RESUMO
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.
Assuntos
Endoftalmite , Infecções por Pseudomonas , Animais , Coelhos , Fluoroquinolonas/farmacologia , Pseudomonas aeruginosa , Serratia marcescens , Comportamento Predatório , Staphylococcus aureus , Proliferação de CélulasRESUMO
Utilization of fluorescent proteins is widespread for the study of microbial pathogenesis and host-pathogen interactions. Here, we discovered that linkage of the 36 N-terminal amino acids of FTL_0580 (a hypothetical protein of Francisella tularensis) to fluorescent proteins increases the fluorescence emission of bacteria that express these recombinant fusions. This N-terminal peptide will be referred to as 580N. Western blotting revealed that the linkage of 580N to Emerald Green Fluorescent Protein (EmGFP) in F. tularensis markedly improved detection of this protein. We therefore hypothesized that transcripts containing 580N may be translated more efficiently than those lacking the coding sequence for this leader peptide. In support, expression of emGFPFt that had been codon-optimized for F. tularensis, yielded significantly enhanced fluorescence than its non-optimized counterpart. Furthermore, fusing emGFP with coding sequence for a small N-terminal peptide (Serine-Lysine-Isoleucine-Lysine), which had previously been shown to inhibit ribosomal stalling, produced robust fluorescence when expressed in F. tularensis. These findings support the interpretation that 580N enhances the translation efficiency of fluorescent proteins in F. tularensis. Interestingly, expression of non-optimized 580N-emGFP produced greater fluorescence intensity than any other construct. Structural predictions suggested that RNA secondary structure also may be influencing translation efficiency. When expressed in Escherichia coli and Klebsiella pneumoniae bacteria, 580N-emGFP produced increased green fluorescence compared to untagged emGFP (neither allele was codon optimized for these bacteria). In conclusion, fusing the coding sequence for the 580N leader peptide to recombinant genes might serve as an economical alternative to codon optimization for enhancing protein expression in bacteria.
Assuntos
Francisella tularensis , Francisella tularensis/genética , Francisella tularensis/química , Francisella tularensis/metabolismo , Lisina/metabolismo , Peptídeos/genética , Códon/genética , Sinais Direcionadores de Proteínas/genéticaRESUMO
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.
Assuntos
Proteoma , Serratia marcescens , Serratia marcescens/genética , Proteoma/metabolismo , Transcriptoma , Proteômica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
In this study, we tested the hypothesis that the conserved bacterial IgaA-family protein, GumB, mediates microbial pathogenesis associated with Serratia marcescens ocular infections through regulation of the Rcs stress response system. The role of the Rcs system and bacterial stress response systems for microbial keratitis is not known, and the role of IgaA proteins in mammalian pathogenesis models has only been tested with partial-function allele variants of Salmonella. Here, we observed that an Rcs-activated gumB mutant had a >50-fold reduction in proliferation compared to the wild type within rabbit corneas at 48 h and demonstrated a notable reduction in inflammation based on inflammatory signs, including the absence of hypopyons, and proinflammatory markers measured at the RNA and protein levels. The gumB mutant phenotypes could be complemented by wild-type gumB on a plasmid. We observed that bacteria with an inactivated Rcs stress response system induced high levels of ocular inflammation and restored corneal virulence to the gumB mutant. The high virulence of the ΔrcsB mutant was dependent upon the ShlA cytolysin transporter ShlB. Similar results were found for testing the cytotoxic effects of wild-type and mutant bacteria on a human corneal epithelial cell line in vitro. Together, these data indicate that GumB regulates virulence factor production through the Rcs system, and this overall stress response system is a key mediator of a bacterium's ability to induce vision-threatening keratitis.
Assuntos
Proteínas de Bactérias/genética , Ceratite/microbiologia , Infecções por Serratia/microbiologia , Serratia marcescens/fisiologia , Estresse Fisiológico , Animais , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno , Mutação , Coelhos , Estresse Fisiológico/genética , Virulência , Fatores de Virulência/genéticaRESUMO
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.
Assuntos
Toxinas Bacterianas/metabolismo , Células Epiteliais/metabolismo , Epitélio Corneano/metabolismo , Infecções por Proteus/metabolismo , Proteus/metabolismo , Infecções por Serratia/metabolismo , Serratia marcescens/metabolismo , Animais , Toxinas Bacterianas/genética , Morte Celular , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Epitélio Corneano/microbiologia , Epitélio Corneano/patologia , Humanos , Camundongos , Perforina/genética , Perforina/metabolismo , Proteus/genética , Infecções por Proteus/genética , Infecções por Proteus/microbiologia , Infecções por Proteus/patologia , Células RAW 264.7 , Infecções por Serratia/genética , Infecções por Serratia/microbiologia , Infecções por Serratia/patologia , Serratia marcescens/genética , Suínos , Sistemas de Secreção Tipo V/genética , Sistemas de Secreção Tipo V/metabolismoRESUMO
There is no approved antiviral therapy for adenovirus (HAdV) ocular infections. Astodrimer sodium (SPL7013) is a polyanionic dendrimer with antiviral activity. The current study evaluated the ocular tolerability and anti-adenoviral efficacy of topical SPL7013 in rabbit ocular models. In a tolerability study, rabbits were treated with 3% SPL7013, vehicle, or 0.5% cidofovir. Their eyes were graded using the Draize scale. In antiviral efficacy studies, HAdV5 inoculated eyes were treated with 3% SPL7013, vehicle, or 0.5% cidofovir. Eyes were cultured for the virus on days 0, 1, 3, 4, 5, 7, 9, 11, and 14. Viral titers were determined. There were no differences in Draize scores between 3% SPL7013 and vehicle on any day. Cidofovir produced significantly higher Draize scores on day 12 than SPL7013 and vehicle. The 3% SPL7013 and 0.5% cidofovir significantly reduced daily viral titers and positive cultures per total compared with vehicle on several different days. The 3% SPL7013 and 0.5% cidofovir significantly reduced the duration of HAdV5 shedding compared to vehicle. The 3% SPL7013 demonstrated significantly more antiviral activity compared with vehicle in the Ad5/NZW rabbit ocular model. The 3% SPL7013 induced "minimal" to "practically non-irritating" Draize scores in the ocular tolerability study. Further development of astodrimer sodium as a topical antiviral therapy for adenoviral ocular infections is indicated.
Assuntos
Infecções por Adenoviridae/tratamento farmacológico , Cidofovir/administração & dosagem , Dendrímeros/administração & dosagem , Infecções Oculares Virais/tratamento farmacológico , Polilisina/administração & dosagem , Células A549 , Adenovírus Humanos/efeitos dos fármacos , Adenovírus Humanos/fisiologia , Administração Tópica , Animais , Cidofovir/farmacologia , Dendrímeros/farmacologia , Modelos Animais de Doenças , Feminino , Humanos , Polilisina/farmacologia , Coelhos , Resultado do Tratamento , Carga Viral/efeitos dos fármacosRESUMO
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.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/genética , Pseudomonas fluorescens/genética , Reepitelização/genética , Sistemas de Secreção Tipo II/genética , Xilose/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Epitélio Corneano/lesões , Proteínas de Membrana/metabolismo , Pseudomonas aeruginosa/metabolismo , Pseudomonas fluorescens/metabolismo , Análise de Sequência de DNA , Sistemas de Secreção Tipo II/metabolismoRESUMO
Serratia marcescens is a facultatively anaerobic bacterium and the most recognized producer of the hydrophobic pigment prodigiosin. Previous work has shown that prodigiosin both increases ATP production during population lag phase and approximately doubles the stationary-phase cell yield. Here, we employed both batch and chemostat culture methods to investigate prodigiosin's role during high rate growth at low cell density as peak cellular ATP levels decline. Batch culture experiments utilizing artificial pigment induction showed an ATP reduction during low cell density growth. In addition, pigment induction during fixed growth rate chemostat culture revealed a negative correlation between cellular levels of prodigiosin and ATP (r = -0.95). Variable growth rate chemostat experiments showed an inverse relationship between ATP per cell and prodigiosin per cell during low-density growth but a direct relationship during high-density growth. Rate modeling of chemostat data quantified the pigment's effect on cellular levels of ATP for both population growth phases. Finally, prodigiosin production in a heterologous bacterium led to ATP decline. These data with intact cells complement the established in vitro proton import function of prodigiosin pigment and may indicate an energy-spilling function during high rate, low cell density growth.
Assuntos
Trifosfato de Adenosina/metabolismo , Prodigiosina/biossíntese , Serratia marcescens/crescimento & desenvolvimento , Serratia marcescens/metabolismo , Filogenia , Serratia marcescens/genéticaRESUMO
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.
Assuntos
Autofagia/efeitos dos fármacos , Proteínas de Bactérias/farmacologia , Epitélio Corneano/patologia , Proteínas Hemolisinas/farmacologia , Limbo da Córnea/citologia , Prodigiosina/farmacologia , Serratia marcescens/patogenicidade , Adenina/análogos & derivados , Adenina/farmacologia , Fenômenos Fisiológicos Bacterianos , Linhagem Celular , Proteínas de Fluorescência Verde , Humanos , Ceratite/microbiologia , Microscopia Confocal , Perforina , Serratia marcescens/genética , Serratia marcescens/isolamento & purificaçãoRESUMO
PURPOSE: The definitive identification of ocular pathogens optimizes effective treatment. Although the types of ocular pathogens are known; there is less definitive information on the prevalence of causative infections including viruses, fungi, and protozoa, which is the focus of this retrospective laboratory review. METHODS: Data used for laboratory certification were reviewed for the detection of bacteria, viruses, fungi, and protozoa, from patients with infectious keratitis, endophthalmitis, and conjunctivitis. The main outcome parameter was laboratory-positive ocular infection. RESULTS: The distribution of infectious agents for keratitis (n=1,387) (2004-2018) was bacteria 72.1% (Staphylococcus aureus 20.3%, Pseudomonas aeruginosa 18%, Streptococcus spp. 8.5%, other gram-positives 12.4%, and other gram-negatives 12.9%), Herpes simplex virus 16%, fungi 6.7%, and Acanthamoeba 5.2%. For endophthalmitis, (n=770) (1993-2018), the bacterial distribution was coagulase-negative Staphylococcus 54%, Streptococcus spp. 21%, S. aureus 10%, other gram-positives 8%, and gram-negatives 7%. The distribution for conjunctivitis (n=847) (2004-2018) was Adenovirus 34%, S. aureus 25.5%, Streptococcus pneumoniae 9%, Haemophilus 9%, other gram-negatives 8.8%, other gram-positives 6%, coagulase-negative Staphylococcus 4.5% and Chlamydia 3.2%. CONCLUSION: An updated monitoring of ocular pathogens creates an awareness of the different infectious etiologies and the importance of laboratory studies. This information can determine treatment needs for infectious ocular diseases.
Assuntos
Acanthamoeba , Conjuntivite , Endoftalmite , Infecções Oculares Bacterianas , Ceratite , Vírus , Antibacterianos/uso terapêutico , Bactérias , Conjuntivite/tratamento farmacológico , Conjuntivite/epidemiologia , Endoftalmite/tratamento farmacológico , Infecções Oculares Bacterianas/tratamento farmacológico , Infecções Oculares Bacterianas/epidemiologia , Fungos , Humanos , Ceratite/diagnóstico , Ceratite/tratamento farmacológico , Ceratite/epidemiologia , Prevalência , Estudos Retrospectivos , Staphylococcus aureusRESUMO
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.
Assuntos
Proteínas de Bactérias/genética , Depsipeptídeos/metabolismo , Proteínas de Membrana/genética , Prodigiosina/metabolismo , Serratia marcescens/genética , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Mutação , Análise de Sequência de DNA , Serratia marcescens/metabolismoRESUMO
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.
Assuntos
Autofagia/fisiologia , Bactérias/patogenicidade , Fenômenos Fisiológicos Bacterianos , Células Epiteliais/fisiologia , Ceratite/microbiologia , Antibacterianos/farmacologia , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Humanos , Limbo da Córnea/citologiaRESUMO
Serratia marcescens is a gram-negative, facultatively-anaerobic bacterium and opportunistic pathogen which produces the red pigment prodigiosin. We employed both batch culture and chemostat growth methods to investigate prodigiosin function in the producing organism. Pigmentation correlated with an increased rate of ATP production during population lag phase. Results with a lacZ transcriptional fusion to the prodigiosin (pig) biosynthetic operon revealed that operon transcription is activated by low cellular levels of ATP at high cell density. Furthermore, these data enabled estimation of the ATP per cell minimum value at which the operon is induced. Pigmented cells were found to accumulate ATP more rapidly and to multiply more quickly than non-pigmented cells during the high density growth phase. Finally, results with both batch and chemostat culture revealed that pigmented cells grow to approximately twice the biomass yield as non-pigmented S. marcescens bacteria. Prodigiosin production may, therefore, provide a growth advantage at ambient temperatures.
Assuntos
Prodigiosina/biossíntese , Serratia marcescens/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biomassa , Óperon , Serratia marcescens/genética , Serratia marcescens/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Ácido Hipocloroso/farmacologia , Oxidantes/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Blefarite/microbiologia , Lentes de Contato/microbiologia , Humanos , Ceratite/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus capitis/efeitos dos fármacos , Staphylococcus epidermidis/efeitos dos fármacosRESUMO
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.
Assuntos
Antibacterianos/administração & dosagem , Antibacterianos/farmacocinética , Bandagens , Colágeno , Lentes de Contato Hidrofílicas , Sistemas de Liberação de Medicamentos/métodos , Moxifloxacina/administração & dosagem , Moxifloxacina/farmacocinética , Soluções Oftálmicas/administração & dosagem , Soluções Oftálmicas/farmacocinética , Endoftalmite/tratamento farmacológico , HumanosRESUMO
PURPOSE: Intravitreal injections of antibiotics and anti-inflammatories are used by some cataract surgeons for surgical prophylaxis. To support this prophylaxis, intravitreal triamcinolone-moxifloxacin (TM) and triamcinolone-moxifloxacin-vancomycin (TMV) were tested for preventing Staphylococcus aureus (SA) endophthalmitis in rabbits. METHODS: Trademark formulations of TM (15/1 mg/mL) and TMV (15/1/10 mg/mL) were intravitreally injected into seven groups of eight rabbits each (A-G). Before intravitreal injection, the vitreous was first challenged with clinical SA endophthalmitis isolates (5,000 colony-forming unit) with varying minimum inhibitory concentrations (MICs in µg/mL) to moxifloxacin (denoted by the MIC at the end of each group listed): A) TMV-10, B) TM-10, C) Saline-10, D) TM-2, E) Saline-2, F) TM-0.032, and G) Saline-0.032. After 24 hr, the rabbit eyes were graded for clinical endophthalmitis and cultured for viable SA. RESULTS: Rabbits treated with TMV and challenged by SA with a moxifloxacin MIC of 10 µg/mL did not present with endophthalmitis (0/8, no eyes with endophthalmitis). For SA with moxifloxacin MICs of 10.0 and 2.0 µg/mL, TM did not prevent endophthalmitis (16/16, 100% of eyes with endophthalmitis). For SA with a moxifloxacin MIC of 0.032 µg/mL, endophthalmitis was prevented with TM (0/8, no eyes with endophthalmitis). All saline-treated eyes developed endophthalmitis (23/23, 100% of eyes with endophthalmitis). CONCLUSIONS: Intravitreal monotherapy with TM did not provide consistent prevention of SA endophthalmitis, whereas intravitreal TMV successfully prevented endophthalmitis because of SA with elevated MIC values to moxifloxacin. Cataract surgeons need to be aware that vancomycin seems to be essential for intravitreal prophylaxis to cover moxifloxacin resistance.
Assuntos
Antibacterianos/administração & dosagem , Antibioticoprofilaxia/métodos , Extração de Catarata , Endoftalmite/prevenção & controle , Infecções Oculares Bacterianas/prevenção & controle , Moxifloxacina/administração & dosagem , Infecções Estafilocócicas/prevenção & controle , Triancinolona/administração & dosagem , Animais , Modelos Animais de Doenças , Quimioterapia Combinada , Injeções Intravítreas , Masculino , Coelhos , Staphylococcus aureusRESUMO
mcr-1 was initially reported as the first plasmid-mediated colistin resistance gene in clinical isolates of Escherichia coli and Klebsiella pneumoniae in China and has subsequently been identified worldwide in various species of the family Enterobacteriaceaemcr-1 encodes a phosphoethanolamine transferase, and its expression has been shown to generate phosphoethanolamine-modified bis-phosphorylated hexa-acylated lipid A in E. coli Here, we investigated the effects of mcr-1 on colistin susceptibility and on lipopolysaccharide structures in laboratory and clinical strains of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, K. pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens, which are often treated clinically by colistin. The effects of mcr-1 on colistin resistance were determined using MIC assays of laboratory and clinical strains of E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa Lipid A structural changes resulting from MCR-1 were analyzed by mass spectrometry. The introduction of mcr-1 led to colistin resistance in E. coli, K. pneumoniae, and A. baumannii but only moderately reduced susceptibility in P. aeruginosa Phosphoethanolamine modification of lipid A was observed consistently for all four species. These findings highlight the risk of colistin resistance as a consequence of mcr-1 expression among ESKAPE pathogens, especially in K. pneumoniae and A. baumannii Furthermore, the observation that lipid A structures were modified despite only modest increases in colistin MICs in some instances suggests more sophisticated surveillance methods may need to be developed to track the dissemination of mcr-1 or plasmid-mediated phosphoethanolamine transferases in general.
Assuntos
Antibacterianos/farmacologia , Lipopolissacarídeos/farmacologia , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Colistina/farmacologia , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Polimixinas/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genéticaRESUMO
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.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/genética , Genes Reguladores/genética , Metaloendopeptidases/biossíntese , Metabolismo Secundário/genética , Serratia marcescens/genética , Serratia marcescens/metabolismo , Fatores de Transcrição/genética , Proteínas de Bactérias/metabolismo , Elementos de DNA Transponíveis/genética , Depsipeptídeos/biossíntese , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/genética , Escherichia coli/metabolismo , Flagelos/metabolismo , Espectrometria de Massas , Prodigiosina/biossíntese , Serratia marcescens/crescimento & desenvolvimento , Fatores de Transcrição/metabolismoRESUMO
Klebsiella pneumoniae remains an important cause of intrapulmonary infection and invasive disease worldwide. K. pneumoniae can evade serum killing and phagocytosis primarily through the expression of a polysaccharide capsule, but its pathogenicity is also influenced by host factors. We examined whether CD36, a scavenger receptor that recognizes pathogen and modified self ligands, is a host determinant of K. pneumoniae pathogenicity. Despite differences in serum sensitivity and virulence of 3 distinct K. pneumoniae (hypermucoviscous K1, research K2, and carbapenemase-producing ST258) strains, the absence of CD36 significantly increased host susceptibility to acute intrapulmonary infection by K. pneumoniae, regardless of strain. We demonstrate that CD36 enhances LPS responsiveness to K. pneumoniae to increase downstream cytokine production and macrophage phagocytosis that is independent of polysaccharide capsular antigen. Our study provides new insights into host determinants of K. pneumoniae pathogenicity and raises the possibility that functional mutations in CD36 may predispose individuals to K. pneumoniae syndromes.