Predatory bacteria prevent the proliferation of intraocular Serratia marcescens and fluoroquinolone-resistant Pseudomonas aeruginosa.

Endogenous endophthalmitis caused by Gram-negative bacteria is an intra-ocular infection that can rapidly progress to irreversible loss of vision. While most endophthalmitis isolates are susceptible to antibiotic therapy, the emergence of resistant bacteria necessitates alternative approaches to combat intraocular bacterial proliferation. In this study the ability of predatory bacteria to limit intraocular growth of Pseudomonas aeruginosa, Serratia marcescens, and Staphylococcus aureus was evaluated in a New Zealand white rabbit endophthalmitis prevention model. Predatory bacteria Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus were able to reduce proliferation of keratitis isolates of P. aeruginosa and to a lesser extent S. marcescens. However, it was not able to significantly reduce the number of intraocular S. aureus, which is not a productive prey for these predatory bacteria, suggesting that the inhibitory effect on P. aeruginosa and S. marcescens requires active predation rather than an antimicrobial immune response. Similarly, UV-inactivated B. bacteriovorus were unable to prevent proliferation of P. aeruginosa. Together, these data indicate in vivo inhibition of Gram-negative bacteria proliferation within the intra-ocular environment by predatory bacteria.

IgaA Protein, GumB, Has a Global Impact on the Transcriptome and Surface Proteome of Serratia marcescens.

Bacterial stress response signaling systems, like the Rcs system are triggered by membrane and cell wall damaging compounds, including antibiotics and immune system factors. These regulatory systems help bacteria survive envelope stress by altering the transcriptome resulting in protective phenotypic changes that may also influence the virulence of the bacterium. This study investigated the role of the Rcs stress response system using a clinical keratitis isolate of Serratia marcescens with a mutation in the gumB gene. GumB, an IgaA ortholog, inhibits activation of the Rcs system, such that mutants have overactive Rcs signaling. Transcriptomic analysis indicated that approximately 15% of all S. marcescens genes were significantly altered with 2-fold or greater changes in expression in the ΔgumB mutant compared to the wild type, indicating a global transcriptional regulatory role for GumB. We further investigated the phenotypic consequences of two classes of genes with altered expression in the ΔgumB mutant expected to contribute to infections: serralysin metalloproteases PrtS, SlpB, and SlpE, and type I pili coded by fimABCD. Secreted fractions from the ΔgumB mutant had reduced cytotoxicity to a corneal cell line, and could be complemented by induced expression of prtS, but not cytolysin shlBA, phospholipase phlAB, or flagellar master regulator flhDC operons. Proteomic analysis, qRT-PCR, and type I pili-dependent yeast agglutination indicated an inhibitory role for the Rcs system in adhesin production. Together these data demonstrate GumB has a global impact on S. marcescens gene expression that had measurable effects on bacterial cytotoxicity and surface adhesin production.

Blowing epithelial cell bubbles with GumB: ShlA-family pore-forming toxins induce blebbing and rapid cellular death in corneal epithelial cells.

Medical devices, such as contact lenses, bring bacteria in direct contact with human cells. Consequences of these host-pathogen interactions include the alteration of mammalian cell surface architecture and induction of cellular death that renders tissues more susceptible to infection. Gram-negative bacteria known to induce cellular blebbing by mammalian cells, Pseudomonas and Vibrio species, do so through a type III secretion system-dependent mechanism. This study demonstrates that a subset of bacteria from the Enterobacteriaceae bacterial family induce cellular death and membrane blebs in a variety of cell types via a type V secretion-system dependent mechanism. Here, we report that ShlA-family cytolysins from Proteus mirabilis and Serratia marcescens were required to induce membrane blebbling and cell death. Blebbing and cellular death were blocked by an antioxidant and RIP-1 and MLKL inhibitors, implicating necroptosis in the observed phenotypes. Additional genetic studies determined that an IgaA family stress-response protein, GumB, was necessary to induce blebs. Data supported a model where GumB and shlBA are in a regulatory circuit through the Rcs stress response phosphorelay system required for bleb formation and pathogenesis in an invertebrate model of infection and proliferation in a phagocytic cell line. This study introduces GumB as a regulator of S. marcescens host-pathogen interactions and demonstrates a common type V secretion system-dependent mechanism by which bacteria elicit surface morphological changes on mammalian cells. This type V secretion-system mechanism likely contributes bacterial damage to the corneal epithelial layer, and enables access to deeper parts of the tissue that are more susceptible to infection.

Xylose-Inducible Promoter Tools for Pseudomonas Species and Their Use in Implicating a Role for the Type II Secretion System Protein XcpQ in the Inhibition of Corneal Epithelial Wound Closure.

Tunable control of gene expression is an invaluable tool for biological experiments. In this study, we describe a new xylose-inducible promoter system and evaluate it in both Pseudomonas aeruginosa and Pseudomonas fluorescens The Pxut promoter, derived from the P. fluorescensxut operon, was incorporated into a broad-host-range pBBR1-based plasmid and was compared to the Escherichia coli-derived PBAD promoter using gfp as a reporter. Green fluorescent protein (GFP) fluorescence from the Pxut promoter was inducible in both Pseudomonas species, but not in E. coli, which may facilitate the cloning of genes toxic to E. coli to generate plasmids. The Pxut promoter was activated at a lower inducer concentration than PBAD in P. fluorescens, and higher gfp levels were achieved using Pxut Flow cytometry analysis indicated that Pxut was leakier than PBAD in the Pseudomonas species tested but was expressed in a higher proportion of cells when induced. d-Xylose as a sole carbon source did not support the growth of P. aeruginosa or P. fluorescens and is less expensive than many other commonly used inducers, which could facilitate large-scale applications. The efficacy of this system was demonstrated by its use to reveal a role for the P. aeruginosa type II secretion system gene xcpQ in bacterial inhibition of corneal epithelial cell wound closure. This study introduces a new inducible promoter system for gene expression for use in Pseudomonas species.IMPORTANCEPseudomonas species are enormously important in human infections, in biotechnology, and as model systems for investigating basic science questions. In this study, we have developed a xylose-inducible promoter system, evaluated it in P. aeruginosa and P. fluorescens, and found it to be suitable for the strong induction of gene expression. Furthermore, we have demonstrated its efficacy in controlled gene expression to show that a type II secretion system protein from P. aeruginosa, XcpQ, is important for host-pathogen interactions in a corneal wound closure model.

Biologically active pigment and ShlA cytolysin of Serratia marcescens induce autophagy in a human ocular surface cell line.

BACKGROUND: The cellular process of autophagy is essential for maintaining the health of ocular tissue. Dysregulation of autophagy is associated with several ocular diseases including keratoconus and macular degeneration. It is known that autophagy can be used to respond to microbial infections and that certain microbes can exploit the autophagic process to their benefit. In this study, a genetic approach was used to identify surface-associated and secreted products generated by the opportunistic pathogen Serratia marcescens involved in activation of autophagy. METHODS: A recombinant human corneal limbal epithelial cell line expressing a LC3-GFP fusion protein was challenged with normalized secretomes from wild-type and mutant S. marcescens derivatives. LC3-GFP fluorescence patterns were used to assess the ability of wild-type and mutant bacteria to influence autophagy. Purified prodigiosin was obtained from stationary phase bacteria and used to challenge ocular cells. RESULTS: Mutations in the global regulators eepR and gumB genes highly reduced the ability of the bacteria to activate autophagy in corneal cells. This effect was further narrowed down to the secreted cytolysin ShlA and the biologically active pigment prodigiosin. Purified prodigiosin and ShlA from Escherichia coli further supported the role of these factors in activating autophagy in human corneal cells. Additional genetic data indicate a role for flagellin and type I pili, but not the nuclease, S-layer protein, or serratamolide biosurfactant in activation of autophagy. CONCLUSIONS: This work identifies specific bacterial components that activate autophagy and give insight into potential host-pathogen interactions or compounds that can be used to therapeutically manipulate autophagy.

An IgaA/UmoB Family Protein from Serratia marcescens Regulates Motility, Capsular Polysaccharide Biosynthesis, and Secondary Metabolite Production.

Secondary metabolites are an important source of pharmaceuticals and key modulators of microbe-microbe interactions. The bacterium Serratia marcescens is part of the Enterobacteriaceae family of eubacteria and produces a number of biologically active secondary metabolites. In this study, we screened for novel regulators of secondary metabolites synthesized by a clinical isolate of S. marcescens and found mutations in a gene for an uncharacterized UmoB/IgaA family member here named gumB Mutation of gumB conferred a severe loss of the secondary metabolites prodigiosin and serratamolide. The gumB mutation conferred pleiotropic phenotypes, including altered biofilm formation, highly increased capsular polysaccharide production, and loss of swimming and swarming motility. These phenotypes corresponded to transcriptional changes in fimA, wecA, and flhD Unlike other UmoB/IgaA family members, gumB was found to be not essential for growth in S. marcescens, yet igaA from Salmonella enterica, yrfF from Escherichia coli, and an uncharacterized predicted ortholog from Klebsiella pneumoniae complemented the gumB mutant secondary metabolite defects, suggesting highly conserved function. These data support the idea that UmoB/IgaA family proteins are functionally conserved and extend the known regulatory influence of UmoB/IgaA family proteins to the control of competition-associated secondary metabolites and biofilm formation.IMPORTANCE IgaA/UmoB family proteins are found in members of the Enterobacteriaceae family of bacteria, which are of environmental and public health importance. IgaA/UmoB family proteins are thought to be inner membrane proteins that report extracellular stresses to intracellular signaling pathways that respond to environmental challenge. This study introduces a new member of the IgaA/UmoB family and demonstrates a high degree of functional similarity between IgaA/UmoB family proteins. Moreover, this study extends the phenomena controlled by IgaA/UmoB family proteins to include the biosynthesis of antimicrobial secondary metabolites.

Bacteria induce autophagy in a human ocular surface cell line.

Autophagy protects cells from intracellular pathogens, but can be exploited by some infectious agents to their benefit. Currently it is not known if bacteria induce autohpagy in cells of the cornea. The goal of this study was to develop an ocular surface autophagy reporter cell line and determine whether ocular bacterial pathogens influence host responses through autophagy induction. The cell line was made using lentivirus transduction of an LC3-GFP fusion protein in human corneal limbal epithelial (HCLE) cells. LC3-GFP puncta in HCLEs were induced by rapamycin and ammonium chloride treatments, and prevented by the autophagy inhibitors 3-methyladenine (3'MA) and bafilomycin. Importantly, secretomes from Escherichia coli, Serratia marcescens, Staphylococcus aureus, methicillin sensitive (MSSA) and resistant (MRSA), were found to induce autophagy, whereas other bacteria, including Acinetobacter baumannii, Achromobacter xylosoxidans, Enterococcus faecalis, Klebsiella pneumoniae, Moraxella sp., and Stenotrophomonas maltophilia, did not. Our data indicates differences between tested ocular isolates of MRSA and MSSA in the activation of autophagy. HCLEs treated with 3'MA were slightly more susceptible to cytotoxic factors produced by S. marcescens and MRSA keratitis isolates, by contrast, bafilomycin A1 treatment caused no difference. This work demonstrates the successful development and validation of an autophagy reporter corneal cell line and indicates differences between ocular bacterial isolates in the activation of autophagy.

Release of Moxifloxacin From Corneal Collagen Shields.

OBJECTIVES: To investigate the diffusion of moxifloxacin through bandage contact lenses (BCLs) versus corneal collagen shields (CSs), the relative ability of BCLs and CSs to release moxifloxacin, and the potential of release of moxifloxacin from CSs in the clinical setting. METHODS: Using an in vitro model, the diffusion of 5% moxifloxacin across BCLs and CSs was compared. Next, the amount of drug release from BCLs and CSs soaked in 0.5% moxifloxacin was measured. Finally, based on a clinical model, CSs were soaked in Vigamox (commercial moxifloxacin) and the total concentration released was detected. Collagen shields remained intact after 24 hr; therefore, enzymatic digestion and mechanical grinding of the CS were performed to determine whether further drug could be released. The concentration of moxifloxacin was measured using a spectrophotometer at set time points up to 24 hr. RESULTS: In the diffusion assay, 35.7±10.5% diffused through the BCLs and 36.2±11.8% diffused through the CSs (P=0.77). The absorption assay demonstrated at 120 min, a total of 33.3±6.77 µg/mL was released from BCLs compared with 45.8±5.2 µg/mL from the CSs (P=0.0008). In vitro experiments to simulate clinical application of Vigamox-soaked CS found the concentration of moxifloxacin released of 127.7±7.25 µg/mL in 2 mL of phosphate-buffered saline over 24 hr. CONCLUSIONS: Moxifloxacin diffuses through BCLs and CSs at similar rates; however, CSs have greater capacity to absorb and release moxifloxacin compared with BCLs. Vigamox-soaked CSs released 250 µg of moxifloxacin and may be a useful method to prevent endophthalmitis.

In Vitro Evaluation of a Hypochlorous Acid Hygiene Solution on Established Biofilms.

OBJECTIVES: The purpose of this study was to determine whether a commercial formulation of hypochlorous acid hygiene solution (0.01%), Avenova, can destroy existing biofilms formed by ocular clinical bacterial isolates, including blepharitis isolates of Staphylococcus aureus and coagulase-negative staphylococci, and a keratitis isolate of Pseudomonas aeruginosa. METHODS: Biofilms grown in bacterial growth media on disposable contact lens cases were challenged with hypochlorous acid hygiene solution. At various time points, surviving bacteria were quantified by serial dilution and colony counts. Staphylococcus aureus biofilms formed on glass were challenged using a hypochlorous acid hygiene solution and imaged using vital staining and confocal laser scanning microscopy. RESULTS: Bactericidal activity (≥3 Log10; 99.9%) was observed for all tested bacterial species after a 30-min exposure. Staphylococcus aureus biofilms had a bactericidal level of killing by 10 min (P<0.01), Staphylococcus capitis by 5 min (P<0.001), Staphylococcus epidermidis by 30 min (P<0.001), and P. aeruginosa by 10 min (P<0.01). Confocal microscopy and crystal violet staining analysis of bacterial biofilms treated with hypochlorous acid solution both demonstrated that biofilm bacteria were readily killed, but biofilm structure was largely maintained. CONCLUSIONS: Hypochlorous acid (0.01%) hygiene solution was able to achieve bactericidal levels of killing of bacteria in biofilms but did not disrupt biofilm structures. Susceptibility of tested staphylococcal blepharitis isolates varied by species, with S. capitis being the most susceptible and S. epidermidis being the least susceptible.

Suppressor analysis of eepR mutant defects reveals coordinate regulation of secondary metabolites and serralysin biosynthesis by EepR and HexS.

The EepR transcription factor positively regulates secondary metabolites and tissue-damaging metalloproteases. To gain insight into mechanisms by which EepR regulates pigment and co-regulated factors, genetic suppressor analysis was performed. Suppressor mutations that restored pigment to the non-pigmented ∆eepR mutant mapped to the hexS ORF. Mutation of hexS also restored haemolysis, swarming motility and protease production to the eepR mutant. HexS is a known direct and negative regulator of secondary metabolites in Serratia marcescens and is a LysR family regulator and an orthologue of LrhA. Here, we demonstrate that HexS directly controls eepR and the serralysin gene prtS. EepR was shown to directly regulate eepR expression but indirectly regulate hexS expression. Together, these data indicate that EepR and HexS oppose each other in controlling stationary phase-associated molecules and enzymes.

Exploitation of a "hockey-puck" phenotype to identify pilus and biofilm regulators in Serratia marcescens through genetic analysis.

Pili are essential adhesive determinants for many bacterial pathogens. A suppressor mutation screen that takes advantage of a pilus-mediated self-aggregative "hockey-puck" colony phenotype was designed to identify novel regulators of type I pili in Serratia marcescens. Mutations that decreased pilus biosynthesis mapped to the fimABCD operon; to the genes alaT, fkpA, and oxyR; upstream of the flagellar master regulator operon flhDC; and to an uncharacterized gene encoding a predicted DUF1401 domain. Biofilm formation and pilus-dependent agglutination assays were used to characterize the relative importance of the identified genes in pilus biosynthesis. Additional mutagenic or complementation analysis was used to verify the role of candidate genes in pilus biosynthesis. Presented data support a model that CRP negatively regulates pilus biosynthesis through increased expression of flhDC and decreased expression of oxyR. Further studies are warranted to determine the mechanism by which these genes mediate pilus biosynthesis or function.

Serratia marcescens Cyclic AMP Receptor Protein Controls Transcription of EepR, a Novel Regulator of Antimicrobial Secondary Metabolites.

UNLABELLED: Serratia marcescens generates secondary metabolites and secreted enzymes, and it causes hospital infections and community-acquired ocular infections. Previous studies identified cyclic AMP (cAMP) receptor protein (CRP) as an indirect inhibitor of antimicrobial secondary metabolites. Here, we identified a putative two-component regulator that suppressed crp mutant phenotypes. Evidence supports that the putative response regulator eepR was directly transcriptionally inhibited by cAMP-CRP. EepR and the putative sensor kinase EepS were necessary for the biosynthesis of secondary metabolites, including prodigiosin- and serratamolide-dependent phenotypes, swarming motility, and hemolysis. Recombinant EepR bound to the prodigiosin and serratamolide promoters in vitro. Together, these data introduce a novel regulator of secondary metabolites that directly connects the broadly conserved metabolism regulator CRP with biosynthetic genes that may contribute to competition with other microbes. IMPORTANCE: This study identifies a new transcription factor that is directly controlled by a broadly conserved transcription factor, CRP. CRP is well studied in its role to help bacteria respond to the amount of nutrients in their environment. The new transcription factor EepR is essential for the bacterium Serratia marcescens to produce two biologically active compounds, prodigiosin and serratamolide. These two compounds are antimicrobial and may allow S. marcescens to compete for limited nutrients with other microorganisms. Results from this study tie together the CRP environmental nutrient sensor with a new regulator of antimicrobial compounds. Beyond microbial ecology, prodigiosin and serratamolide have therapeutic potential; therefore, understanding their regulation is important for both applied and basic science.

EepR Mediates Secreted-Protein Production, Desiccation Survival, and Proliferation in a Corneal Infection Model.

Serratia marcescens is a soil- and water-derived bacterium that secretes several host-directed factors and causes hospital infections and community-acquired ocular infections. The putative two-component regulatory system composed of EepR and EepS regulates hemolysis and swarming motility through transcriptional control of the swrW gene and pigment production through control of the pigA-pigN operon. Here, we identify and characterize a role for EepR in regulation of exoenzyme production, stress survival, cytotoxicity to human epithelial cells, and virulence. Genetic analysis supports the model that EepR is in a common pathway with the widely conserved cyclic-AMP receptor protein that regulates protease production. Together, these data introduce a novel regulator of host-pathogen interactions and secreted-protein production.

Identification of SlpB, a Cytotoxic Protease from Serratia marcescens.

The Gram-negative bacterium and opportunistic pathogen Serratia marcescens causes ocular infections in healthy individuals. Secreted protease activity was characterized from 44 ocular clinical isolates, and a higher frequency of protease-positive strains was observed among keratitis isolates than among conjunctivitis isolates. A positive correlation between protease activity and cytotoxicity to human corneal epithelial cells in vitro was determined. Deletion of prtS in clinical keratitis isolate K904 reduced, but did not eliminate, cytotoxicity and secreted protease production. This indicated that PrtS is necessary for full cytotoxicity to ocular cells and implied the existence of another secreted protease(s) and cytotoxic factors. Bioinformatic analysis of the S. marcescens Db11 genome revealed three additional open reading frames predicted to code for serralysin-like proteases noted here as slpB, slpC, and slpD. Induced expression of prtS and slpB, but not slpC and slpD, in strain PIC3611 rendered the strain cytotoxic to a lung carcinoma cell line; however, only prtS induction was sufficient for cytotoxicity to a corneal cell line. Strain K904 with deletion of both prtS and slpB genes was defective in secreted protease activity and cytotoxicity to human cell lines. PAGE analysis suggests that SlpB is produced at lower levels than PrtS. Purified SlpB demonstrated calcium-dependent and AprI-inhibited protease activity and cytotoxicity to airway and ocular cell lines in vitro. Lastly, genetic analysis indicated that the type I secretion system gene, lipD, is required for SlpB secretion. These genetic data introduce SlpB as a new cytotoxic protease from S. marcescens.

NADPH oxidase-driven phagocyte recruitment controls Candida albicans filamentous growth and prevents mortality.

Candida albicans is a human commensal and clinically important fungal pathogen that grows as both yeast and hyphal forms during human, mouse and zebrafish infection. Reactive oxygen species (ROS) produced by NADPH oxidases play diverse roles in immunity, including their long-appreciated function as microbicidal oxidants. Here we demonstrate a non-traditional mechanistic role of NADPH oxidase in promoting phagocyte chemotaxis and intracellular containment of fungi to limit filamentous growth. We exploit the transparent zebrafish model to show that failed NADPH oxidase-dependent phagocyte recruitment to C. albicans in the first four hours post-infection permits fungi to germinate extracellularly and kill the host. We combine chemical and genetic tools with high-resolution time-lapse microscopy to implicate both phagocyte oxidase and dual-specific oxidase in recruitment, suggesting that both myeloid and non-myeloid cells promote chemotaxis. We show that early non-invasive imaging provides a robust tool for prognosis, strongly connecting effective early immune response with survival. Finally, we demonstrate a new role of a key regulator of the yeast-to-hyphal switching program in phagocyte-mediated containment, suggesting that there are species-specific methods for modulation of NADPH oxidase-independent immune responses. These novel links between ROS-driven chemotaxis and fungal dimorphism expand our view of a key host defense mechanism and have important implications for pathogenesis.

Visualizing Bdellovibrio bacteriovorus by Using the tdTomato Fluorescent Protein.

Bdellovibrio bacteriovorus is a Gram-negative bacterium that belongs to the delta subgroup of proteobacteria and is characterized by a predatory life cycle. In recent years, work has highlighted the potential use of this predator to control bacteria and biofilms. Traditionally, the reduction in prey cells was used to monitor predation dynamics. In this study, we introduced pMQ414, a plasmid that expresses the tdTomato fluorescent reporter protein, into a host-independent strain and a host-dependent strain of B. bacteriovorus 109J. The new construct was used to conveniently monitor predator proliferation in real time, in different growth conditions, in the presence of lytic enzymes, and on several prey bacteria, replicating previous studies that used plaque analysis to quantify B. bacteriovorus. The new fluorescent plasmid also enabled us to visualize the predator in liquid cultures, in the context of a biofilm, and in association with human epithelial cells.

Diffusion of Antimicrobials Across Silicone Hydrogel Contact Lenses.

OBJECTIVES: To measure the diffusion of topical preparations of moxifloxacin, amphotericin B (AmB), and polyhexamethylene biguanide (PHMB) through silicone hydrogel (SH) contact lenses (CLs) in vitro. METHODS: Using an in vitro model, the diffusion of three antimicrobials through SH CLs was measured. Diffused compounds were measured using a spectrophotometer at set time points over a period of 4 hr. The amount of each diffused antimicrobial was determined by comparing the experimental value with a standard curve. A biological assay was performed to validate the CL diffusion assay by testing antimicrobial activity of diffused material against lawns of susceptible bacteria (Staphylococcus epidermidis) and yeast (Saccharomyces cerevisiae). Experiments were repeated at least two times with a total of at least four independent replicates. RESULTS: Our data show detectable moxifloxacin and PHMB diffusion through SH CLs at 30 min, whereas AmB diffusion remained below the limit of detection within the 4-hr experimental period. In the biological assay, diffused moxifloxacin demonstrated microbial killing starting at 20 min on bacterial lawns, whereas PHMB and AmB failed to demonstrate killing on microbial lawns over the course of the 60-min experiment. CONCLUSIONS: In vitro diffusion assays demonstrate limited penetration of certain anti-infective agents through SH CLs. Further studies regarding the clinical benefit of using these agents along with bandage CL for corneal pathologic condition are warranted.

Candida albicans induces arginine biosynthetic genes in response to host-derived reactive oxygen species.

The interaction of Candida albicans with phagocytes of the host's innate immune system is highly dynamic, and its outcome directly impacts the progression of infection. While the switch to hyphal growth within the macrophage is the most obvious physiological response, much of the genetic response reflects nutrient starvation: translational repression and induction of alternative carbon metabolism. Changes in amino acid metabolism are not seen, with the striking exception of arginine biosynthesis, which is upregulated in its entirety during coculture with macrophages. Using single-cell reporters, we showed here that arginine biosynthetic genes are induced specifically in phagocytosed cells. This induction is lower in magnitude than during arginine starvation in vitro and is driven not by an arginine deficiency within the phagocyte but instead by exposure to reactive oxygen species (ROS). Curiously, these genes are induced in a narrow window of sublethal ROS concentrations. C. albicans cells phagocytosed by primary macrophages deficient in the gp91(phox) subunit of the phagocyte oxidase do not express the ARG pathway, indicating that the induction is dependent on the phagocyte oxidative burst. C. albicans arg pathway mutants are retarded in germ tube and hypha formation within macrophages but are not notably more sensitive to ROS. We also find that the ARG pathway is regulated not by the general amino acid control response but by transcriptional regulators similar to the Saccharomyces cerevisiae ArgR complex. In summary, phagocytosis induces this single amino acid biosynthetic pathway in an ROS-dependent manner.

Dose optimization in surgical prophylaxis: sub-inhibitory dosing of vancomycin increases rates of biofilm formation and the rates of surgical site infection.

Antibiotic stewardship is viewed as having great public health benefit with limited direct benefit to the patient at the time of administration. The objective of our study was to determine if inappropriate administration of antibiotics could create conditions that would increase the rates of surgical infection. We hypothesized that sub-MIC levels of vancomycin would increase Staphylococcus aureus growth, biofilm formation, and rates of infection. S. aureus MRSA and MSSA strains were used for all experiments. Bacteria were grown planktonically and monitored using spectrophotometry. Quantitative agar culture was used to measure planktonic and biofilm bacterial burden. A mouse abscess model was used to confirm phenotypes in vivo. In the planktonic growth assay, increases in bacterial burden at » MIC vancomycin were observed in USA300 JE2 by 72 h. Similar findings were observed with ½ MIC in Newman and SH1000. For biofilm formation, USA300 JE2 at » and ½ MIC vancomycin increased biofilm formation by approximately 1.3- and 2.3-fold respectively at 72 h as compared to untreated controls. Similar findings were observed with Newman and SH1000 with a 2.4-fold increase in biofilm formation at ½ MIC vancomycin. In a mouse abscess model, there was a 1.2-fold increase with sub-MIC vancomycin at 3 days post infection. Our study showed that Sub-optimal vancomycin dosing promoted S. aureus planktonic growth and biofilm formation, phenotypic measures of bacterial virulence. This phenotype induced by sub-MIC levels of vancomycin was also observed to increase rates of infection and pathogenesis in our mouse model. Risks of exposure to sub-MIC concentrations with vancomycin in surgical procedures are greater as there is decreased bioavailability in tissue in comparison to other antibiotics. This highlights the importance of proper antibiotic selection, stewardship, and dosing for both surgical prophylaxis and treatment of infection.

Intra-ocular Predation of Fluoroquinolone-Resistant Pseudomonas aeruginosa and Serratia marcescens by Predatory Bacteria.

Endogenous endophthalmitis caused by Gram-negative bacteria is an intra-ocular infection that can rapidly progress to irreversible loss of vision. While most endophthalmitis isolates are susceptible to antibiotic therapy, the emergence of resistant bacteria necessitates alternative approaches to combat intraocular bacterial proliferation. In this study the ability of predatory bacteria to limit intraocular growth of Pseudomonas aeruginosa, Serratia marcescens, and Staphylococcus aureus was evaluated in a New Zealand White rabbit endophthalmitis prevention model. Predatory bacteria Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus were able to reduce proliferation of keratitis isolates of P. aeruginosa and S. marcescens. However, it was not able to significantly reduce S. aureus, which is not a productive prey for these predatory bacteria, suggesting that the inhibitory effect on P. aeruginosa requires active predation rather than an antimicrobial immune response. Similarly, UV-inactivated B. bacteriovorus were unable to prevent proliferation of P. aeruginosa. Together, these data suggest in vivo predation of Gram-negative bacteria within the intra-ocular environment.