nuclear shells in bacteriophage-infected cells and cryo-electron microscopy of a minimal chimallin compartment in vitro, we show that chimallin self-assembles as a flexible sheet into closed micrometre-scale compartments. The architecture and assembly dynamics of the chimallin shell suggest mechanisms for its nucleation and growth, and its role as a scaffold for phage-encoded factors mediating macromolecular transport, cytoskeletal interactions, and viral maturation.

HORMA Domain Proteins and a Trip13-like ATPase Regulate Bacterial cGAS-like Enzymes to Mediate Bacteriophage Immunity.

Bacteria are continually challenged by foreign invaders, including bacteriophages, and have evolved a variety of defenses against these invaders. Here, we describe the structural and biochemical mechanisms of a bacteriophage immunity pathway found in a broad array of bacteria, including E. coli and Pseudomonas aeruginosa. This pathway uses eukaryotic-like HORMA domain proteins that recognize specific peptides, then bind and activate a cGAS/DncV-like nucleotidyltransferase (CD-NTase) to generate a cyclic triadenylate (cAAA) second messenger; cAAA in turn activates an endonuclease effector, NucC. Signaling is attenuated by a homolog of the AAA+ ATPase Pch2/TRIP13, which binds and disassembles the active HORMA-CD-NTase complex. When expressed in non-pathogenic E. coli, this pathway confers immunity against bacteriophage λ through an abortive infection mechanism. Our findings reveal the molecular mechanisms of a bacterial defense pathway integrating a cGAS-like nucleotidyltransferase with HORMA domain proteins for threat sensing through protein detection and negative regulation by a Trip13 ATPase.

Structure and Mechanism of a Cyclic Trinucleotide-Activated Bacterial Endonuclease Mediating Bacteriophage Immunity.

Bacteria possess an array of defenses against foreign invaders, including a broadly distributed bacteriophage defense system termed CBASS (cyclic oligonucleotide-based anti-phage signaling system). In CBASS systems, a cGAS/DncV-like nucleotidyltransferase synthesizes cyclic di- or tri-nucleotide second messengers in response to infection, and these molecules activate diverse effectors to mediate bacteriophage immunity via abortive infection. Here, we show that the CBASS effector NucC is related to restriction enzymes but uniquely assembles into a homotrimer. Binding of NucC trimers to a cyclic tri-adenylate second messenger promotes assembly of a NucC homohexamer competent for non-specific double-strand DNA cleavage. In infected cells, NucC activation leads to complete destruction of the bacterial chromosome, causing cell death prior to completion of phage replication. In addition to CBASS systems, we identify NucC homologs in over 30 type III CRISPR/Cas systems, where they likely function as accessory nucleases activated by cyclic oligoadenylate second messengers synthesized by these systems' effector complexes.

A phage tubulin assembles dynamic filaments by an atypical mechanism to center viral DNA within the host cell.

Tubulins are essential for the reproduction of many eukaryotic viruses, but historically, bacteriophage were assumed not to require a cytoskeleton. Here, we identify a tubulin-like protein, PhuZ, from bacteriophage 201φ2-1 and show that it forms filaments in vivo and in vitro. The PhuZ structure has a conserved tubulin fold, with an unusual, extended C terminus that we demonstrate to be critical for polymerization in vitro and in vivo. Longitudinal packing in the crystal lattice mimics packing observed by EM of in-vitro-formed filaments, indicating how interactions between the C terminus and the following monomer drive polymerization. PhuZ forms a filamentous array that is required for positioning phage DNA within the bacterial cell. Correct positioning to the cell center and optimal phage reproduction only occur when the PhuZ filament is dynamic. Thus, we show that PhuZ assembles a spindle-like array that functions analogously to the microtubule-based spindles of eukaryotes.

An essential and highly selective protein import pathway encoded by nucleus-forming phage.

Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here, we identify two components of this protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA (Protein importer of chimalliviruses A), that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together, our results allow us to propose a multistep model for the Protein Import Chimallivirus pathway, where proteins are targeted to PicA by amino acids on their surface and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts.

A bacteriophage nucleus-like compartment shields DNA from CRISPR nucleases.

All viruses require strategies to inhibit or evade the immune pathways of cells that they infect. The viruses that infect bacteria, bacteriophages (phages), must avoid immune pathways that target nucleic acids, such as CRISPR-Cas and restriction-modification systems, to replicate efficiently1. Here we show that jumbo phage ΦKZ segregates its DNA from immunity nucleases of its host, Pseudomonas aeruginosa, by constructing a proteinaceous nucleus-like compartment. ΦKZ is resistant to many immunity mechanisms that target DNA in vivo, including two subtypes of CRISPR-Cas3, Cas9, Cas12a and the restriction enzymes HsdRMS and EcoRI. Cas proteins and restriction enzymes are unable to access the phage DNA throughout the infection, but engineering the relocalization of EcoRI inside the compartment enables targeting of the phage and protection of host cells. Moreover, ΦKZ is sensitive to Cas13a-a CRISPR-Cas enzyme that targets RNA-probably owing to phage mRNA localizing to the cytoplasm. Collectively, we propose that Pseudomonas jumbo phages evade a broad spectrum of DNA-targeting nucleases through the assembly of a protein barrier around their genome.

A phage nucleus-associated RNA-binding protein is required for jumbo phage infection.

Large-genome bacteriophages (jumbo phages) of the proposed family Chimalliviridae assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and DNA-targeting CRISPR-Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here, we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d results in accumulation of phage-encoded mRNAs in the phage nucleus, reduces phage protein production, and compromises virion assembly. Taken together, our data show that the conserved ChmC protein plays crucial roles in the viral life cycle, potentially by facilitating phage mRNA translocation through the nuclear shell to promote protein production and virion development.

Machine learning from Pseudomonas aeruginosa transcriptomes identifies independently modulated sets of genes associated with known transcriptional regulators.

The transcriptional regulatory network (TRN) of Pseudomonas aeruginosa coordinates cellular processes in response to stimuli. We used 364 transcriptomes (281 publicly available + 83 in-house generated) to reconstruct the TRN of P. aeruginosa using independent component analysis. We identified 104 independently modulated sets of genes (iModulons) among which 81 reflect the effects of known transcriptional regulators. We identified iModulons that (i) play an important role in defining the genomic boundaries of biosynthetic gene clusters (BGCs), (ii) show increased expression of the BGCs and associated secretion systems in nutrient conditions that are important in cystic fibrosis, (iii) show the presence of a novel ribosomally synthesized and post-translationally modified peptide (RiPP) BGC which might have a role in P. aeruginosa virulence, (iv) exhibit interplay of amino acid metabolism regulation and central metabolism across different carbon sources and (v) clustered according to their activity changes to define iron and sulfur stimulons. Finally, we compared the identified iModulons of P. aeruginosa with those previously described in Escherichia coli to observe conserved regulons across two Gram-negative species. This comprehensive TRN framework encompasses the majority of the transcriptional regulatory machinery in P. aeruginosa, and thus should prove foundational for future research into its physiological functions.

Advanced transcriptomic analysis reveals the role of efflux pumps and media composition in antibiotic responses of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen and major cause of hospital-acquired infections. The virulence of P. aeruginosa is largely determined by its transcriptional regulatory network (TRN). We used 411 transcription profiles of P. aeruginosa from diverse growth conditions to construct a quantitative TRN by identifying independently modulated sets of genes (called iModulons) and their condition-specific activity levels. The current study focused on the use of iModulons to analyze the biofilm production and antibiotic resistance of P. aeruginosa. Our analysis revealed: (i) 116 iModulons, 81 of which show strong association with known regulators; (ii) novel roles of regulators in modulating antibiotics efflux pumps; (iii) substrate-efflux pump associations; (iv) differential iModulon activity in response to beta-lactam antibiotics in bacteriological and physiological media; (v) differential activation of 'Cell Division' iModulon resulting from exposure to different beta-lactam antibiotics and (vi) a role of the PprB iModulon in the stress-induced transition from planktonic to biofilm lifestyle. In light of these results, the construction of an iModulon-based TRN provides a transcriptional regulatory basis for key aspects of P. aeruginosa infection, such as antibiotic stress responses and biofilm formation. Taken together, our results offer a novel mechanistic understanding of P. aeruginosa virulence.

High-Resolution Bacterial Cytological Profiling Reveals Intrapopulation Morphological Variations upon Antibiotic Exposure.

Phenotypic heterogeneity is crucial to bacterial survival and could provide insights into the mechanism of action (MOA) of antibiotics, especially those with polypharmacological actions. Although phenotypic changes among individual cells could be detected by existing profiling methods, due to the data complexity, only population average data were commonly used, thereby overlooking the heterogeneity. In this study, we developed a high-resolution bacterial cytological profiling method that can capture morphological variations of bacteria upon antibiotic treatment. With an unprecedented single-cell resolution, this method classifies morphological changes of individual cells into known MOAs with an overall accuracy above 90%. We next showed that combinations of two antibiotics induce altered cell morphologies that are either unique or similar to that of an antibiotic in the combinations. With these combinatorial profiles, this method successfully revealed multiple cytological changes caused by a natural product-derived compound that, by itself, is inactive against Acinetobacter baumannii but synergistically exerts its multiple antibacterial activities in the presence of colistin. The findings have paved the way for future single-cell profiling in bacteria and have highlighted previously underappreciated intrapopulation variations caused by antibiotic perturbation.

Jumbo phages are active against extensively drug-resistant eyedrop-associated Pseudomonas aeruginosa infections.

Antibiotic-resistant bacteria present an emerging challenge to human health. Their prevalence has been increasing across the globe due in part to the liberal use of antibiotics that has pressured them to develop resistance. Those bacteria that acquire mobile genetic elements are especially concerning because those plasmids may be shared readily with other microbes that can then also become antibiotic resistant. Serious infections have recently been related to the contamination of preservative-free eyedrops with extensively drug-resistant (XDR) isolates of Pseudomonas aeruginosa, already resulting in three deaths. These drug-resistant isolates cannot be managed with most conventional antibiotics. We sought to identify alternatives to conventional antibiotics for the lysis of these XDR isolates and identified multiple bacteriophages (viruses that attack bacteria) that killed them efficiently. We found both jumbo phages (>200 kb in genome size) and non-jumbo phages that were active against these isolates, the former killing more efficiently. Jumbo phages effectively killed the three separate XDR P. aeruginosa isolates both on solid and liquid medium. Given the ongoing nature of the XDR P. aeruginosa eyedrop outbreak, the identification of phages active against them provides physicians with several novel potential alternatives for treatment.

Revealing 29 sets of independently modulated genes in Staphylococcus aureus, their regulators, and role in key physiological response.

The ability of Staphylococcus aureus to infect many different tissue sites is enabled, in part, by its transcriptional regulatory network (TRN) that coordinates its gene expression to respond to different environments. We elucidated the organization and activity of this TRN by applying independent component analysis to a compendium of 108 RNA-sequencing expression profiles from two S. aureus clinical strains (TCH1516 and LAC). ICA decomposed the S. aureus transcriptome into 29 independently modulated sets of genes (i-modulons) that revealed: 1) High confidence associations between 21 i-modulons and known regulators; 2) an association between an i-modulon and σS, whose regulatory role was previously undefined; 3) the regulatory organization of 65 virulence factors in the form of three i-modulons associated with AgrR, SaeR, and Vim-3; 4) the roles of three key transcription factors (CodY, Fur, and CcpA) in coordinating the metabolic and regulatory networks; and 5) a low-dimensional representation, involving the function of few transcription factors of changes in gene expression between two laboratory media (RPMI, cation adjust Mueller Hinton broth) and two physiological media (blood and serum). This representation of the TRN covers 842 genes representing 76% of the variance in gene expression that provides a quantitative reconstruction of transcriptional modules in S. aureus, and a platform enabling its full elucidation.

Intrinsic Antibacterial Activity of Xeruborbactam In Vitro: Assessing Spectrum and Mode of Action.

Xeruborbactam (formerly QPX7728) is a cyclic boronate inhibitor of numerous serine and metallo-beta-lactamases. At concentrations generally higher than those required for beta-lactamase inhibition, xeruborbactam has direct antibacterial activity against some Gram-negative bacteria, with MIC50/MIC90 values of 16/32 µg/mL and 16/64 µg/mL against carbapenem-resistant Enterobacterales and carbapenem-resistant Acinetobacter baumannii, respectively (the MIC50/MIC90 values against Pseudomonas aeruginosa are >64 µg/mL). In Klebsiella pneumoniae, inactivation of OmpK36 alone or in combination with OmpK35 resulted in 2- to 4-fold increases in the xeruborbactam MIC. In A. baumannii and P. aeruginosa, AdeIJK and MexAB-OprM, respectively, affected xeruborbactam's antibacterial potency (the MICs were 4- to 16-fold higher in efflux-proficient strains). In Escherichia coli and K. pneumoniae, the 50% inhibitory concentrations (IC50s) of xeruborbactam's binding to penicillin-binding proteins (PBPs) PBP1a/PBP1b, PBP2, and PBP3 were in the 40 to 70 µM range; in A. baumannii, xeruborbactam bound to PBP1a, PBP2, and PBP3 with IC50s of 1.4 µM, 23 µM, and 140 µM, respectively. Treating K. pneumoniae and P. aeruginosa with xeruborbactam at 1× and 2× MIC resulted in changes of cellular morphology similar to those observed with meropenem; the morphological changes observed after treatment of A. baumannii were consistent with inhibition of multiple PBPs but were unique to xeruborbactam compared to the results for control beta-lactams. No single-step xeruborbactam resistance mutants were obtained after selection at 4× MIC of xeruborbactam using wild-type strains of E. coli, K. pneumoniae, and A. baumannii; mutations selected at 2× MIC in K. pneumoniae did not affect antibiotic potentiation by xeruborbactam through beta-lactamase inhibition. Consistent with inhibition of PBPs, xeruborbactam enhanced the potencies of beta-lactam antibiotics even against strains that lacked beta-lactamase. In a large panel of KPC-producing clinical isolates, the MIC90 values of meropenem tested with xeruborbactam (8 µg/mL) were at least 4-fold lower than those in combination with vaborbactam at 64 µg/mL, the concentration of vaborbactam that is associated with complete inhibition of KPC. The additional enhancement of the potency of beta-lactam antibiotics beyond beta-lactamase inhibition may contribute to the potentiation of beta-lactam antibiotics by xeruborbactam.

Bacterial Cytological Profiling Identifies Rhodanine-Containing PAINS Analogs as Specific Inhibitors of Escherichia coli Thymidylate Kinase In Vivo.

In this study, we sought to determine whether an in vivo assay for studying antibiotic mechanisms of action could provide insight into the activity of compounds that may inhibit multiple targets. Thus, we conducted an activity screen of 31 structural analogs of rhodanine-containing pan-assay interference compounds (PAINS). We identified nine active molecules against Escherichia coli and classified them according to their in vivo mechanisms of action. The mechanisms of action of PAINS are generally difficult to identify due to their promiscuity. However, we leveraged bacterial cytological profiling, a fluorescence microscopy technique, to study these complex mechanisms. Ultimately, we found that although some of our molecules promiscuously inhibit multiple cellular pathways, a few molecules specifically inhibit DNA replication despite structural similarity to related PAINS. A genetic analysis of resistant mutants revealed thymidylate kinase (essential for DNA synthesis) as an intracellular target of some of these rhodanine-containing antibiotics. This finding was supported by in vitro activity assays, as well as experiments utilizing a thymidylate kinase overexpression system. The analog that demonstrated the half-maximal inhibitory concentration in vitro and MIC in vivo displayed the greatest specificity for inhibition of the DNA replication pathway, despite containing a rhodamine moiety. Although it is thought that PAINS cannot be developed as antibiotics, this work showcases novel inhibitors of E. coli thymidylate kinase. Moreover, perhaps more importantly, this work highlights the utility of bacterial cytological profiling for studying the in vivo specificity of antibiotics and demonstrates that bacterial cytological profiling can identify multiple pathways that are inhibited by an individual molecule. IMPORTANCE We demonstrate that bacterial cytological profiling is a powerful tool for directing antibiotic discovery efforts because it can be used to determine the specificity of an antibiotic's in vivo mechanism of action. By assaying analogs of PAINS, molecules that are notoriously intractable and nonspecific, we (surprisingly) identify molecules with specific activity against E. coli thymidylate kinase. This suggests that structural modifications to PAINS can confer stronger inhibition by targeting a specific cellular pathway. While in vitro inhibition assays are susceptible to false-positive results (especially from PAINS), bacterial cytological profiling provides the resolution to identify molecules with specific in vivo activity.

Avibactam Sensitizes Carbapenem-Resistant NDM-1-Producing Klebsiella pneumoniae to Innate Immune Clearance.

Infections caused by New Delhi metallo-ß-lactamase (NDM)-producing strains of multidrug-resistant Klebsiella pneumoniae are a global public health threat lacking reliable therapies. NDM is impervious to all existing ß-lactamase inhibitor (BLI) drugs, including the non-ß-lactam BLI avibactam (AVI). Though lacking direct activity against NDMs, AVI can interact with penicillin-binding protein 2 in a manner that may influence cell wall dynamics. We found that exposure of NDM-1-producing K. pneumoniae to AVI led to striking bactericidal interactions with human cathelicidin antimicrobial peptide LL-37, a frontline component of host innate immunity. Moreover, AVI markedly sensitized NDM-1-producing K. pneumoniae to killing by freshly isolated human neutrophils, platelets, and serum when complement was active. Finally, AVI monotherapy reduced lung counts of NDM-1-producing K. pneumoniae in a murine pulmonary challenge model. AVI sensitizes NDM-1-producing K. pneumoniae to innate immune clearance in ways that are not appreciated by standard antibiotic testing and that merit further study.

Bacterial Cytological Profiling as a Tool To Study Mechanisms of Action of Antibiotics That Are Active against Acinetobacter baumannii.

An increasing number of multidrug-resistant Acinetobacter baumannii (MDR-AB) infections have been reported worldwide, posing a threat to public health. The establishment of methods to elucidate the mechanism of action (MOA) of A. baumannii-specific antibiotics is needed to develop novel antimicrobial therapeutics with activity against MDR-AB We previously developed bacterial cytological profiling (BCP) to understand the MOA of compounds in Escherichia coli and Bacillus subtilis Given how distantly related A. baumannii is to these species, it was unclear to what extent it could be applied. Here, we implemented BCP as an antibiotic MOA discovery platform for A. baumannii We found that the BCP platform can distinguish among six major antibiotic classes and can also subclassify antibiotics that inhibit the same cellular pathway but have different molecular targets. We used BCP to show that the compound NSC145612 inhibits the growth of A. baumannii via targeting RNA transcription. We confirmed this result by isolating and characterizing resistant mutants with mutations in the rpoB gene. Altogether, we conclude that BCP provides a useful tool for MOA studies of antibacterial compounds that are active against A. baumannii.

Phenotypic consequences of RNA polymerase dysregulation in Escherichia coli.

Many bacterial adaptive responses to changes in growth conditions due to biotic and abiotic factors involve reprogramming of gene expression at the transcription level. The bacterial RNA polymerase (RNAP), which catalyzes transcription, can thus be considered as the major mediator of cellular adaptive strategies. But how do bacteria respond if a stress factor directly compromises the activity of the RNAP? We used a phage-derived small protein to specifically perturb bacterial RNAP activity in exponentially growing Escherichia coli. Using cytological profiling, tracking RNAP behavior at single-molecule level and transcriptome analysis, we reveal that adaptation to conditions that directly perturb bacterial RNAP performance can result in a biphasic growth behavior and thereby confer the 'adapted' bacterial cells an enhanced ability to tolerate diverse antibacterial stresses. The results imply that while synthetic transcriptional rewiring may confer bacteria with the intended desirable properties, such approaches may also collaterally allow them to acquire undesirable traits.

Fosfomycin Enhances the Activity of Daptomycin against Vancomycin-Resistant Enterococci in an In Vitro Pharmacokinetic-Pharmacodynamic Model.

Daptomycin (DAP) is being used more frequently to treat infections caused by vancomycin-resistant enterococcus (VRE). DAP tends to be less active against enterococci than staphylococci and may require high doses or combination therapy to be bactericidal. Fosfomycin (FOF) has activity against VRE and has demonstrated synergistic bactericidal activity with DAP in vitro The objective of this study was to evaluate the activity of DAP alone and in combination with FOF against VRE in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model. The activity of DAP at 8 and 12 mg/kg of body weight/day (DAP 8 and DAP 12, respectively) and FOF of 40 mg/kg intravenously every 8 h, alone and in combination, were evaluated against 2 vancomycin-resistant Enterococcus faecium strains (8019 and 5938) and 2 vancomycin-resistant E. faecalis strains (V583 and R7302) in an in vitro PK/PD model over 72 h. Cell surface charge in the presence and absence of FOF was evaluated by zeta potential analysis. Daptomycin-boron-dipyrromethene (bodipy) binding was assessed by fluorescence microscopy. The addition of FOF to DAP 8 and DAP 12 resulted in significantly increased killing over DAP alone at 72 h for 8019, V583, and R7302 (P < 0.05). Therapeutic enhancement was observed with DAP 12 plus FOF against 8019, V583, and R7302. Cell surface charge became more negative after exposure to FOF by ∼2 to 8mV in all 4 strains. Daptomycin-bodipy binding increased by 2.6 times in the presence of fosfomycin (P < 0.0001). The combination of DAP plus FOF may provide improved killing against VRE (including DAP-resistant strains) through modulation of cell surface charge. Further studies to clarify the role of intravenous FOF are warranted.

Standard susceptibility testing overlooks potent azithromycin activity and cationic peptide synergy against MDR Stenotrophomonas maltophilia.

OBJECTIVES: The Gram-negative bacillus Stenotrophomonas maltophilia (SM) is an emerging MDR opportunistic pathogen. Recent studies identify a potentially relevant activity of azithromycin against Gram-negative bacteria overlooked in standard bacteriological testing. We investigated azithromycin activity against SM in testing conditions incorporating mammalian tissue culture medium and host defence factors. METHODS: MIC testing, chequerboard assays, time-kill assays and fluorescence microscopy were performed for azithromycin, the cationic peptide antibiotic colistin and the human defence peptide cathelicidin LL-37 alone or in combination in cation-adjusted Mueller-Hinton broth or mammalian tissue culture media. Azithromycin sensitization of SM to host immune clearance was tested in a human neutrophil killing assay and a murine pneumonia model. RESULTS: We observed potent bactericidal activity of azithromycin against SM in mammalian tissue culture medium absent in bacteriological medium. Colistin and LL-37 strongly potentiated azithromycin killing of SM by increasing drug entry. Additionally, azithromycin sensitized SM to neutrophil killing and increased SM clearance in the murine pneumonia model. CONCLUSIONS: Despite lack of activity in standard MIC testing, azithromycin synergizes with cationic peptide antibiotics to kill SM in medium mimicking tissue fluid conditions. Azithromycin, alone or in combination with colistin, merits further exploration in therapy of drug-resistant SM infections.