Intermittent alternating magnetic fields diminish metal-associated biofilm in vivo.

Prosthetic joint infection (PJI) is a complication of arthroplasty that results in significant morbidity. The presence of biofilm makes treatment difficult, and removal of the prosthesis is frequently required. We have developed a non-invasive approach for biofilm eradication from metal implants using intermittent alternating magnetic fields (iAMF) to generate targeted heating at the implant surface. The goal of this study was to determine whether iAMF demonstrated efficacy in an in vivo implant biofilm infection model. iAMF combined with antibiotics led to enhanced reduction of biofilm on metallic implants in vivo compared to antibiotics or untreated control. iAMF-antibiotic combinations resulted in a > 1 - log further reduction in biofilm burden compared to antibiotics or iAMF alone. This combination effect was seen in both S. aureus and P. aeruginosa and seen with multiple antibiotics used to treat infections with these pathogens. In addition, efficacy was temperature dependent with increasing temperatures resulting in a greater reduction of biofilm. Tissue damage was limited (< 1 mm from implant-tissue interface). This non-invasive approach to eradicating biofilm could serve as a new paradigm in treating PJI.

Comparative Evaluation of Pseudomonas aeruginosa Adhesion to a Poly-(2-Methacryloyloxyethyl Phosphorylcholine)-Modified Silicone Hydrogel Contact Lens.

Pseudomonas aeruginosa is the most common causative agent associated with microbial keratitis. During contact lens wear, pathogens may be introduced into the ocular environment, which might cause adverse events. Lehfilcon A is a recently developed contact lens with a water gradient surface composed of polymeric 2-methacryloyloxyethyl phosphorylcholine (MPC). MPC is re-ported to impart anti-biofouling properties onto modified substrates. Therefore, in this in vitro experimental study, we tested the capability of lehfilcon A to resist adhesion by P. aeruginosa. Quantitative bacterial adhesion assays using five strains of P. aeruginosa were conducted to compare the adherence properties of lehfilcon A to five currently marketed silicone hydrogel (SiHy) contact lenses (comfilcon A, fanfilcon A, senofilcon A, senofilcon C, and samfilcon A). Compared to lehfilcon A, we observed 26.7 ± 8.8 times (p = 0.0028) more P. aeruginosa binding to comfilcon A, 30.0 ± 10.8 times (p = 0.0038) more binding to fanfilcon A, 18.2 ± 6.2 times (p = 0.0034) more binding to senofilcon A, 13.6 ± 3.9 times (p = 0.0019) more binding to senofilcon C, and 29.5 ± 11.8 times (p = 0.0057) more binding to samfilcon A. These results demonstrate that, for various strains of P. aeruginosa, lehfilcon A reduces bacterial adhesion compared to other contact lens materials.

Evaluation of Serratia marcescens Adherence to Contact Lens Materials.

Bacterial keratitis is a risk associated with the use of contact lenses for cosmetic purposes or vision correction. In this in vitro experimental study, we examined the ability of the ocular pathogen Serratia marcescens to adhere to monthly or biweekly replacement contact lenses. We performed quantitative adhesion assays to evaluate the adherence of S. marcescens to seven contact lens materials: comfilcon A, senofilcon A, omafilcon B, fanfilcon A, balafilcon A, senofilcon C, and lehfilcon A. Lehfilcon A is a newly marketed silicon hydrogel contact lens with a surface modification of poly-(2-methacryloyloxyethyl phosphorylcholine) (PMPC). PMPC has previously been demonstrated to be an effective anti-biofouling treatment for numerous surfaces. We observed low S. marcescens adherence to lehfilcon A compared to other materials. We demonstrate the use of the fluorescent dye 5(6)-Carboxytetramethylrhodamine succinimidyl ester to covalently stain live cells prior to material adhesion studies.

Microbial Adherence to Contact Lenses and Pseudomonas aeruginosa as a Model Organism for Microbial Keratitis.

Microbial keratitis (MK), the infection of the cornea, is a devastating disease and the fifth leading cause of blindness and visual impairment around the world. The overwhelming majority of MK cases are linked to contact lens wear combined with factors which promote infection such as corneal abrasion, an immunocompromised state, improper contact lens use, or failing to routinely disinfect lenses after wear. Contact lens-related MK involves the adherence of microorganisms to the contact lens. Therefore, this review discusses the information currently available regarding the disease pathophysiology, the common types of microorganisms causing MK, physical and organic mechanisms of adhesion, material properties which are involved in adhesion, and current antimicrobial strategies. This review also concludes that Pseudomonas aeruginosa is a model organism for the investigation of contact lens microbial adherence due to its prevalence in MK cases, its extremely robust adhesion, antimicrobial-resistant properties, and the severity of the disease it causes.

Acanthamoeba spp. aggregate and encyst on contact lens material increasing resistance to disinfection.

Introduction: Acanthamoeba keratitis is often caused when Acanthamoeba contaminate contact lenses and infect the cornea. Acanthamoeba is pervasive in the environment as a motile, foraging trophozoite or biocide-resistant and persistent cyst. As contact lens contamination is a potential first step in infection, we studied Acanthamoeba's behavior and interactions on different contact lens materials. We hypothesized that contact lenses may induce aggregation, which is a precursor to encystment, and that aggregated encystment would be more difficult to disinfect than motile trophozoites. Methods: Six clinically and/or scientifically relevant strains of Acanthamoeba (ATCC 30010, ATCC 30461, ATCC 50370, ATCC 50702, ATCC 50703, and ATCC PRA-115) were investigated on seven different common silicone hydrogel contact lenses, and a no-lens control, for aggregation and encystment for 72 h. Cell count and size were used to determine aggregation, and fluorescent staining was used to understand encystment. RNA seq was performed to describe the genome of Acanthamoeba which was individually motile or aggregated on different lens materials. Disinfection efficacy using three common multi-purpose solutions was calculated to describe the potential disinfection resistance of trophozoites, individual cysts, or spheroids. Results: Acanthamoeba trophozoites of all strains examined demonstrated significantly more aggregation on specific contact lens materials than others, or the no-lens control. Fluorescent staining demonstrated encystment in as little as 4 hours on contact lens materials, which is substantially faster than previously reported in natural or laboratory settings. Gene expression profiles corroborated encystment, with significantly differentially expressed pathways involving actin arrangement and membrane complexes. High disinfection resistance of cysts and spheroids with multi-purpose solutions was observed. Discussion: Aggregation/encystment is a protective mechanism which may enable Acanthamoeba to be more disinfection resistant than individual trophozoites. This study demonstrates that some contact lens materials promote Acanthamoeba aggregation and encystment, and Acanthamoeba spheroids obstruct multi-purpose solutions from disinfecting Acanthamoeba.

The Canonical Long-Chain Fatty Acid Sensing Machinery Processes Arachidonic Acid To Inhibit Virulence in Enterohemorrhagic Escherichia coli.

The mammalian gastrointestinal tract is a complex biochemical organ that generates a diverse milieu of host- and microbe-derived metabolites. In this environment, bacterial pathogens sense and respond to specific stimuli, which are integrated into the regulation of their virulence programs. Previously, we identified the transcription factor FadR, a long-chain fatty acid (LCFA) acyl coenzyme A (acyl-CoA) sensor, as a novel virulence regulator in the human foodborne pathogen enterohemorrhagic Escherichia coli (EHEC). Here, we demonstrate that exogenous LCFAs directly inhibit the locus of enterocyte effacement (LEE) pathogenicity island in EHEC through sensing by FadR. Moreover, in addition to LCFAs that are 18 carbons in length or shorter, we introduce host-derived arachidonic acid (C20:4) as an additional LCFA that is recognized by the FadR system in EHEC. We show that arachidonic acid is processed by the acyl-CoA synthetase FadD, which permits binding to FadR and decreases FadR affinity for its target DNA sequences. This interaction enables the transcriptional regulation of FadR-responsive operons by arachidonic acid in EHEC, including the LEE. Finally, we show that arachidonic acid inhibits hallmarks of EHEC disease in a FadR-dependent manner, including EHEC attachment to epithelial cells and the formation of attaching and effacing lesions. Together, our findings delineate a molecular mechanism demonstrating how LCFAs can directly inhibit the virulence of an enteric bacterial pathogen. More broadly, our findings expand the repertoire of ligands sensed by the canonical LFCA sensing machinery in EHEC to include arachidonic acid, an important bioactive lipid that is ubiquitous within host environments.IMPORTANCE Polyunsaturated fatty acids (PUFAs) play important roles in host immunity. Manipulation of lipid content in host tissues through diet or pharmacological interventions is associated with altered severity of various inflammatory diseases. Our work introduces a defined host-pathogen interaction by which arachidonic acid, a host-derived and dietary PUFA, can impact the outcome of enteric infection with the human pathogen enterohemorrhagic Escherichia coli (EHEC). We show that long-chain fatty acids including arachidonic acid act as signaling molecules that directly suppress a key pathogenicity island in EHEC following recognition by the fatty acyl-CoA-responsive transcription factor FadR. Thus, in addition to its established effects on host immunity and its bactericidal activities against other pathogens, we demonstrate that arachidonic acid also acts as a signaling molecule that inhibits virulence in an enteric pathogen.

IS26-mediated amplification of blaOXA-1 and blaCTX-M-15 with concurrent outer membrane porin disruption associated with de novo carbapenem resistance in a recurrent bacteraemia cohort.

BACKGROUND: Approximately half of clinical carbapenem-resistant Enterobacterales (CRE) isolates lack carbapenem-hydrolysing enzymes and develop carbapenem resistance through alternative mechanisms. OBJECTIVES: To elucidate development of carbapenem resistance mechanisms from clonal, recurrent ESBL-positive Enterobacterales (ESBL-E) bacteraemia isolates in a vulnerable patient population. METHODS: This study investigated a cohort of ESBL-E bacteraemia cases in Houston, TX, USA. Oxford Nanopore Technologies long-read and Illumina short-read sequencing data were used for comparative genomic analysis. Serial passaging experiments were performed on a set of clinical ST131 Escherichia coli isolates to recapitulate in vivo observations. Quantitative PCR (qPCR) and qRT-PCR were used to determine copy number and transcript levels of ß-lactamase genes, respectively. RESULTS: Non-carbapenemase-producing CRE (non-CP-CRE) clinical isolates emerged from an ESBL-E background through a concurrence of primarily IS26-mediated amplifications of blaOXA-1 and blaCTX-M-1 group genes coupled with porin inactivation. The discrete, modular translocatable units (TUs) that carried and amplified ß-lactamase genes mobilized intracellularly from a chromosomal, IS26-bound transposon and inserted within porin genes, thereby increasing ß-lactamase gene copy number and inactivating porins concurrently. The carbapenem resistance phenotype and TU-mediated ß-lactamase gene amplification were recapitulated by passaging a clinical ESBL-E isolate in the presence of ertapenem. Clinical non-CP-CRE isolates had stable carbapenem resistance phenotypes in the absence of ertapenem exposure. CONCLUSIONS: These data demonstrate IS26-mediated mechanisms underlying ß-lactamase gene amplification with concurrent outer membrane porin disruption driving emergence of clinical non-CP-CRE. Furthermore, these amplifications were stable in the absence of antimicrobial pressure. Long-read sequencing can be utilized to identify unique mobile genetic element mechanisms that drive antimicrobial resistance.

Antisense antibacterial compounds.

Extensive antibiotic use combined with poor historical drug stewardship practices have created a medical crisis in which once treatable bacterial infections are now increasingly unmanageable. To combat this, new antibiotics will need to be developed and safeguarded. An emerging class of antibiotics based upon nuclease-stable antisense technologies has proven valuable in preclinical testing against a variety of bacterial pathogens. This review describes the current state of development of antisense-based antibiotics, the mechanisms thus far employed by these compounds, and possible future avenues of research.

The Serotonin Neurotransmitter Modulates Virulence of Enteric Pathogens.

The gut-brain axis is crucial to microbial-host interactions. The neurotransmitter serotonin is primarily synthesized in the gastrointestinal (GI) tract, where it is secreted into the lumen and subsequently removed by the serotonin transporter, SERT. Here, we show that serotonin decreases virulence gene expression by enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium, a murine model for EHEC. The membrane-bound histidine sensor kinase, CpxA, is a bacterial serotonin receptor. Serotonin induces dephosphorylation of CpxA, which inactivates the transcriptional factor CpxR controlling expression of virulence genes, notably those within the locus of enterocyte effacement (LEE). Increasing intestinal serotonin by genetically or pharmacologically inhibiting SERT decreases LEE expression and reduces C. rodentium loads. Conversely, inhibiting serotonin synthesis increases pathogenesis and decreases host survival. As other enteric bacteria contain CpxA, this signal exploitation may be engaged by other pathogens. Additionally, repurposing serotonin agonists to inhibit CpxA may represent a potential therapeutic intervention for enteric bacteria.

VAMPr: VAriant Mapping and Prediction of antibiotic resistance via explainable features and machine learning.

Antimicrobial resistance (AMR) is an increasing threat to public health. Current methods of determining AMR rely on inefficient phenotypic approaches, and there remains incomplete understanding of AMR mechanisms for many pathogen-antimicrobial combinations. Given the rapid, ongoing increase in availability of high-density genomic data for a diverse array of bacteria, development of algorithms that could utilize genomic information to predict phenotype could both be useful clinically and assist with discovery of heretofore unrecognized AMR pathways. To facilitate understanding of the connections between DNA variation and phenotypic AMR, we developed a new bioinformatics tool, variant mapping and prediction of antibiotic resistance (VAMPr), to (1) derive gene ortholog-based sequence features for protein variants; (2) interrogate these explainable gene-level variants for their known or novel associations with AMR; and (3) build accurate models to predict AMR based on whole genome sequencing data. We curated the publicly available sequencing data for 3,393 bacterial isolates from 9 species that contained AMR phenotypes for 29 antibiotics. We detected 14,615 variant genotypes and built 93 association and prediction models. The association models confirmed known genetic antibiotic resistance mechanisms, such as blaKPC and carbapenem resistance consistent with the accurate nature of our approach. The prediction models achieved high accuracies (mean accuracy of 91.1% for all antibiotic-pathogen combinations) internally through nested cross validation and were also validated using external clinical datasets. The VAMPr variant detection method, association and prediction models will be valuable tools for AMR research for basic scientists with potential for clinical applicability.

AcrAB-TolC Inhibition by Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers Restores Antibiotic Activity in Vitro and in Vivo.

Overexpression of bacterial efflux pumps is a driver of increasing antibiotic resistance in Gram-negative pathogens. The AcrAB-TolC efflux pump has been implicated in resistance to a number of important antibiotic classes including fluoroquinolones, macrolides, and ß-lactams. Antisense technology, such as peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), can be utilized to inhibit expression of efflux pumps and restore susceptibility to antibiotics. Targeting of the AcrAB-TolC components with PPMOs revealed a sequence for acrA, which was the most effective at reducing antibiotic efflux. This acrA-PPMO enhances the antimicrobial effects of the levofloxacin and azithromycin in a panel of clinical Enterobacteriaceae strains. Additionally, acrA-PPMO enhanced azithromycin in vivo in a K. pneumoniae septicemia model. PPMOs targeting the homologous resistance-nodulation-division (RND)-efflux system in P. aeruginosa, MexAB-OprM, also enhanced potency to several classes of antibiotics in a panel of strains and in a cell culture infection model. These data suggest that PPMOs can be used as an adjuvant in antibiotic therapy to increase the efficacy or extend the spectrum of useful antibiotics against a variety of Gram-negative infections.

Redox, amino acid, and fatty acid metabolism intersect with bacterial virulence in the gut.

The gut metabolic landscape is complex and is influenced by the microbiota, host physiology, and enteric pathogens. Pathogens have to exquisitely monitor the biogeography of the gastrointestinal tract to find a suitable niche for colonization. To dissect the important metabolic pathways that influence virulence of enterohemorrhagic Escherichia coli (EHEC), we conducted a high-throughput screen. We generated a dataset of regulatory pathways that control EHEC virulence expression under anaerobic conditions. This unraveled that the cysteine-responsive regulator, CutR, converges with the YhaO serine import pump and the fatty acid metabolism regulator FadR to optimally control virulence expression in EHEC. CutR activates expression of YhaO to increase activity of the YhaJ transcription factor that has been previously shown to directly activate the EHEC virulence genes. CutR enhances FadL, which is a pump for fatty acids that represses inhibition of virulence expression by FadR, unmasking a feedback mechanism responsive to metabolite fluctuations. Moreover, CutR and FadR also augment murine infection by Citrobacter rodentium, which is a murine pathogen extensively employed as a surrogate animal model for EHEC. This high-throughput approach proved to be a powerful tool to map the web of cellular circuits that allows an enteric pathogen to monitor the gut environment and adjust the levels of expression of its virulence repertoire toward successful infection of the host.

The Interplay between the Microbiota and Enterohemorrhagic Escherichia coli.

The gastrointestinal tract of mammals is home to a plethora of microbial species that comprise the microbiota. The role of the microbiota in human health is at the forefront of science in recent years, because it is now appreciated that this intricate microbe-host association shapes the host's immune response and physiology. Many diseases are associated with changes in the microbiota, called dysbiosis. Dysbiosis is associated with obesity, metabolic syndromes, inflammatory bowel-disease, inflammatory bowel syndrome, cancer, diabetes, allergies, and autism. The microbiota is largely regarded as a barrier to enteric infections, such as with enterohemorrhagic Escherichia coli (EHEC). However, the interactions between pathogens and the microbiota are largely unknown, as is how these interactions influence the outcome of enteric disease. The microbial composition of the gastrointestinal tract shapes the landscape in which EHEC survives within the host. This organism competes for nutrients derived from the host diet, liberates additional resources from dietary and host sources, and produces signaling molecules sensed by EHEC to direct gene expression. To successfully colonize the recto-anal junction of a ruminant, the EHEC reservoir, or the colon of a human, an accidental host, EHEC must alter its physiology to survive within the host digestive tract. In this article, we explore the classes of molecules produced or modified by the microbiota that appear to be instrumental in governing virulence gene expression of EHEC. We also explore how interaction with different microbiotas influences EHEC infectivity and host interaction.

Cooperation of TLR12 and TLR11 in the IRF8-dependent IL-12 response to Toxoplasma gondii profilin.

TLRs play a central role in the innate recognition of pathogens and the activation of dendritic cells (DCs). In this study, we establish that, in addition to TLR11, TLR12 recognizes the profilin protein of the protozoan parasite Toxoplasma gondii and regulates IL-12 production by DCs in response to the parasite. Similar to TLR11, TLR12 is an endolysosomal innate immune receptor that colocalizes and interacts with UNC93B1. Biochemical experiments revealed that TLR11 and TLR12 directly bind to T. gondii profilin and are capable of forming a heterodimer complex. We also establish that the transcription factor IFN regulatory factor 8, not NF-κB, plays a central role in the regulation of the TLR11- and TLR12-dependent IL-12 response of DCs. These results suggest a central role for IFN regulatory factor 8-expressing CD8(+) DCs in governing the TLR11- and TLR12-mediated host defense against T. gondii.

B cell-intrinsic MyD88 signaling prevents the lethal dissemination of commensal bacteria during colonic damage.

The Toll-like receptor adaptor protein MyD88 is essential for the regulation of intestinal homeostasis in mammals. In this study, we determined that Myd88-deficient mice are susceptible to colonic damage that is induced by dextran sulfate sodium (DSS) administration resulting from uncontrolled dissemination of intestinal commensal bacteria. The DSS-induced mortality of Myd88-deficient mice was completely prevented by antibiotic treatment to deplete commensal bacteria. By using cell type-specific Myd88-deficient mice, we established that B cell-intrinsic MyD88 signaling plays a central role in the resistance to DSS-induced colonic damage via the production of IgM and complement-mediated control of intestinal bacteria. Our results indicate that the lack of intact MyD88 signaling in B cells, coupled with impaired epithelial integrity, enables commensal bacteria to function as highly pathogenic organisms, causing rapid host death.

Microbial infection-induced expansion of effector T cells overcomes the suppressive effects of regulatory T cells via an IL-2 deprivation mechanism.

Foxp3(+) regulatory T (Treg) cells are a critical cell population that suppresses T cell activation in response to microbial and viral pathogens. We identify a cell-intrinsic mechanism by which effector CD4(+) T cells overcome the suppressive effects of Treg cells in the context of three distinct infections: Toxoplasma gondii, Listeria monocytogenes, and vaccinia virus. The acute responses to the parasitic, bacterial, and viral pathogens resulted in a transient reduction in frequency and absolute number of Treg cells. The infection-induced partial loss of Treg cells was essential for the initiation of potent Th1 responses and host protection against the pathogens. The observed disappearance of Treg cells was a result of insufficiency in IL-2 caused by the expansion of pathogen-specific CD4(+) T cells with a limited capacity of IL-2 production. Exogenous IL-2 treatment during the parasitic, bacterial, and viral infections completely prevented the loss of Treg cells, but restoration of Treg cells resulted in a greatly enhanced susceptibility to the pathogens. These results demonstrate that the transient reduction in Treg cells induced by pathogens via IL-2 deprivation is essential for optimal T cell responses and host resistance to microbial and viral pathogens.

Innate responses to Toxoplasma gondii in mice and humans.

Primary infection with Toxoplasma gondii stimulates production of high levels of interleukin 12 (IL-12) and interferon γ (IFN-γ) by cells of the innate immune system. These two cytokines are central to resistance to T. gondii. Signaling through the Toll-like receptor (TLR) adaptor protein MyD88 is indispensible for activating early innate immune responses. Recent studies have established that TLR11 plays a dominant role in sensing T. gondii. At the same time, TLR11 is represented in humans only by a pseudogene, and the major question of how innate and adaptive immune responses occur in the absence of TLR11 remains unanswered. In this article, similarities and differences in sensors and effector molecules that determine host resistance to the parasite in humans and mice are discussed.

UNC93B1 is essential for TLR11 activation and IL-12-dependent host resistance to Toxoplasma gondii.

Toll-like receptor (TLR) activation relies on biochemical recognition of microbial molecules and localization of the TLR within specific cellular compartments. Cell surface TLRs largely recognize bacterial membrane components, and intracellular TLRs are exclusively involved in sensing nucleic acids. Here we show that TLR11, an innate sensor for the Toxoplasma protein profilin, is an intracellular receptor that resides in the endoplasmic reticulum. The 12 membrane-spanning endoplasmic reticulum-resident protein UNC93B1 interacts directly with TLR11 and regulates the activation of dendritic cells in response to Toxoplasma gondii profilin and parasitic infection in vivo. A deficiency in functional UNC93B1 protein abolished TLR11-dependent IL-12 secretion by dendritic cells, attenuated Th1 responses against T. gondii, and dramatically enhanced susceptibility to the parasite. Our results reveal that the association with UNC93B1 and the intracellular localization of TLRs are not unique features of nucleic acid-sensing TLRs but is also essential for TLR11-dependent recognition of T. gondii profilin and for host protection against this parasite.

Gut commensal bacteria direct a protective immune response against Toxoplasma gondii.

Toxoplasma gondii is a universally distributed pathogen that infects over one billion people worldwide. Host resistance to this protozoan parasite depends on a Th1 immune response with potent production of the cytokines interleukin-12 and interferon gamma. Although Toll-like receptor 11 (TLR11) plays a major role in controlling Th1 immunity to this pathogen in mice, this innate immune receptor is nonfunctional in humans, and the mechanisms of TLR11-independent sensing of T. gondii remain elusive. Here, we show that oral infection by T. gondii triggers a TLR11-independent but MyD88-dependent Th1 response that is impaired in TLR2xTLR4 double knockout and TLR9 single knockout mice. These mucosal innate and adaptive immune responses to T. gondii rely on the indirect stimulation of dendritic cells by normal gut microflora. Thus, our results reveal that gut commensal bacteria can serve as molecular adjuvants during parasitic infection, providing indirect immunostimulation that protects against T. gondii in the absence of TLR11.