Gentamicin Survival Research Articles

Novel Quorum Quenching YtnP Lactonase From Bacillus paralicheniformis Reduces Pseudomonas aeruginosa Virulence and Increases Antibiotic Efficacy in vivo.

Bacterial infections have become increasingly difficult to treat due to the occurrence of antibiotic-resistant strains. A promising strategy to increase the efficacy of therapy is to combine antibacterials with agents that decrease pathogen virulence via the modulation of the quorum sensing (QS). Lactonases inhibit acylated homoserine lactone (AHL)-mediated QS in Gram-negative bacteria, including the leading nosocomial pathogen Pseudomonas aeruginosa. Here we describe the characteristics of heterologously expressed YtnP lactonase from Bacillus paralicheniformis ZP1 (YtnP-ZP1) isolated from agricultural soil using the culture enrichment method. Purified YtnP-ZP1 hydrolyzed different AHLs with preference to substrates with long acyl residues as evaluated in assays with biosensors and HPLC. The enzyme showed good thermostability and activity in a wide temperature range. YtnP-ZP1 in 50 μg mL–1 concentration reduced the amount of P. aeruginosa-produced long-chain AHLs by 85%, while it hydrolyzed 50% of short-chain AHLs. Incubation of P. aeruginosa PAO1 with YtnP-ZP1 reduced its swarming motility and elastolytic activity without bactericidal effect. YtnP-ZP1 caused the inhibition of biofilm formation and disintegration of mature biofilms in P. aeruginosa PAO1 and multiresistant clinical strain BR5H that was visualized by crystal violet staining. The treatment with YtnP-ZP1 in concentrations higher than 25 μg mL–1 improved the survival of P. aeruginosa PAO1-infected zebrafish (Danio rerio), rescuing 80% of embryos, while in combination with tobramycin or gentamicin survival rate increased to 100%. The treatment of P. aeruginosa PAO1 biofilms on infected zebrafish tail wounds with 50 μg mL–1 YtnP-ZP1 and 2 × MIC tobramycin led to infection clearing in 2 days. The extensive toxicity studies proved YtnP-ZP1 was non-toxic to human cells and zebrafish. In conclusion, novel YtnP-ZP1 lactonase with its effective anti-virulence activity could be used to increase the efficacy of clinically approved antibiotics in clearing both systemic and biofilm-associated P. aeruginosa infections.

Molecular Biology and Genetic Tools to Investigate Functional Redundancy Among Fe-S Cluster Carriers in E. coli.

Iron-sulfur (Fe-S) clusters are among the oldest protein cofactors, and Fe-S cluster-based chemistry has shaped the cellular metabolism of all living organisms. Over the last 30years, thanks to molecular biology and genetic approaches, numerous actors for Fe-S cluster assembly and delivery to apotargets have been uncovered. In prokaryotes, Escherichia coli is the best-studied for its convenience of growth and its genetic amenability. During evolution, redundant ways to secure the supply of Fe-S clusters to the client proteins have emerged in E. coli. Disrupting gene expression is essential for gene function exploration, but redundancy can blur the interpretations as it can mask the role of important biogenesis components. This chapter describes molecular biology and genetic strategies that have permitted to reveal the E. coli Fe-S cluster conveying component network, composition, organization, and plasticity. In this chapter, we will describe the following genetic methods to investigate the importance of E. coli Fe-S cluster carriers: one-step inactivation of chromosomal genes in E. coli using polymerase chain reaction (PCR) products, P1 transduction, arabinose-inducible expression system, mevalonate (MVA) genetic by-pass, sensitivity tests to oxidative stress and iron starvation, β-galactosidase assay, gentamicin survival test, and Hot Fusion cloning method.

Mycoplasma hyopneumoniae evades phagocytic uptake by porcine alveolar macrophages in vitro

Mycoplasma hyopneumoniae, the agent of porcine enzootic pneumonia (EP), is able to persist in the lung tissue and evade destruction by the host for several weeks. To understand the mechanism of pathogen survival, phagocytic uptake of M. hyopneumoniae by primary porcine alveolar macrophages was investigated. Intracellular location and survival of the pathogen were explored using gentamicin survival assays, flow cytometry and confocal microscopy of M. hyopneumoniae 232 labelled with green fluorescent protein (GFP). Following 1 h and 16 h of co-incubation, few viable M. hyopneumoniae were recovered from inside macrophages. Flow cytometric analysis of macrophages incubated with M. hyopneumoniae expressing GFP indicated that the mycoplasmas became associated with macrophages, but were shown to be extracellular when actin-dependent phagocytosis was blocked with cytochalasin D. Confocal microscopy detected GFP-labelled M. hyopneumoniae inside macrophages and the numbers increased modestly with time of incubation. Neither the addition of porcine serum complement or convalescent serum from EP-recovered pigs was able to enhance engulfment of M. hyopneumoniae. This investigation suggests that M. hyopneumoniae evades significant uptake by porcine alveolar macrophages and this may be a mechanism of immune escape by M. hyopneumoniae in the porcine respiratory tract.

Bacillus licheniformis in geogenic dust induces inflammation in respiratory epithelium

Exposure to environmental geogenic (or earth-derived) dust can lead to more frequent and severe infections in the human airway. Particulate matter < 10 µm (PM10) is the component of air pollution that is commonly associated with the exacerbation of respiratory diseases. We have previously demonstrated that mice exposed to geogenic dust PM10 experienced an exacerbation of inflammatory responses to influenza A virus. Whether geogenic dust PM10 also exacerbates respiratory bacterial infection is not yet known, nor are the components of the dust that drive these responses.We treated airway bronchial epithelial cells (NuLi-1) with UV-irradiated geogenic dust PM10 from six remote Western Australian towns. High levels of IL-6 and IL-8 production were observed, as well as persistent microbial growth. 16 S rRNA sequencing of the growth identified the microbe as Bacillus licheniformis, a spore-forming, environmentally abundant bacterium. We next investigated the interaction of B. licheniformis with respiratory epithelium in vitro to determine whether this exacerbated infection with a bacterial respiratory pathogen (non-typeable Haemophilus influenzae, NTHi).Heat treatment (100 °C) of all PM10 samples eliminated B. licheniformis contamination and reduced epithelial inflammatory responses, suggesting that heat-labile and/or microbial factors were involved in the host response to geogenic dust PM10. We then exposed NuLi-1 epithelium to increasing doses of the isolated Bacillus licheniformis (multiplicity of infection of 10:1, 1:1 or 0.1:1 bacteria: cells) for 1, 3, and 24 h. B. licheniformis and NTHi infection (association and invasion) was assessed using a standard gentamicin survival assay, and epithelial release of IL-6 and IL-8 was measured using a bead based immunoassay.B. licheniformis was cytotoxic to NuLi-1 cells at 24 h. At 3 h post-challenge, B. licheniformis elicited high IL-6 and IL-8 inflammatory responses from NuLi-1 cells compared with cells treated with heat-treated geogenic dust PM10 (p < 0.0001). Whilst treatment of cells with B. licheniformis increased inflammation, this did not make the cells more susceptible to NTHi infection. This study highlights that geogenic dust PM10 can harbour viable bacterial spores that induce inflammation in respiratory epithelium. The impact on respiratory health from inhalation of bacterial spores in PM10 in arid environments may be underestimated. Further investigation into the contribution of B. licheniformis and the wider dust microbiome to respiratory infection is warranted.

Effect of epithelial cell type on in vitro invasion of non-typeable Haemophilus influenzae

Non-typeable Haemophilus influenzae (NTHi) have been shown to have variable ability for in vitro invasion with a range of epithelial cells, and increased invasion of BEAS-2B cells has been associated with altered penicillin binding protein3 (PBP3), which is concerning as these strains are increasing worldwide. The aim of the study was to investigate the effect of respiratory cell type and the presence of altered PBP3 on the in vitro invasion of NTHi. A collection of 16 clinical NTHi isolates was established, 7 had normal PBP3, and 9 had altered PBP3 as defined by an N526K substitution. The isolates were tested for invasion of BEAS-2B, NHBE, A549 and NCI-H292 respiratory epithelial cells in vitro using a gentamicin survival assay, with invasion measured as the percentage of intracellular organisms relative to the initial inoculum. The overall median invasion for the 16 NTHi isolates for cell types BEAS-2B, NHBE, A549 and NCI-H292 cells were 3.17, 2.31, 0.11 and 1.52 respectively. The differences were statistically significant for BEAS-2B compared to A549 (P=0.015) and A549 compared to NCI-H292 (P=0.015), and there were also very marked differences in invasion for some individual isolates depending on the cell type used. There was a consistent bias for invasion of isolates with normal versus abnormal PBP3: and this was statistically significant for BEAS-2B (0.07 to 9.90, P=0.031) and A549 cells (0.02 to 1.68, P=0.037). These results show that NTHi invasion of respiratory epithelial cells in vitro is both strain dependant and influenced significantly by the cell line used, and that the association between altered PBP3 and increased invasion is conserved across multiple cell lines.

How type 1 fimbriae help Escherichia coli to evade extracellular antibiotics.

To survive antibiotics, bacteria use two different strategies: counteracting antibiotic effects by expression of resistance genes or evading their effects e.g. by persisting inside host cells. Since bacterial adhesins provide access to the shielded, intracellular niche and the adhesin type 1 fimbriae increases bacterial survival chances inside macrophages, we asked if fimbriae also influenced survival by antibiotic evasion. Combined gentamicin survival assays, flow cytometry, single cell microscopy and kinetic modeling of dose response curves showed that type 1 fimbriae increased the adhesion and internalization by macrophages. This was caused by strongly decreased off-rates and affected the number of intracellular bacteria but not the macrophage viability and morphology. Fimbriae thus promote antibiotic evasion which is particularly relevant in the context of chronic infections.

Genotypic and phenotypic characterization of Pseudomonas aeruginosa isolates from post-cataract endophthalmitis patients

Endophthalmitis caused by Pseudomonas aeruginosa is associated with rapid disease progression and poor visual outcome due to high virulence of the organism. Our aim was to characterize the virulence determinants of P. aeruginosa causing post-operative endophthalmitis. Repetitive sequence analysis (ERIC PCR) was done to study the clonal relatedness of the 17 P. aeruginosa isolates. Type 3 secretion system (T3SS) genotypes were determined and the isolates were further classified as invasive or cytotoxic based on gentamicin survival, trypan blue dye exclusion and MTT assays. Phenotypically, the strains were characterized based on bacterial motility patterns, biofilm formation, phospholipase production and antibiotic susceptibility patterns. Most of our ocular isolates were invasive in nature and nearly half of them were multi-drug resistant. 47% of the isolates formed a strong biofilm, whereas the rest formed moderate to weak biofilms, which may account for an increased colonization and antibiotic resistance. Although the T3SS genotypes correlated well with the invasive/cytotoxic nature of the strains, none of the genotypes were associated with any particular phenotypic trait. To the best of our knowledge, this is the first report on the phenotypic characteristics of P. aeruginosa strains causing post-operative endophthalmitis. Our findings demonstrate that these strains have higher invasive potential and an ability to form biofilm which possibly contributes to an increased ocular virulence.

Genotypically defined β-lactamase-negative ampicillin-resistant isolates of non-typable Haemophilus influenzae are associated with increased invasion of bronchial epithelial cells in vitro.

The aim of the study was to investigate the association between the presence of altered penicillin-binding protein 3 (PBP3) in non-typable Haemophilus influenzae (NTHi) and an increased capacity to invade bronchial epithelial cells in vitro. A collection of 40 clinical isolates of NTHi comprised of 20 with normal PBP3 and 20 with altered PBP3 (defined by an N526K substitution) was established. The isolates were tested for the ability to invade bronchial epithelial cells in vitro using a 4 h gentamicin survival assay. Invasion was measured as the percentage of intracellular organisms relative to the initial inoculum. The mean invasion rate was 0.00-14.79 % in the normal PBP3 isolates and 0.02-36.69 % in the altered PBP3 isolates. The altered PBP3 isolates had a higher (P = 0.003) mean invasion rate (6.86 %, n = 20) than the normal PBP3 isolates (1.31 %, n = 20). Subsequently, two variants of altered PBP3 (transformant 1, N526K; transformant 2, M377I, S385T, L389F and N526K) were cloned into three of the initial isolates (parents) with normal PBP3 and relatively low invasive ability, and the parents and transformants tested for invasion as above. There was no difference (P = 0.89) in the mean invasion rates for the parents (0.81 %, n = 3), transformants 1 (0.90 %, n = 3) and transformants 2 (1.38 %, n = 3). There was an association between the presence of altered PBP3 in NTHi and an increased capacity to invade BEAS-2B cells in vitro, but cloning experiments suggested that the altered PBP3 was not involved directly in enhanced invasion.

Simvastatin represses translocation of &lt;I&gt;Pseudomonas aeruginosa&lt;/I&gt; across Madin-Darby canine kidney cell monolayers

Pseudomonas aeruginosa causes both invasive (bacteremic) and chronic noninvasive infections. An increase in intestinal epithelial permeability is a characteristic of severe sepsis. Alterations in the normal barrier function of the gut mucosa may result in the translocation of microbial cells and products. On the otherhand, it has been demonstrated that statin use is associated with a lower risk of mortality from bloodstream infections. Therefore, we investigated the ability of P. aeruginosa PAO1 to translocate across the Madin-Darby canine kidney (MDCK) cell monolayers in the presence and absence of simvastatin. The bacteria readily translocated across MDCK cell monolayers after 3 h of infection irrespective of the presence or absence of the drug in the medium. However, the bacteria were less able to penetrate the MDCK monolayers in the presence of simvastatin than in its absence. A gentamicin survival assay demonstrated that simvastatin did not affect the bacteria's invasive behavior in the MDCK cells.

Disruption of Contact Lens–AssociatedPseudomonas aeruginosaBiofilms Formed in the Presence of Neutrophils

To evaluate the capacity of neutrophils to enhance biofilm formation on contact lenses by an infectious Pseudomonas aeruginosa (PA) corneal isolate. Agents that target F-actin and DNA were tested as a therapeutic strategy for disrupting biofilms formed in the setting of neutrophils in vitro and for limiting the infectious bioburden in vivo. Biofilm formation by infectious PA strain 6294 was assessed in the presence of neutrophils on a static biofilm plate and on unworn etafilcon A soft contact lenses. A d-isomer of poly(aspartic acid) was used alone and with DNase to reduce biofilm formation on test contact lenses. The gentamicin survival assay was used to determine the effectiveness of the test compound in reducing subsequent intracellular bacterial load in the corneal epithelium in a contact lens infection model in the rabbit. In a static reactor and on hydrogel lenses, PA biofilm density was enhanced 30-fold at 24 hours in the presence of neutrophils (P < 0.0001). The combination of DNase and anionic poly(aspartic acid) reduced the PA biofilms formed in the presence of activated neutrophils by 79.2% on hydrogel contact lenses (P < 0.001). An identical treatment resulted in a 41% reduction in internalized PA in the rabbit corneal epithelium after 24 hours (P = 0.03). These results demonstrate that PA can exploit the presence of neutrophils to form biofilm on contact lenses within a short time. Incorporation of F-actin and DNA represent a mechanism for neutrophil-induced biofilm enhancement and are targets for available agents to disrupt pathogenic biofilms formed on contact lenses and as a treatment for established corneal infections.

Single-Cell Techniques Using Chromosomally Tagged Fluorescent Bacteria To Study Listeria monocytogenes Infection Processes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen which invades different cell types, including nonphagocytic cells, where it is able to replicate and survive. The different steps of the cellular infectious process have been well described and consist of bacterial entry, lysis of the endocytic vacuole, intracellular replication, and spreading to neighboring cells. To study the listerial infectious process, gentamicin survival assays, plaque formation, and direct microscopy observations are typically used; however, there are some caveats with each of these techniques. In this study we describe new single-cell techniques based on use of an array of integrative fluorescent plasmids (green, cyan, and yellow fluorescent proteins) to easily, rapidly, and quantitatively detect L. monocytogenes in vitro and in vivo. We describe construction of 13 integrative and multicopy plasmids which can be used for detecting intracellular bacteria, for measuring invasion, cell-to-cell spreading, and intracellular replication, for monitoring in vivo infections, and for generating transcriptional or translational reporters. Furthermore, we tested these plasmids in a variety of epifluorescence- and flow cytometry-based assays. We showed that we could (i) determine the expression of a particular promoter during the cell cycle, (ii) establish in one rapid experiment at which step in the cell cycle a particular mutant is defective, and (iii) easily measure the number of infected cells in vitro and in mouse organs. The plasmids that are described and the methods to detect them are new powerful tools to study host-Listeria interactions in a fast, robust, and high-throughput manner.

The Role of Twitching Motility inPseudomonas aeruginosaExit from and Translocation of Corneal Epithelial Cells

The authors have shown that twitching motility, a pilus-mediated form of bacterial surface movement, is required for Pseudomonas aeruginosa virulence in a murine model of keratitis. To study the role of twitching motility in virulence, Pseudomonas traversal of multilayered corneal epithelia in vitro was investigated. Translocation of multilayered corneal epithelia was investigated with the invasive strain PAK and isogenic twitching motility mutants. Rabbit corneal epithelial cells were grown to multilayers with filters and inoculated on their apical surfaces with 10(6) colony-forming unit bacteria, and translocating bacteria were quantified by viable counts of the basal chamber. Transepithelial resistance (TER) was recorded. Cellular exit of P. aeruginosa after invasion was quantified with modified gentamicin survival assays, and the role of apoptosis in exit was explored. PAK translocated the epithelia as early as 1 hour after infection, and by 8 hours apical and basal numbers of bacteria were similar. Bacterial translocation did not reduce TER. Each twitching motility mutant (pilU, pilT with pili, pilA lacking pili) was defective in translocation (>2 log reduction vs. PAK; P < 0.005). All twitching mutants were competent for cell invasion but defective in cellular exit, accumulating intracellularly to numbers exceeding those of PAK. Inhibiting apoptosis reduced the cellular exit of PAK. These results show that twitching motility enables P. aeruginosa to translocate corneal epithelial layers and suggest that it contributes to epithelial cell exit by a mechanism involving apoptosis. The relationship between these in vitro findings and the role of twitching motility in P. aeruginosa virulence in vivo remains to be determined.

Pathogenic Phenotype and Genotype ofPseudomonas aeruginosaIsolates from Spontaneous Canine Ocular Infections

This study was designed to determine whether the ability to adversely affect corneal epithelial cell health is a factor common to Pseudomonas aeruginosa keratitis strains and to assess the prevalence of each pathogenic phenotype and genotype in a canine model of naturally-acquired P. aeruginosa ocular infection. P. aeruginosa ocular isolates were collected by sampling 100 dogs without disease (six isolates collected) and by sampling dogs with conjunctivitis (two isolates), endophthalmitis (one isolate), active keratitis (12 isolates), and resolved P. aeruginosa keratitis (four isolates). Phenotype was determined in vitro by quantifying corneal epithelial cell invasion by gentamicin survival assays, and cytotoxic activity by Trypan blue exclusion assays. Genotyping was performed for genes encoding the type III secreted effectors. The ratio of invasive to cytotoxic strains with 95% confidence intervals (CI) was 0.83 (CI, 0.42-0.99) for conjunctival microflora isolates, 0.80 (CI, 0.54-0.94) for ocular infection isolates, and 1.0 (CI, 0.45-1.0) for strains isolated post-resolution of keratitis. Among ocular infection isolates, invasive and cytotoxic strains were significantly (P <or= 0.02) associated with older and younger dogs, respectively. Visible adverse effects on epithelial cells were significantly (P <or= 0.03) more frequent for keratitis strains (6/12) than other strains (1/13), but only three of these keratitis strains and the single non-keratitis strain possessed ExoU. Invasive strains predominated in the dogs of this study. Only keratitis strains had visible adverse effects on epithelial cells without overt cytotoxicity, suggesting virulence strategies affecting live corneal epithelial cell health are selected for among keratitis strains.

Regulation ofPseudomonas aeruginosaInternalization after Contact Lens Wear In Vivo and in Serum-Free Culture by Ocular Surface Cells

To determine the effects of contact lenses (CLs) and Pseudomonas aeruginosa (PA) infection on localization of cystic fibrosis transmembrane conductance regulator (CFTR) on corneal surface epithelial cells and the association between lipid raft formation and CFTR in mediating PA binding and internalization in ocular surface epithelium. CFTR immunolocalization was evaluated in vivo in rabbit corneal-conjunctival epithelium (with/without CL wear) before and after PA exposure and in serum-free human corneal epithelial cell culture (hTCEpi). Lipid raft formation was visualized with Alexa555-conjugated cholera toxin beta-subunit. Lipid raft involvement in PA internalization was assayed in vivo by gentamicin survival assays after topical filipin pretreatment. Involvement of CFTR in PA binding and internalization was evaluated by blockade with CFTR peptides or LPS. CL wear in vivo enhanced anti-CFTR staining, but CFTR localization did not correlate with the PA binding by ocular surface cells. Conjunctival epithelial cells stained for CFTR but did not bind or internalize PA. Corneal epithelial cells in vivo did not stain for CFTR unless challenged by contact lens-induced hypoxia. PA internalization by hTCEpi was significantly inhibited by LPS (P < 0.01), but not by CFTR peptides. Remarkably, normal conjunctival epithelial cells showed lipid raft formation and CFTR staining but did not bind PA. Inhibition of raft formation by filipin blocked PA internalization in vivo after CL wear. CFTR is not the predominant receptor for ocular surface PA infection, and after hypoxic CL challenge, neither lipid rafts nor CFTR localization alone predicts PA binding; however, lipid rafts are critical to CL-mediated PA internalization.

Prolonged Hypoxia Induces Lipid Raft Formation and Increases Pseudomonas Internalization in vivo After Contact Lens Wear and Lid Closure

To investigate the effects of hypoxia on lipid raft formation and Pseudomonas aeruginosa internalization by the corneal epithelium with and without the physical effects of contact lens wear. One eye of each rabbit was randomly fitted with a low-Dk rigid gas-permeable contact lens (LDCTL) or closed with sutures, with the other as a control. After 1 day or 3 days, the rabbits were killed and bacterial invasion was assessed by gentamicin survival assay. Lipid rafts were identified by staining with FITC-conjugated beta subunit of cholera toxin. Corneal epithelial Bcl-2 expression was detected by Western blotting; surface epithelial cell size and thickness (epithelium and stroma) were measured by confocal microscopy. One-day hypoxia induced no significant changes in P. aeruginosa internalization, Bcl-2 expression, or lipid raft formation except in one of four eyelid-closed eyes. After 3 days, P. aeruginosa internalization was increased significantly (P < 0.05) in LDCTL-wearing eyes and not significantly (P = 0.10) increased in eyelid-closed eyes. Both 3-day test conditions also induced lipid raft-forming cells that bound P. aeruginosa, albeit in different regions of the cornea (peripherally in LDCTL-wearing eyes and centrally in closed eyes); did not alter epithelial thickness or surface cell size; and appeared to decrease epithelial Bcl-2 expression. This is the first direct comparison in vivo between two different methods inducing hypoxia on the corneal surface. Association of P. aeruginosa internalization with lipid raft formation in both conditions suggests a critical link among prolonged hypoxia, lipid raft formation, and susceptibility to P. aeruginosa infection. However, different distribution patterns of lipid raft-forming cells suggest physical effects of contact lens wear may direct localization of lipid raft-associated P. aeruginosa internalization on the corneal surface.

Alternative methods to limit extracellular bacterial activity for enumeration of intracellular bacteria

The gentamicin survival assay, a method routinely used to estimate bacterial infection of eukaryotic host cells, depends on the presumed limited penetration of gentamicin across the eukaryotic cell membrane. However, some studies have suggested that gentamicin may in fact enter eukaryotic cells and kill intracellular bacteria. In this study we devised alternative methods to enumerate intracellular Salmonellae using a lytic bacteriophage, SP6, and an amino acid auxotroph, Pro- mutant, which replicates selectively within host cells in the presence of its uptake inhibitor, 3,4-dehydro-L-proline. The conventional gentamicin survival assay was systematically compared with the alternative methods for the enumeration of intracellular Salmonellae. We found that gentamicin decreases the survival of intracellular Salmonellae when added to extracellular media at concentrations above 20 microg/ml. The alternative methods do not suffer from this disadvantage, suggesting that they should be used to replace the gentamicin survival assay. In addition, the proline auxotroph method could be applied to detect bacterial release from host cells.

Internalization ofPseudomonas aeruginosaIs Mediated by Lipid Rafts in Contact Lens–Wearing Rabbit and Cultured Human Corneal Epithelial Cells

The internalization of Pseudomonas aeruginosa (PA) in nasal and tracheal epithelium has recently been shown to involve the formation of cholesterol- and sphingolipid-rich plasma membrane domains (lipid rafts). The purpose of this study was to investigate the role of lipid rafts in PA internalization by corneal epithelium in vivo, in vitro, and after contact lens wear. Lipid raft formation was evaluated in rabbit corneas with and without contact lens wear and a human corneal epithelial (hTCEpi) cell line before and after PA infection with cornea-pathogenic strains by staining with FITC-conjugated cholera toxin beta-subunit, known to bind the lipid raft component GM1. Bacterial internalization was assessed by gentamicin survival assay. The role of lipid rafts in PA internalization was evaluated by pretreatment of hTCEpi cells with cholesterol metabolism inhibitors. The interaction of PA with lipid rafts was confirmed by flow cytometry. Contact lens wear in rabbits induced lipid raft formation in occasional surface corneal epithelial cells. Subsequent PA exposure showed preferential binding to lipid raft-forming cells, leading to lipid raft aggregation and PA internalization. A similar sequence of lipid raft formation and PA internalization was also observed in hTCEpi for all PA strains. Internalization of all PA strains was blocked by three cholesterol metabolism inhibitors (P < 0.01). Flow cytometry showed an association of PA with rafts. These findings demonstrate that contact-lens-mediated PA internalization involves lipid raft formation. Also, hTCEpi cells may be used as an experimental model for studying further the molecular mechanism(s) of PA infection in the corneal epithelium.

Interaction of Pseudomonas aeruginosa with Human Tear Fluid Components

Purpose: Reflex human tears bind Pseudomonas aeruginosa bacteria and prevent them from invading corneal epithelial cells. In this study, we assessed the effect of eye closure and the role of sialoglycoprotein (SG) in tears on bacterial binding and invasion. Methods: Human tears (reflex and closed-eye) were collected using a microcapillary tube. Reflex tears were separated into 13 fractions by high-performance liquid chromatography while high-molecular-weight components from closed-eye tears were separated into an SG/mucin fraction and a nonmucin fraction. Bacterial binding was quantified by viable counts and bacterial invasion was tested using the gentamicin survival technique. Results: Closed-eye tears bound significantly more bacteria than open-eye tears. Fractionation of reflex tears showed that 11 out of the 13 fractions bound bacteria, while all 13 fractions significantly reduced bacterial invasion of corneal epithelial cells. Surprisingly, the SG/mucin component of closed-eye tears resisted bacterial binding and had no significant effect on bacterial invasion. Conclusions: P. aeruginosa bacteria bind more efficiently to closed-eye tears than to open-eye tears. The mechanism by which tears bind bacteria and protect against invasion does not require SG/mucin, as this fraction of closed-eye tears does not contain either activity.

Modification of Pseudomonas aeruginosa interactions with corneal epithelial cells by human tear fluid.

Both cytotoxic and invasive strains of Pseudomonas aeruginosa can damage corneal epithelial cells in vitro, but neither can infect healthy corneas in vivo. We tested the hypothesis that whole human tear fluid can protect corneal epithelia against P. aeruginosa virulence mechanisms. Cultured corneal epithelial cells were inoculated with 10(6) CFU of one of 10 strains of P. aeruginosa (five cytotoxic, five invasive)/ml with or without reflex tear fluid collected from the conjunctival sacs of human volunteers. Cytotoxicity was assessed by observation of trypan blue staining and measurement of lactate dehydrogenase release; invasion was quantified by using gentamicin survival assays. Tear fluid retarded growth of only 50% of the P. aeruginosa strains (three of five invasive strains, two of five cytotoxic strains) yet protected corneal cells against invasion by or cytotoxicity of 9 of 10 strains. The only strain resistant to the tear cytoprotective effects was susceptible to tear bacteriostatic activity. Dilution of tear fluid threefold significantly reduced cytoprotection, while bacteriostatic activity prevailed with dilutions beyond 100-fold. Sulfacetamide (1 mg/ml) with bacteriostatic activity matching that of tear fluid was less cytoprotective than tear fluid (80% protection with tear fluid, 48% with sulfacetamide). Video microscopy revealed bacterial chain formation in both tear fluid and sulfacetamide, but tear fluid also blocked bacterial swimming motility. After prolonged tear contact, bacteria regained normal growth rates, swimming motility, and cytotoxic activity, suggesting a breakdown of protective tear factors. Boiled tear fluid lost bacteriostatic activity and effects on bacterial motility but retained cytoprotective function. These results suggest that human tear fluid can protect corneal epithelial cells against P. aeruginosa virulence mechanisms in a manner not dependent upon bacteriostatic activity or effects on bacterial motility. Whether overlapping tear film components are involved in these defense functions is to be determined.

Antibiotics improve survival and alter the inflammatory profile in a murine model of sepsis from Pseudomonas aeruginosa pneumonia.

Differing antibiotic regimens can influence both survival and the inflammatory state in sepsis. We investigated whether the addition and/or type of antimicrobial agent could effect mortality in a murine model of Pseudomonas aeruginosa pneumonia-induced sepsis and if antibiotics altered systemic levels of cytokines. FVB/N mice were subjected to intratracheal injection of pathogenic bacteria and were given gentamicin, imipenem, or 0.9% NaCl 2 h after surgery, which continued every 12 h for a total of six doses. Survival at 7 days (n = 24 in each group) was 100% for mice given gentamicin, 88% for mice given imipenem, and 8% for sham mice treated with 0.9% NaCl (P < 0.0001). Systemic interleukin (IL) 6 levels were assayed 6 h postoperatively on all mice to see if they were predictive of outcome. Plasma IL-6 levels above 3,600 pg/mL were associated with a 100% mortality, levels under 1,200 pg/mL were associated with a 100% survival, and levels between 1,200 and 3,600 pg/mL had no utility in predicting mortality. In a separate experiment, mice were sacrificed at 3, 6, 12 or 24 h after instillation of P. aeruginosa and were assayed for levels of TNF-alpha, IL-6, IL-10, and IL-12. Significant alterations in the proinflammatory cytokines TNF-alpha and IL-6 were present at all time points except 3 h between mice treated with antibiotics and sham controls. In contrast, statistically significant differences in the anti-inflammatory cytokine IL-10 were present between the groups only at 6 h, and levels of IL-12 were similar at all time points. These results indicate that both gentamicin and imipenem increase survival at least 10-fold in a model of pneumonia-induced monomicrobial sepsis, and this is predominantly associated with a down-regulation of proinflammatory cytokines.