Murine Abscess Model Research Articles

Using host-mimicking conditions and a murine cutaneous abscess model to identify synergistic antibiotic combinations effective against Pseudomonas aeruginosa.

Antibiotic drug combination therapy is critical for the successful treatment of infections caused by multidrug resistant pathogens. We investigated the efficacy of β-lactam and β-lactam/β-lactamase inhibitor combinations with other antibiotics, against the hypervirulent, ceftazidime/avibactam resistant Pseudomonas aeruginosa Liverpool epidemic strain (LES) B58. Although minimum inhibitory concentrations in vitro differed by up to eighty-fold between standard and host-mimicking media, combinatorial effects only marginally changed between conditions for some combinations. Effective combinations in vitro were further tested in a chronic, high-density murine infection model. Colistin and azithromycin demonstrated combinatorial effects with ceftazidime and ceftazidime/avibactam both in vitro and in vivo. Conversely, while tobramycin and tigecycline exhibited strong synergy in vitro, this effect was not observed in vivo. Our approach of using host-mimicking conditions and a sophisticated animal model to evaluate drug synergy against bacterial pathogens represents a promising approach. This methodology may offer insights into the prediction of combination therapy outcomes and the identification of potential treatment failures.

Drug repurposing: insights into the antimicrobial effects of AKBA against MRSA

Staphylococcus aureus is a major threat in infectious diseases due to its varied infection types and increased resistance. S. aureus could form persister cells under certain condition and could also attach on medical apparatus to form biofilms, which exhibited extremely high resistance to antibiotics. 3-Acetyl-11-keto-beta-boswellic acid (AKBA) is a well-studied anti-tumor and antioxidant drug. This study is aimed to determine the antimicrobial effects of AKBA against S. aureus and its persister cells and biofilms. The in vitro antimicrobial susceptibility of AKBA was assessed by micro-dilution assay, disc diffusion assay and time-killing assay. Drug combination between AKBA and conventional antibiotics was detected by checkerboard assay. And the antibiofilm effects of AKBA against S. aureus were explored by crystal violet staining combined with SYTO/PI probes staining. Next, RBC lysis activity and CCK-8 kit were used to determine the cytotoxicity of AKBA. In addition, murine subcutaneous abscess model was used to assess the antimicrobial effects of AKBA in vivo. Our results revealed that AKBA was found to show effective antimicrobial activity against methicillin-resistant S. aureus (MRSA) with the minimal inhibitory concentration of 4–8 µg/mL with undetectable cytotoxicity. And no resistant mutation was induced by AKBA after 20 days of consecutive passage. Further, we found that AKBA could be synergy with gentamycin or amikacin against S. aureus and its clinical isolates. By crystal violet and SYTO9/PI staining, AKBA exhibited strong biofilm inhibitory and eradication effects at the concentration of 1 ~ 4 µg/mL. In addition, the effective antimicrobial effect was verified in vivo in a mouse model. And no detectable in vivo toxicity was found. These results indicated that AKBA has great potential to development as an alternative treatment for the refractory S. aureus infections.

Simeprevir restores the anti-Staphylococcus activity of polymyxins

Methicillin-resistant Staphylococcus aureus (MRSA) infection poses a severe threat to global public health due to its high mortality. Currently, polymyxins are mainly used for the treatment of Gram-negative bacterial-related infection, while exhibiting limited antibacterial activities against Staphylococcus aureus (S. aureus). However, the combination of antibiotics with antibiotic adjuvants is a feasible strategy for the hard-treated infection and toxicity reducing. We will investigate the antibacterial activity of simeprevir (SIM), which treated for genotype 1 and 4 chronic hepatitis C, combined with polymyxins against MRSA through high-throughput screening technology. In our study, the synergistic antibacterial effect of SIM and polymyxins against S. aureus in vitro was found by checkerboard assay and time-growth curve. The cytotoxicity of SIM combined with polymyxin B sulfate [PB(S)] or polymyxin E (PE) in vitro was evaluated using CCK-8, human RBC hemolysis and scratch assays. In addition, we investigated the eradication of biofilm formation of S. aureus by biofilm inhibition assay and the killing of persister cells. Moreover, we evaluated the therapeutic effect and in vivo toxicity of the combination against MRSA in murine subcutaneous abscess model. Furthermore, it was preliminarily found that SIM significantly enhanced the destruction of MRSA membrane by SYTOX Green and DISC3(5) probes. In summary, these results reveal that the therapy of SIM combined with polymyxins (especially PE) is promising for the treatment of MRSA infection.

Uncovering the anti-biofilm activity of Ilicicolin B against Staphylococcus aureus

The formation of bacterial biofilms reduces the entry of antibiotics into bacteria and helps bacteria tolerate otherwise lethal concentrations of antimicrobials, leading to antibiotic resistance. Therefore, clearing bacterial biofilm is an effective strategy to tackle drug resistance. Currently, there are no approved antibiotics for inhibiting bacterial biofilm formation. We found that Ilicicolin B had excellent antibacterial activity against MRSA without obvious hemolytic activity. More importantly, Ilicicolin B effectively inhibited the biofilm formation in a concentration-dependent manner by crystal violet colorimetric assay and fluorescence microscopy analysis. Exposure of Staphylococcus aureus to Ilicicolin B for 24 h reduced the protein and polysaccharide components in EPS, suggesting that Ilicicolin B disintegrated the biofilms by dissociating the EPS in a matrix. In addition, Ilicicolin B demonstrated strong antibacterial effects in a murine abscess model of S. aureus. Our findings suggest that Ilicicolin B has the potential to treat S. aureus infection by inhibiting biofilm formation.

Effective treatment of a broad-host-range lytic phage SapYZU15 in eliminating Staphylococcus aureus from subcutaneous infection

Multidrug resistance (MDR) Staphylococcus aureus is frequently isolated from food products, and can cause severe clinical infection. Bacteriophage (phage) therapy is a promising biocontrol agent against MDR S. aureus in food contamination and clinical infections. In this study, the antimicrobial susceptibility of 47 S. aureus isolates from three swine farms, two slaughterhouses, and four markets (Yangzhou, China) were evaluated. The biological characteristics of four lytic S. aureus phages were compared and the lytic activity of phage SapYZU15 against MDR S. aureus was assessed using milk, fresh pork and a mouse model of subcutaneous abscess. The results showed that 28 S. aureus isolates (59.6%, 28/47) exhibited multiple antibiotic resistance to at least three different classes of antibiotics. Compared to SapYZU01, SapYZU02, and SapYZU03, SapYZU15 had a shorter latent period (10 min), larger burst size (322.00 PFU/cell), broader host range, wider temperature stability (−80 to 50 °C), and pH stability. Furthermore, SapYZU15 significantly reduces the counts of S. aureus in milk and pork (5.69 and 1.16 log colony-forming unit/mL, respectively) at 25 °C and controls the growth of S. aureus at 4 °C. Compared to the mice infected with S. aureus MRSA JCSC 4744 and cocktail (S. aureus YZUsa1, YZUsa4, YZUsa12, YZUsa14, and MRSA JCSC 4744), treatment with SapYZU15 led to faster tissue healing, less weight loss, and lower viable S. aureus counts in the murine abscess model. Moreover, prevention with SapYZU15 effectively inhibited abscess formation through a synergistic effect with pro-inflammatory cytokines. Consequently, our results suggest that SapYZU15 is an effective strategy for controlling S. aureus contamination in food products, and possesses an immense potential to treat and prevent clinic infection caused by MDR S. aureus strains. The interactions and mechanisms between SapYZU15 and its bacterial host differed depending on the model, temperature, and multiplicity of infection (MOI).

A Molecular Switch-Integrated Nanoplatform Enables Photo-Unlocked Antibacterial Drug Delivery for Synergistic Abscess Therapy.

Drug delivery systems (DDSs) capable of sequential multistage drug release are urgently needed for antibacterial applications. Herein, a molecular switch-integrated, photo-responsive nanoplatform is reported based on hollow mesoporous silica nanospheres (HMSN) loaded with silver nanoparticles(Ag NPs), vancomycin (Van), and hemin (HAVH) for bacteria elimination and abscess therapy. Upon near-infrared (NIR) light irradiation, the molecular switch, hemin, can effuse from the mesopores of HMSN, triggering the release of pre-loaded Ag+ and Van, which enables photothermal-modulated drug release and synergistic photothermal-chemo therapy (PTT-CHT). The HAVH_NIR irreversibly disrupts the bacterial cell membrane, facilitating the penetration of Ag+ and Van. It is found that these compounds restrain the transcription and translation of ribosomes and lead to rapid bacterial death. Furthermore, hemin can effectively inhibit excessive inflammatory responses associated with the treatment, promoting accelerated wound healing in a murine abscess model. This work presents a new strategy for antibacterial drug delivery with high controllability and extendibility, which may benefit the development of smart multifunctional nanomedicine for diseases not limited to bacterial infections.

Impact of Polymicrobial Infection on Fitness of Streptococcus gordonii In Vivo.

Pathogenic microbial ecosystems are often polymicrobial, and interbacterial interactions drive emergent properties of these communities. In the oral cavity, Streptococcus gordonii is a foundational species in the development of plaque biofilms, which can contribute to periodontal disease and, after gaining access to the bloodstream, target remote sites such as heart valves. Here, we used a transposon sequencing (Tn-Seq) library of S. gordonii to identify genes that influence fitness in a murine abscess model, both as a monoinfection and as a coinfection with an oral partner species, Porphyromonas gingivalis. In the context of a monoinfection, conditionally essential genes were widely distributed among functional pathways. Coinfection with P. gingivalis almost completely changed the nature of in vivo gene essentiality. Community-dependent essential (CoDE) genes under the coinfection condition were primarily related to DNA replication, transcription, and translation, indicating that robust growth and replication are required to survive with P. gingivalis in vivo. Interestingly, a group of genes in an operon encoding streptococcal receptor polysaccharide (RPS) were associated with decreased fitness of S. gordonii in a coinfection with P. gingivalis. Individual deletion of two of these genes (SGO_2020 and SGO_2024) resulted in the loss of RPS production by S. gordonii and increased susceptibility to killing by neutrophils. P. gingivalis protected the RPS mutants by inhibiting neutrophil recruitment, degranulation, and neutrophil extracellular trap (NET) formation. These results provide insight into genes and functions that are important for S. gordonii survival in vivo and the nature of polymicrobial synergy with P. gingivalis. Furthermore, we show that RPS-mediated immune protection in S. gordonii is dispensable and detrimental in the presence of a synergistic partner species that can interfere with neutrophil killing mechanisms. IMPORTANCE Bacteria responsible for diseases originating at oral mucosal membranes assemble into polymicrobial communities. However, we know little regarding the fitness determinants of the organisms that initiate community formation. Here, we show that the extracellular polysaccharide of Streptococcus gordonii, while important for streptococcal survival as a monoinfection, is detrimental to survival in the context of a coinfection with Porphyromonas gingivalis. We found that the presence of P. gingivalis compensates for immune protective functions of extracellular polysaccharide, rendering production unnecessary. The results show that fitness determinants of bacteria in communities differ substantially from those of individual species in isolation. Furthermore, constituents of communities can undertake activities that relieve the burden of energetically costly biosynthetic reactions on partner species.

Multifunctional ZnFeO4-Based Antibiotic Cross-Linked Nanoplatform for Magnetically Targeted Treatment of Microbial Biofilms

During the infections of wounds or medical device surfaces, bacteria such as Pseudomonas aeruginosa are prone to forming biofilms which prevent the penetration of antimicrobial agents. Herein, a combined therapy using bactericidal nano-micelles Ce6-ZnFeO4@ABCsi-micelles was developed that employed colistin cross-linked Pluronic micelles as a carrier, with the magnetic metal oxide ZnFeO4 and photosensitizer dihydroporphyrin (Ce6) encapsulated in the core of micelles. Driven by an external magnetic field, the nano-micelles could penetrate the external barrier of the biofilm. Reactive oxygen species (ROS) generated by the photosensitizer Ce6 disrupted the barrier of the biofilm, which facilitated the distribution of the Ce6 and colistin antibiotics into the biofilm, realizing a 99.5% clearance rate of bacteria. Using such a combination of chemotherapy and photodynamic therapy, Ce6-ZnFeO4@ABCsi-micelles could clear wound infection in a murine abscess model, owing to the targeted driving effect of the external magnetic field, along with the chemotherapeutic effect of colistin and the photodynamic effect of Ce6. This design provides an effective targeted therapeutic strategy to address biofilm infections.

Surviving the host: Microbial metabolic genes required for growth of Pseudomonas aeruginosa in physiologically-relevant conditions.

Pseudomonas aeruginosa, like other pathogens, adapts to the limiting nutritional environment of the host by altering patterns of gene expression and utilizing alternative pathways required for survival. Understanding the genes essential for survival in the host gives insight into pathways that this organism requires during infection and has the potential to identify better ways to treat infections. Here, we used a saturated transposon insertion mutant pool of P. aeruginosa strain PAO1 and transposon insertion sequencing (Tn-Seq), to identify genes conditionally important for survival under conditions mimicking the environment of a nosocomial infection. Conditions tested included tissue culture medium with and without human serum, a murine abscess model, and a human skin organoid model. Genes known to be upregulated during infections, as well as those involved in nucleotide metabolism, and cobalamin (vitamin B12) biosynthesis, etc., were required for survival in vivo- and in host mimicking conditions, but not in nutrient rich lab medium, Mueller Hinton broth (MHB). Correspondingly, mutants in genes encoding proteins of nucleotide and cobalamin metabolism pathways were shown to have growth defects under physiologically-relevant media conditions, in vivo, and in vivo-like models, and were downregulated in expression under these conditions, when compared to MHB. This study provides evidence for the relevance of studying P. aeruginosa fitness in physiologically-relevant host mimicking conditions and identified metabolic pathways that represent potential novel targets for alternative therapies.

The ColR/S two-component system is a conserved determinant of host association across Pseudomonas species.

Members of the bacterial genus Pseudomonas form mutualistic, commensal, and pathogenic associations with diverse hosts. The prevalence of host association across the genus suggests that symbiosis may be a conserved ancestral trait and that distinct symbiotic lifestyles may be more recently evolved. Here we show that the ColR/S two-component system, part of the Pseudomonas core genome, is functionally conserved between Pseudomonas aeruginosa and Pseudomonas fluorescens. Using plant rhizosphere colonization and virulence in a murine abscess model, we show that colR is required for commensalism with plants and virulence in animals. Comparative transcriptomics revealed that the ColR regulon has diverged between P. aeruginosa and P. fluorescens and deleting components of the ColR regulon revealed strain-specific, but not host-specific, requirements for ColR-dependent genes. Collectively, our results suggest that ColR/S allows Pseudomonas to sense and respond to a host, but that the ColR-regulon has diverged between Pseudomonas strains with distinct lifestyles. This suggests that conservation of two-component systems, coupled with life-style dependent diversification of the regulon, may play a role in host association and lifestyle transitions.

Novel Alligator Cathelicidin As-CATH8 Demonstrates Anti-Infective Activity against Clinically Relevant and Crocodylian Bacterial Pathogens

Host defense peptides (HDPs) represent an alternative way to address the emergence of antibiotic resistance. Crocodylians are interesting species for the study of these molecules because of their potent immune system, which confers high resistance to infection. Profile hidden Markov models were used to screen the genomes of four crocodylian species for encoded cathelicidins and eighteen novel sequences were identified. Synthetic cathelicidins showed broad spectrum antimicrobial and antibiofilm activity against several clinically important antibiotic-resistant bacteria. In particular, the As-CATH8 cathelicidin showed potent in vitro activity profiles similar to the last-resort antibiotics vancomycin and polymyxin B. In addition, As-CATH8 demonstrated rapid killing of planktonic and biofilm cells, which correlated with its ability to cause cytoplasmic membrane depolarization and permeabilization as well as binding to DNA. As-CATH8 displayed greater antibiofilm activity than the human cathelicidin LL-37 against methicillin-resistant Staphylococcus aureus in a human organoid model of biofilm skin infection. Furthermore, As-CATH8 demonstrated strong antibacterial effects in a murine abscess model of high-density bacterial infections against clinical isolates of S. aureus and Acinetobacter baumannii, two of the most common bacterial species causing skin infections globally. Overall, this work expands the repertoire of cathelicidin peptides known in crocodylians, including one with considerable therapeutic promise for treating common skin infections.

The Small RNA Teg41 Is a Pleiotropic Regulator of Virulence in Staphylococcus aureus.

Previously, our group demonstrated a role for the small RNA (sRNA) Teg41 in regulating production of the alpha phenol-soluble modulin toxins (αPSMs) in Staphylococcus aureus. Overexpressing Teg41 increased αPSM production while deleting the 3' end of Teg41 (Teg41Δ3' strain) resulted in a decrease in αPSM production, reduced hemolytic activity of S. aureus culture supernatants, and attenuated virulence in a murine abscess model of infection. In this study, we further explore the attenuation of virulence in the Teg41Δ3' strain. Using both localized and systemic models of infection, we demonstrate that the Teg41Δ3' strain is more severely attenuated than an ΔαPSM mutant, strongly suggesting that Teg41 influences more than the αPSMs. Proteomic and transcriptomic analysis of the wild-type and Teg41Δ3' strains reveals widespread alterations in transcript abundance and protein production in the absence of Teg41, confirming that Teg41 has pleiotropic effects in the cell. We go on to investigate the molecular mechanism underlying Teg41-mediated gene regulation. Surprisingly, results demonstrate that certain Teg41 target genes, including the αPSMs and βPSMs, are transcriptionally altered in the Teg41Δ3' strain, while other targets, specifically spa (encoding surface protein A), are regulated at the level of transcript stability. Collectively, these data demonstrate that Teg41 is a pleiotropic RNA regulator in S. aureus that influences expression of a variety of genes using multiple different mechanisms.

Development of Resistance to Eravacycline by Klebsiella pneumoniae and Collateral Sensitivity-Guided Design of Combination Therapies.

ABSTRACTThe evolution of bacterial antibiotic resistance is exhausting the list of currently used antibiotics and endangers those in the pipeline. The combination of antibiotics is a promising strategy that may suppress resistance development and/or achieve synergistic therapeutic effects. Eravacycline is a newly approved antibiotic that is effective against a variety of multidrug-resistant (MDR) pathogens. However, the evolution of resistance to eravacycline and strategies to suppress the evolution remain unexplored. Here, we demonstrated that a carbapenem-resistant Klebsiella pneumoniae clinical isolate quickly developed resistance to eravacycline, which is mainly caused by mutations in the gene encoding the Lon protease. The evolved resistant mutants display collateral sensitivities to β-lactam/β-lactamase inhibitor (BLBLI) combinations aztreonam/avibactam and ceftazidime-avibactam. Proteomic analysis revealed upregulation of the multidrug efflux system AcrA-AcrB-TolC and porin proteins OmpA and OmpU, which contributed to the increased resistance to eravacycline and susceptibility to BLBLIs, respectively. The combination of eravacycline with aztreonam/avibactam or ceftazidime-avibactam suppresses resistance development. We further demonstrated that eravacycline-resistant mutants evolved from an NDM-1-containing K. pneumoniae strain display collateral sensitivity to aztreonam/avibactam, and the combination of eravacycline with aztreonam/avibactam suppresses resistance development. In addition, the combination of eravacycline with aztreonam/avibactam or ceftazidime-avibactam displayed synergistic therapeutic effects in a murine cutaneous abscess model. Overall, our results revealed mechanisms of resistance to eravacycline and collateral sensitivities to BLBLIs and provided promising antibiotic combinations in the treatment of multidrug-resistant K. pneumoniae infections.IMPORTANCE The increasing bacterial antibiotic resistance is a serious threat to global public health, which demands novel antimicrobial medicines and treatment strategies. Eravacycline is a newly approved antibiotic that belongs to the tetracycline antibiotics. Here, we found that a multidrug-resistant Klebsiella pneumoniae clinical isolate rapidly developed resistance to eravacycline and the evolved resistant mutants displayed collateral sensitivity to antibiotics aztreonam/avibactam and ceftazidime-avibactam. We demonstrated that the combination of eravacycline with aztreonam/avibactam or ceftazidime-avibactam repressed resistance development and improved the treatment efficacies. We also elucidated the mechanisms that contribute to the increased resistance to eravacycline and susceptibility to aztreonam/avibactam and ceftazidime-avibactam. This work demonstrated the mechanisms of antibiotic resistance and collateral sensitivity and provided a new therapeutically option for effective antibiotic combinations.

A Brominated Furanone Inhibits Pseudomonas aeruginosa Quorum Sensing and Type III Secretion, Attenuating Its Virulence in a Murine Cutaneous Abscess Model.

Quorum sensing (QS) and type III secretion systems (T3SSs) are among the most attractive anti-virulence targets for combating multidrug-resistant pathogenic bacteria. Some halogenated furanones reduce QS-associated virulence, but their role in T3SS inhibition remains unclear. This study aimed to assess the inhibition of these two systems on Pseudomonas aeruginosa virulence. The halogenated furanones (Z)-4-bromo-5-(bromomethylene)-2(5H) (C-30) and 5-(dibromomethylene)-2(5H) (named hereafter GBr) were synthesized, and their ability to inhibit the secretion of type III exoenzymes and QS-controlled virulence factors was analyzed in P. aeruginosa PA14 and two clinical isolates. Furthermore, their ability to prevent bacterial establishment was determined in a murine cutaneous abscess model. The GBr furanone reduced pyocyanin production, biofilm formation, and swarming motility in the same manner or more effectively than C-30. Moreover, both furanones inhibited the secretion of ExoS, ExoT, or ExoU effectors in all tested strains. The administration of GBr (25 and 50 µM) to CD1 mice infected with the PA14 strain significantly decreased necrosis formation in the inoculation zone and the systemic spread of bacteria more efficiently than C-30 (50 µM). Molecular docking analysis suggested that the gem position of bromine in GBr increases its affinity for the active site of the QS LasR regulator. Overall, our findings showed that the GBr furanone displayed efficient multi-target properties that may favor the development of more effective anti-virulence therapies.

Competition between Pseudomonas aeruginosa and Staphylococcus aureus is dependent on intercellular signaling and regulated by the NtrBC two-component system

Pseudomonas aeruginosa and Staphylococcus aureus are often comorbid human pathogens, isolated from expectorated sputum of cystic fibrosis patients and chronically infected wounds. Prior studies revealed a competitive advantage of P. aeruginosa over S. aureus in vitro that was slightly muted in vivo. Here, we demonstrated that the two-component regulatory system NtrBC influences the competitive advantage of P. aeruginosa over S. aureus in skin organoid and mouse models of co-infection. Expression of ntrBC was induced during co-culture of the two species and could be recapitulated in monoculture by the addition of the metabolite N-acetylglucosamine that is released from S. aureus following lysis. P. aeruginosa LESB58 WT, but not mutant (ΔntrC and ΔntrBC) strains, induced lysis of S. aureus USA300 LAC during planktonic growth and outcompeted S. aureus USA300 LAC during biofilm formation in vitro. We confirmed these findings in a murine abscess model of high-density infection. Accordingly, the secretory profile of P. aeruginosa LESB58 mutants revealed reduced production of anti-staphylococcal virulence factors including pyoverdine, pyocyanin and elastase. These phenotypes of LESB58 ΔntrBC could be at least partly complemented by overexpression of quorum sensing molecules including homoserine lactones or alkylquinolone signaling molecules. These data implicate the NtrBC two-component system in the complex regulatory cascade triggered by interspecies signaling that gives P. aeruginosa LESB58 a competitive edge over S. aureus USA300 LAC.

Porphyromonas gingivalis Tyrosine Kinase Is a Fitness Determinant in Polymicrobial Infections.

Many pathogenic microbial ecosystems are polymicrobial, and community function can be shaped by interbacterial interactions. Little is known, however, regarding the genetic determinants required for fitness in heterotypic community environments. In periodontal diseases, Porphyromonas gingivalis is a primary pathogen, but only within polymicrobial communities. Here, we used a transposon sequencing (Tn-Seq) library of P. gingivalis to screen for genes that influence fitness of the organism in a coinfection murine abscess model with the oral partner species Streptococcus gordonii and Fusobacterium nucleatum. Genes impacting fitness with either organism were involved in diverse processes, including metabolism and energy production, along with cell wall and membrane biogenesis. Despite the overall similarity of function, the majority of identified genes were specific to the partner species, indicating that synergistic mechanisms of P. gingivalis vary to a large extent according to community composition. Only two genes were identified as essential for P. gingivalis fitness in abscess development with both S. gordonii and F. nucleatum: ptk1, encoding a tyrosine kinase, and inlJ, encoding an internalin family surface protein. Ptk1, but not InlJ, is required for community development with S. gordonii, and we found that the action of this kinase is similarly required for P. gingivalis to accumulate in a community with F. nucleatum. A limited number of P. gingivalis genes are therefore required for species-independent synergy, and the Ptk1 tyrosine kinase network may integrate and coordinate input from multiple organisms.

Sustained Release of a Synthetic Autoinducing Peptide Mimetic Blocks Bacterial Communication and Virulence In Vivo.

AbstractThere is significant interest in approaches to the treatment of bacterial infections that block virulence without creating selective pressures that lead to resistance. Here, we report the development of an “anti‐virulence” strategy that exploits the activity of potent synthetic inhibitors of quorum sensing (QS) in Staphylococcus aureus. We identify peptide‐based inhibitors of QS that are resistant to sequestration or degradation by components of murine tissue and demonstrate that encapsulation of a lead inhibitor in degradable polymer microparticles provides materials that substantially inhibit QS in vitro. Using a murine abscess model, we show that this inhibitor attenuates methicillin‐resistant S. aureus (MRSA) skin infections in vivo, and that sustained release of the inhibitor from microparticles significantly improved outcomes compared to mice that received a single‐dose bolus. Our results present an effective and modular approach to controlling bacterial virulence in vivo and could advance the development of new strategies for skin infection control.

Targeting the Pseudomonas aeruginosa Virulence Factor Phospholipase C With Engineered Liposomes.

Engineered liposomes composed of the naturally occurring lipids sphingomyelin (Sm) and cholesterol (Ch) have been demonstrated to efficiently neutralize toxins secreted by Gram-positive bacteria such as Streptococcus pneumoniae and Staphylococcus aureus. Here, we hypothesized that liposomes are capable of neutralizing cytolytic virulence factors secreted by the Gram-negative pathogen Pseudomonas aeruginosa. We used the highly virulent cystic fibrosis P. aeruginosa Liverpool Epidemic Strain LESB58 and showed that sphingomyelin (Sm) and a combination of sphingomyelin with cholesterol (Ch:Sm; 66 mol/% Ch and 34 mol/% Sm) liposomes reduced lysis of human bronchial and red blood cells upon challenge with the Pseudomonas secretome. Mass spectrometry of liposome-sequestered Pseudomonas proteins identified the virulence-promoting hemolytic phospholipase C (PlcH) as having been neutralized. Pseudomonas aeruginosa supernatants incubated with liposomes demonstrated reduced PlcH activity as assessed by the p-nitrophenylphosphorylcholine (NPPC) assay. Testing the in vivo efficacy of the liposomes in a murine cutaneous abscess model revealed that Sm and Ch:Sm, as single dose treatments, attenuated abscesses by >30%, demonstrating a similar effect to that of a mutant lacking plcH in this infection model. Thus, sphingomyelin-containing liposome therapy offers an interesting approach to treat and reduce virulence of complex infections caused by P. aeruginosa and potentially other Gram-negative pathogens expressing PlcH.

Titanium carbide MXene-based hybrid hydrogel for chemo-photothermal combinational treatment of localized bacterial infection

With the increased emergence and threat of multi-drug resistant microorganisms, MXenes have become not only an emerging class of two-dimensional functional nanomaterials, but also potential nanomedicines (i.e., antimicrobial agents) that deserve further exploration. Very recently, Ti3C2 MXene was observed to offer a unique membrane-disruption effect and superior light-to-heat conversion efficiency, but its antibacterial property remains unsatisfactory due to poor MXene-bacteria interactions, low photothermal therapy efficiency, and occurrence of bacterial rebound in vivo. Herein, the cationic antibiotic ciprofloxacin (Cip) is combined with Ti3C2 MXene, and a hybrid hydrogel was constructed by incorporating Cip-Ti3C2 nanocomposites into the network structure of a Cip-loaded hydrogels to effectively trap and kill bacteria. We found that the Cip-Ti3C2 nanocomposites achieved an impressive in vitro bactericidal efficiency of >99.99999% (7.03 log10) for the inhibition of methicillin-resistant Staphylococcus aureus (MRSA) by combining chemotherapy with photothermal therapy. In an MRSA-induced murine abscess model, the hybrid hydrogel simultaneously achieved high-efficiency sterilization and long-term inhibition effects, avoiding the rebound of bacteria after photothermal therapy, and thus maximized the in vivo therapeutic efficacy of Ti3C2 MXene-based systems. Overall, this work provides a strategy for efficiently combating localized bacterial infection by rationally designing MXene-based hybrid hydrogels. Statement of significanceTwo-dimensional Ti3C2 MXene was recently regarded as a promising functional nanomaterial, however, its antibacterial applications are limited by the poor MXene-bacteria interactions, low photothermal therapy efficiency, and the occurrence of bacterial rebound in vivo. This work aims to construct a Ti3C2 MXene-based hybrid hydrogel for chemo-photothermal therapy and enhance the antimicrobial performance via a combination of the high-efficiency sterilization of ciprofloxacin-Ti3C2 nanocomposites with the long-term inhibition effect of ciprofloxacin hydrogel. The present study provides an example of efficient MXene-based antimicrobials to treat localized bacterial infection such as methicillin-resistant Staphylococcus aureus (MRSA)-induced skin abscess.

Staquorsin: A Novel Staphylococcus aureus Agr-Mediated Quorum Sensing Inhibitor Impairing Virulence in vivo Without Notable Resistance Development.

The emergence of microbial resistance to the available antibiotics is a major public health concern, especially with the limited rate of developing new antibiotics. The utilization of anti-virulence agents is a non-conventional approach that can be used to combat microbial infection. In Staphylococcus aureus, many virulence factors are regulated by the Agr-mediated quorum sensing (QS). We developed a chemical compound that acts a potential Agr-inhibitor without reducing bacterial viability. The compound was designated staquorsin for Staphylococcus aureus QS inhibitor. In silico analyses confirmed the binding of staquorsin to the AgrA active site with an absolute binding score comparable to savirin, a previously described AgrA inhibitor. However, staquorsin turned out to be superior over savarin in not affecting the S. aureus viability in concentrations up to 600 μM. On the other hand, savirin inhibited S. aureus growth in concentrations as low as 25 μM. Moreover, staquorsin proved to be a potent inhibitor of the Agr system by inhibiting hemolysins, lipase production, and affecting biofilms formation and detachment. On the molecular level it significantly inhibited the effector transcript RNA III. In vivo testing, using the murine skin abscess model, confirmed the ability of staquorsin to modulate S. aureus virulence by effectively controlling the infection. Twenty passages of S. aureus in the presence of 40 μM staquorsin have not resulted in loss of activity as evidenced by maintaining its ability to reduce hemolysin production and RNA III transcript levels. In conclusion, we hereby describe a novel anti-virulence compound inhibiting the S. aureus Agr-system and its associated virulence factors. It is active both in vitro and in vivo, and its frequent use does not lead to the development of resistance. These findings model staquorsin as a promising drug candidate to join the fierce battle against the formidable pathogen S. aureus.