Pseudomonas aeruginosa Outer Membrane Vesicles Triggered by Human Mucosal Fluid and Lysozyme Can Prime Host Tissue Surfaces for Bacterial Adhesion.

Pseudomonas aeruginosa is a leading cause of human morbidity and mortality that often targets epithelial surfaces. Host immunocompromise, or the presence of indwelling medical devices, including contact lenses, can predispose to infection. While medical devices are known to accumulate bacterial biofilms, it is not well understood why resistant epithelial surfaces become susceptible to P. aeruginosa. Many bacteria, including P. aeruginosa, release outer membrane vesicles (OMVs) in response to stress that can fuse with host cells to alter their function. Here, we tested the hypothesis that mucosal fluid can trigger OMV release to compromise an epithelial barrier. This was tested using tear fluid and corneal epithelial cells in vitro and in vivo. After 1 h both human tear fluid, and the tear component lysozyme, greatly enhanced OMV release from P. aeruginosa strain PAO1 compared to phosphate buffered saline (PBS) controls (∼100-fold). Transmission electron microscopy (TEM) and SDS-PAGE showed tear fluid and lysozyme-induced OMVs were similar in size and protein composition, but differed from biofilm-harvested OMVs, the latter smaller with fewer proteins. Lysozyme-induced OMVs were cytotoxic to human corneal epithelial cells in vitro and murine corneal epithelium in vivo. OMV exposure in vivo enhanced Ly6G/C expression at the corneal surface, suggesting myeloid cell recruitment, and primed the cornea for bacterial adhesion (∼4-fold, P < 0.01). Sonication disrupted OMVs retained cytotoxic activity, but did not promote adhesion, suggesting the latter required OMV-mediated events beyond cell killing. These data suggest that mucosal fluid induced P. aeruginosa OMVs could contribute to loss of epithelial barrier function during medical device-related infections.

Inactivation of human enterovirus 71 and coxsackie virus A16 and hand, foot, and mouth disease.

Human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the two major etiological agents in major outbreaks of hand, foot, and mouth disease. Transmission of these viruses is facilitated by prolonged environmental survival and their resistance to biocides, and effective disinfection is crucial to interrupt the cycle of environmental spread. We tested the virucidal efficacy of sodium hypochlorite against both EV71 and CVA16, performed according to the Association of Official Analytical Chemists (AOAC) test criteria and methods approved by the US Environmental Protection Agency. Our results indicated the complete inactivation of infectivity of EV71 and CVA16 after a 5-minute exposure to 3120 ppm sodium hypochlorite.

Development of real-time in vivo imaging of device-related Staphylococcus epidermidis infection in mice and influence of animal immune status on susceptibility to infection.

Staphylococcus epidermidis is the leading cause of hospital-acquired device-related infections, but there is a lack of suitable methods to investigate the pathogenesis of S. epidermidis infection. We created a bioluminescent strain of S. epidermidis and developed a subcutaneous catheter-related murine infection model for real-time monitoring of biofilm-associated infection. Additionally, we compared severely immunocompromised and immunocompetent mice, demonstrating the substantial effect of animal immune status on susceptibility to experimentally induced S. epidermidis disease. This study presents a novel approach for investigating the in vivo details of the pathogenesis of S. epidermidis infection.

Bioluminescent imaging of bacterial biofilm infections in vivo.

Whole body biophotonic imaging (BPI) is a technique that has contributed significantly to the way researchers study bacterial pathogens and develop pre-clinical treatments to combat their ensuing infections in vivo. Not only does this approach allow disease profiles and drug efficacy studies to be conducted non-destructively in live animals over the entire course of the disease, but in many cases, it enables investigators to observe disease profiles that could otherwise easily be missed using conventional methodologies. The principles of this technique are that bacterial pathogens engineered to express bioluminescence (visible light) can be readily monitored from outside of the living animal using specialized low-light imaging equipment, enabling their movement, expansion and treatment to be seen completely non-invasively. Moreover, because the same group of animals can be imaged at each time-point throughout the study, the overall number of animals used is dramatically reduced, saving lives, time, and money. Also, as each animal acts as its own control over time, the issues associated with animal-to-animal variation are circumvented, thus improving the quality of the biostatistical data generated. The ability to monitor infections in vivo in a longitudinal fashion is especially appealing to assess chronic infections such as those involving implanted devices. Typically, bacteria grow as biofilms on these foreign bodies and are reputably difficult to monitor with conventional methods. Because of the non-destructive and non-invasive nature of BPI, the procedure can be performed repeatedly in the same animal, allowing the biofilm to be studied in situ without detachment or disturbance. This ability not only allows unique patterns of disease relapse to be seen following termination of antibiotic therapy but also in vivo resistance development during prolonged treatment, both of which are common occurrences with device-related infections. This chapter describes the bioluminescent engineering of both Gram-positive and Gram-negative bacteria and overviews their use in device-associated infections in several anatomical sites in a variety of animal models.

Reduction of astrogliosis by early treatment of pneumococcal meningitis measured by simultaneous imaging, in vivo, of the pathogen and host response.

We developed a method for simultaneous in vivo biophotonic monitoring of pneumococcal meningitis and the accompanying neuronal injury in live transgenic mice. Streptococcus pneumoniae engineered for bioluminescence (lux) was used for direct visualization of disease progression and antibiotic treatment in a mouse model of meningitis. The host response was monitored in transgenic mice containing an inducible firefly luciferase (luc) reporter gene under transcriptional control of the mouse glial fibrillary acidic protein (GFAP) promoter. Based on the different spectra of light emission and substrate requirements for lux and luc, we were able to separately monitor the two reporters using a highly sensitive in vivo imaging system. The level of neuronal damage and recovery following antibiotic treatment was dependent on the time of treatment. This model has potential for simultaneous multiparameter monitoring and testing of therapies that target the pathogen or host response to prevent neuronal injury and recovery.

Noninvasive monitoring of pneumococcal meningitis and evaluation of treatment efficacy in an experimental mouse model.

Noninvasive real-time in vivo bioluminescent imaging was used to assess the spread of Streptococcus pneumoniae throughout the spinal cord and brain during the acute stages of bacterial meningitis. A mouse model was established by lumbar (LP) or intracisternal (IC) injection of bioluminescent S. pneumoniae into the subarachnoid space. Bacteria replicated initially at the site of inoculation and spread progressively from the spinal cord to the brain or from the brain down to the cervical part of the spinal column and to the lower vertebral levels. After 24 hr, animals showed strong bioluminescent signals throughout the spinal canal, indicating acute meningitis of the intracranial and intraspinal meninges. A decline in bacterial cell viability, as judged by a reduction in the bioluminescent signal, was observed over time in animals treated with ceftriaxone, but not in untreated groups. Mice treated with the antibiotic survived infection, whereas all mice in untreated groups became moribund, first in the IC group then in the LP group. No untreated animal survived beyond 48 hr after induction of infection. Colony counts of infected cerebrospinal fluid (CSF) correlated positively with bioluminescent signals. This methodology is especially appealing because it allows detecting infected mice as early as 3 hr after inoculation, provide temporal, sequential, and spatial distribution of bacteria within the brain and spinal cord throughout the entire disease process and the rapid monitoring of treatment efficacy in a nondestructive manner. Moreover, it avoids the need to sacrifice the animals for CSF sampling and the potential manipulative damage that can occur with other conventional methods.

Noninvasive biophotonic imaging for monitoring of catheter-associated urinary tract infections and therapy in mice.

Urinary tract infections (UTIs) are among the most common bacterial infections acquired by humans, particularly in catheterized patients. A major problem with catheterization is the formation of bacterial biofilms on catheter material and the risk of developing persistent UTIs that are difficult to monitor and eradicate. To better understand the course of UTIs and allow more accurate studies of in vivo antibiotic efficacy, we developed a catheter-based biofilm infection model with mice, using bioluminescently engineered bacteria. Two important urinary tract pathogens, Pseudomonas aeruginosa and Proteus mirabilis, were made bioluminescent by stable insertion of a complete lux operon. Segments of catheter material (precolonized or postimplant infected) with either pathogen were placed transurethrally in the lumen of the bladder by using a metal stylet without surgical manipulation. The bioluminescent strains were sufficiently bright to be readily monitored from the outside of infected animals, using a low-light optical imaging system, including the ability to trace the ascending pattern of light-emitting bacteria through ureters to the kidneys. Placement of the catheter in the bladder not only resulted in the development of strong cystitis that persisted significantly longer than in mice challenged with bacterial suspensions alone but also required prolonged antibiotic treatment to reduce the level of infection. Treatment of infected mice for 4 days with ciprofloxacin at 30 mg/kg of body weight twice a day cured cystitis and renal infection in noncatheterized mice. Similarly, ciprofloxacin reduced the bacterial burden to undetectable levels in catheterized mice but did not inhibit rebound of the infection upon cessation of antibiotic therapy. This methodology easily allows spatial information to be monitored sequentially throughout the entire disease process, including ascending UTI, treatment efficacy, and relapse, all without exogenous sampling, which is not possible with conventional methods.

Monitoring in vivo fitness of rifampicin-resistant Staphylococcus aureus mutants in a mouse biofilm infection model.

OBJECTIVES: To investigate in vivo fitness of rifampicin-resistant Staphylococcus aureus mutants in a mouse biofilm model using bioluminescence imaging. MATERIALS AND METHODS: S. aureus was engineered with a luciferase operon to emit bioluminescence that can be detected in vivo using an IVIS imaging system. Two rifampicin-resistant strains of S. aureus that were previously isolated from animals undergoing rifampicin treatment, S464P (resistant to low concentrations of rifampicin) and H481Y (resistant to high concentrations of rifampicin), were characterized and then compared with their parental strain for in vivo fitness to form biofilm infections in the absence of rifampicin. RESULTS: The mutant S464P showed better adaptation to in vivo growth than either the parental strain or H481Y without selective pressure. Six days after implanting pre-colonized catheters, bioluminescent signals were seen from 100% of the catheters coated by the mutant S464P. In comparison, only 83% and 61% of the catheters coated by the parental strain and H481Y, respectively, maintained a signal in vivo. Rifampicin treatment of S464P biofilms in vivo resulted in a slight decline, but earlier rebound in bioluminescence from these catheters compared with the parental signal, whereas rifampicin had no affect on bioluminescence in mice infected with mutant H481Y. CONCLUSIONS: The mutant with low-level rifampicin resistance appears to be better adapted to in vivo growth than the mutant that has high-level rifampicin resistance. Moreover, the former mutant may actually have a slight competitive advantage over the rifampicin-susceptible strain (parental), raising awareness for the occurrence of such strains in clinical environments.

Real-time in vivo bioluminescent imaging for evaluating the efficacy of antibiotics in a rat Staphylococcus aureus endocarditis model.

Therapeutic options for invasive Staphylococcus aureus infections have become limited due to rising antimicrobial resistance, making relevant animal model testing of new candidate agents more crucial than ever. In the present studies, a rat model of aortic infective endocarditis (IE) caused by a bioluminescently engineered, biofilm-positive S. aureus strain was used to evaluate real-time antibiotic efficacy directly. This strain was vancomycin and cefazolin susceptible but gentamicin resistant. Bioluminescence was detected and quantified daily in antibiotic-treated and control animals with IE, using a highly sensitive in vivo imaging system (IVIS). Persistent and increasing cardiac bioluminescent signals (BLS) were observed in untreated animals. Three days of vancomycin therapy caused significant reductions in both cardiac BLS (>10-fold versus control) and S. aureus densities in cardiac vegetations (P < 0.005 versus control). However, 3 days after discontinuation of vancomycin therapy, a greater than threefold increase in cardiac BLS was observed, indicating relapsing IE (which was confirmed by quantitative culture). Cefazolin resulted in modest decreases in cardiac BLS and bacterial densities. These microbiologic and cardiac BLS differences during therapy correlated with a longer time-above-MIC for vancomycin (>12 h) than for cefazolin ( approximately 4 h). Gentamicin caused neither a reduction in cardiac S. aureus densities nor a reduction in BLS. There were significant correlations between cardiac BLS and S. aureus densities in vegetations in all treatment groups. These data suggest that bioluminescent imaging provides a substantial advance in the real-time monitoring of the efficacy of therapy of invasive S. aureus infections in live animals.

Noninvasive optical imaging method to evaluate postantibiotic effects on biofilm infection in vivo.

Eradication of Staphylococcus aureus biofilms after rifampin treatment was tested in a mouse model of device-related infection by using biophotonic imaging. Following treatment, the bioluminescent signals decreased to undetectable levels, irrespective of the age of the biofilm. After the final treatment, the signals rebounded in a time-dependent manner and reached those for the untreated mice. Readministration of rifampin was unsuccessful in eradicating reestablished infections, with the rifampin MICs for such bacteria being increased and with the bacteria having point mutations in the rpoB gene.

Rapid direct method for monitoring antibiotics in a mouse model of bacterial biofilm infection.

We have developed a rapid, continuous method for monitoring the effectiveness of several antibacterial agents in real time, noninvasively, by using a recently described mouse model of chronic biofilm infection (J. L. Kadurugamuwa et al., Infect. Immun. 71:882-890, 2003), which relies on biophotonic imaging of bioluminescent bacteria. To facilitate real-time monitoring of infection, we used a Staphylococcus aureus isolate that was made bioluminescent by inserting a modified lux operon into the bacterial chromosome. This bioluminescent reporter bacterium was used to study the antimicrobial effects of several antibiotics belonging to different molecular families. Treatment with rifampin, tobramycin, and ciprofloxacin was started 7 days after subcutaneous implantation of catheters precolonized with 10(4) CFU of S. aureus. Three different doses of antibiotics were administered twice a day for 4 consecutive days. The number of metabolically active bacteria in untreated mice and the tobramycin- and ciprofloxacin-treated groups remained relatively unchanged over the 4-week observation period, indicating poor efficacies for tobramycin and ciprofloxacin. A rapid dose-dependent decline in metabolic activity in rifampin-treated groups was observed, with almost a 90% reduction after two doses and nearly undetectable levels after three doses. The disappearance of light emission correlated with colony counts. After the final treatment, cell numbers rebounded as a function of concentration in a time-dependent manner. The staphylococci isolated from the catheters of mice treated with rifampin were uniformly resistant to rifampin but retained their in vitro susceptibilities to tobramycin and ciprofloxacin. Since the metabolic activities of viable cells and a postantibiotic effect could be detected directly on the support matrix nondestructively and noninvasively, the methodology is specifically appealing for investigating the effects of antibiotics on biofilms in vivo. Moreover, our study points to the possible use of biophotonic imaging for the detection of the development of resistance to therapeutic agents during treatment of chronic infections in vivo.

Direct continuous method for monitoring biofilm infection in a mouse model.

We have developed a rapid, continuous method for real-time monitoring of biofilms, both in vitro and in a mouse infection model, through noninvasive imaging of bioluminescent bacteria colonized on Teflon catheters. Two important biofilm-forming bacterial pathogens, Staphylococcus aureus and Pseudomonas aeruginosa, were made bioluminescent by insertion of a complete lux operon. These bacteria produced significant bioluminescent signals for both in vitro studies and the development of an in vivo model, allowing effective real-time assessment of the physiological state of the biofilms. In vitro viable counts and light output were parallel and highly correlated (S. aureus r = 0.98; P. aeruginosa r = 0.99) and could be maintained for 10 days or longer, provided that growth medium was replenished every 12 h. In the murine model, subcutaneous implantation of the catheters (precolonized or postimplant infected) was well tolerated. An infecting dose of 10 (3) to 10 (5) CFU/catheter for S. aureus and P. aeruginosa resulted in a reproducible, localized infection surrounding the catheter that persisted until the termination of the experiment on day 20. Recovery of the bacteria from the catheters of infected animals showed that the bioluminescent signal corresponded to the CFU and that the lux constructs were highly stable even after many days in vivo. Since the metabolic activity of viable cells could be detected directly on the support matrix, nondestructively, and noninvasively, this method is especially appealing for the study of chronic biofilm infections and drug efficacy studies in vivo.

Membrane vesicles derived from Pseudomonas aeruginosa and Shigella flexneri can be integrated into the surfaces of other gram-negative bacteria.

Incubation of intact Salmonella typhi Ty21a, Salmonella enterica serovar Typhimurium (Salmonella typhimurium) aroA or Escherichia coli DH5 alpha with membrane vesicles (MVs) derived from either Shigella flexneri M90T or Pseudomonas aeruginosa dsp89 resulted in a significant incorporation of vesicle antigens into the outer membrane of the bacteria; each recipient strain possessed a surface mosaic of new Shigella and Pseudomonas antigens intermixed with the native antigens of the Salmonella or Escherichia strains. Electron microscopy of preparations during the integration of vesicle antigens revealed that the MVs rapidly fused with the outer membrane of the host strains. Western blot analysis of host bacteria confirmed the integration of foreign antigens. Quantitative analysis for binding and fusion of antigens using an ELISA showed that approximately 78.7 +/- 12.8 ng of the Pseudomonas and 67.5 +/- 13.8 ng of the Shigella LPSs (microgram host protein)-1 were integrated into the Sal. typhimurium strain. Similar integrations of the Shigella or Pseudomonas vesicles were found with the E. coli or Sal. typhi strains. There was no loss of viability in the recipient bacteria after incorporation of the MVs, although vesicle antigens became diluted during continued growth as daughter cells shared the vesicle antigens. The new antigens were highly stable after being incorporated into recipient strains, being able to withstand storage of several months at 4 degrees C as well as several cycles of freezing and thawing. Since the recipient bacteria are common vaccine strains, the procedure described here offers a simple efficient means of introducing exogenous surface antigens, in their native form, into the outer membranes of Gram-negative bacteria for possible vaccine use.