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 bioluminescent Staphylococcus aureus infections in living mice using a novel luxABCDE construct

Strains of Staphylococcus aureus were transformed with plasmid DNA containing a Photorhabdus luminescens lux operon (luxABCDE) that was genetically modified to be functional in both gram-positive and gram-negative bacteria. S. aureus cells containing this novel lux construct, downstream of an appropriate promoter sequence, are highly bioluminescent, allowing the detection of fewer than 100 CFU in vitro (direct detection of exponentially dividing cells in liquid culture). Furthermore, these bacteria produce light stably at 37 degrees C and do not require exogenous aldehyde substrate, thus allowing S. aureus infections in living animals to be monitored by bioluminescence. Two strains of S. aureus 8325-4 that produce high levels of constitutive bioluminescence were injected into the thigh muscles of mice, and the animals were then either treated with the antibiotic amoxicillin or left untreated. Bioluminescence from bacteria present in the thighs of the mice was monitored in vivo over a period of 24 h. The effectiveness of the antibiotic in the treated animals could be measured by a decrease in the light signal. At 8 h, the infection in both groups of treated animals had begun to clear, as judged by a decrease in bioluminescence, and by 24 h no light signal could be detected. In contrast, both groups of untreated mice had strong bioluminescent signals at 24 h. Quantification of CFU from bacteria extracted from the thigh muscles of the mice correlated well with the bioluminescence data. This paper shows for the first time that bioluminescence offers a method for monitoring S. aureus infections in vivo that is sensitive and noninvasive and requires fewer animals than conventional methodologies.     

Distinctin improves the efficacies of glycopeptides and betalactams against staphylococcal biofilm in an experimental model of central venous catheter infection

The ability of microorganisms to adhere to medical implants is a problem of high visibility and has been focused in numerous investigations. To assess the efficacy of distinctin and conventional antibiotics in the treatment of central venous catheter in vitro and in vivo studies were performed. The in vitro susceptibility assay was performed against S. aureus biofilms developed on 96-well polystyrene tissue culture plates. Efficacy studies were performed in a rat model of CVC infection. Twenty-four hours after implantation, the catheters were filled with distinctin. Thirty minutes later, rats were challenged via the CVC with S. aureus. Administration of antibiotics into the CVC at a concentration equal to the MBC for adherent cells, or at 1024 microg/mL began 24 h later. The killing activities of all antibiotics against adherent bacteria were at least four- to eightfold lower than against freely growing cells. When antibiotics were used in distinctin pretreated wells, they showed a significant increase of activity. The in vivo studies showed that when CVCs were pretreated with distinctin biofilm bacterial load was further decreased to 10(1) CFU/mL and bacteremia was not detected. Distinctin displays potential as an adjunctive agent to antibiotics in the treatment of CVC-related infections.     

Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram-positive lux transposon

Animal studies with Streptococcus pneumoniae have provided valuable models for drug development. In order to monitor long-term pneumococcal infections noninvasively in living mice, a novel gram-positive lux transposon cassette, Tn4001 luxABCDE Km(r), that allows random integration of lux genes onto the bacterial chromosome was constructed. The cassette was designed so that the luxABCDE and kanamycin resistance genes were linked to form a single promoterless operon. Bioluminescence and kanamycin resistance only occur in a bacterial cell if this operon has transposed downstream of a promoter on the bacterium's chromosome. S. pneumoniae D39 was transformed with plasmid pAUL-A Tn4001 luxABCDE Km(r), and a number of highly bioluminescent colonies were recovered. Genomic DNA from the brightest D39 strain was used to transform a number of clinical S. pneumoniae isolates, and several of these strains were tested in animal models, including a pneumococcal lung infection model. Strong bioluminescent signals were seen in the lungs of the animals containing these pneumococci, allowing the course and antibiotic treatment of the infections to be readily monitored in real time in the living animals. Recovery of the bacteria from the animals showed that the bioluminescent signal corresponded to the number of CFU and that the lux construct was highly stable even after several days in vivo. We believe that this lux transposon will greatly expand the ability to evaluate drug efficacy against gram-positive bacteria in living animals using bioluminescence.     

Three-year experience with sonicated vascular catheter cultures in a clinical microbiology laboratory.

Using a quantitative sonication method, we cultured 1,681 consecutive vascular catheters submitted to a clinical microbiology laboratory in a 36-month period. A total of 46% of the cultures were positive; the most common organisms isolated were coagulase-negative staphylococci (36.4%), Pseudomonas aeruginosa (13.9%), enterococci (10.0%), yeasts (9.2%), Staphylococcus aureus (5.8%), and Enterobacter species (4.4%). The frequencies of positive blood cultures within 48 h prior to a positive catheter culture result were as follows: Candida albicans (68.4%), S. aureus (60%), Enterobacter cloacae (42.9%), Staphylococcus epidermidis (32.1%), P. aeruginosa (27.7%), and enterococci (23.3%). The sonication method allowed quantification of the number of CFU removed from a catheter for between 10(2) and 10(7) CFU. For catheter cultures in which greater than or equal to 10(2) CFU grew, a linear regression equation could be calculated: (risk of positive blood culture for the same organism) = 14 [log10 (number of organisms removed from the catheter)] -21 (r = 0.93). For catheter cultures in which less than 10(2) CFU grew, positive blood cultures for the same organism were strongly associated with a proven infection at a site distant from the catheter (P = 0.001) or probable contamination (S. epidermidis). Our findings indicate that this technique has considerable potential for use in clinical microbiology laboratories to aid in the diagnosis of vascular catheter infections and for clinical investigations into the pathogenesis of these infections.     

Endothelial cells and renal epithelial cells do not express the Wegener''s autoantigen, proteinase 3.

In vitro and in vivo T cell responses were determined during the course of bronchopulmonary infection with mucoid Pseudomonas aeruginosa. T cell responses were compared in two inbred mouse strains, namely BALB/c mice, which are resistant to the establishment of chronic bronchopulmonary Ps. aeruginosa infection, and C57Bl/6 mice, which have high numbers of bacteria in the lungs through 14 days post-infection. Unseparated lung cells and lung T cells from BALB/c mice exhibited significantly higher in vitro proliferative responses to both heat-killed Ps. aeruginosa and concanavalin A (Con A) than cells from C57Bl/6 mice through 20 days post-intratracheal infection with 10(4) colony-forming units (CFU) Ps. aeruginosa. Proliferation of unseparated lung cells but not lung T cells from BALB/c mice infected 6 days previously with 10(5) CFU Ps. aeruginosa was suppressed in response to Con A; these cells were unresponsive to specific antigen. Suppression of lymphocyte proliferation in the lungs of C57Bl/6 mice infected with 10(4) CFU Ps. aeruginosa and in BALB/c mice infected with 10(5) CFU was found to be mediated by adherent lung cells via the production of nitric oxide and prostaglandins. Determination of in vivo T cell-mediated responses in infected mice demonstrated that resistant BALB/c mice had high DTH and low Pseudomonas-specific antibody responses, while C57Bl/6 mice had low DTH and high antibody levels, in particular, IgG2b and IgM.     

Real time noninvasive monitoring of contaminating bacteria in a soft tissue implant infection model

Infection is the main cause of biomaterials-related failure. A simple technique to test in-vivo new antimicrobial and/or nonadhesive implant coatings is unavailable. Current in vitro methods for studying bacterial adhesion and growth on biomaterial surfaces lack the influence of the host immune system. Most in vivo methods to study biomaterials-related infections routinely involve implant-removal, preventing comprehensive longitudinal monitoring. In vivo imaging circumvents these drawbacks and is based on the use of noninvasive optical imaging of bioluminescent bacteria. Staphylococcus aureus Xen29 is genetically modified to be stably bioluminescent, by the introduction of a modified full lux operon onto its chromosome. Surgical meshes with adhering S. aureus Xen29 were implanted in mice and bacterial growth and spread into the surrounding tissue was monitored longitudinally from bioluminescence with a highly sensitive CCD camera. Distinct spatiotemporal bioluminescence patterns, extending beyond the mesh area into surrounding tissues were observed. After 10 days, the number of living organisms isolated from explanted meshes was found to correlate with bioluminescence prior to sacrifice of the animals. Therefore, it is concluded that in vivo imaging using bioluminescent bacteria is ideally suited to study antimicrobial coatings taking into account the host immune system. In addition, longitudinal monitoring of infection in one animal will significantly reduce the number of experiments and animals.     

In vitro quantitative model of catheter infection during simulated parenteral nutrition.

We developed a quantitative in vitro model of catheter infection. Colonization was initiated by inoculating the catheter lumen with a small number of bacteria (approximately 5 x 10(3) viable organisms). Then the inoculated catheters were used for simulated total parenteral nutrition therapy consisting of infusions for 9 h a day, and bacteria were counted in the effluent fluid against time, enabling us to monitor catheter colonization quantitatively. Bacterial colonization of prosthetic devices is a progressive process, as evidenced by the slow day-to-day increase of bacterial release seen here. On the other hand, bacterial strains of various representative species exhibited significant differences in their ability to infect catheters. These results suggest that the in vitro model presented here is a reliable tool for monitoring the degree of catheter colonization under standardized conditions and could be used for further studying the main factors of catheter-related sepsis or the treatment of this information.     

Development of an "early warning" sensor for encrustation of urinary catheters following Proteus infection

Biofilm formation in long-term urinary catheterized patients can lead to encrustation and blockage of urinary catheters with serious clinical complication. Catheter encrustation stems from infection with urease-producing bacteria, particularly Proteus mirabilis. Urease generates ammonia from urea, and the elevated pH of the urine results in crystallization of calcium and magnesium phosphates, which block the flow of urine. The aim of this research is to develop an "early warning" silicone sensor for catheter encrustation following bacterial infection of an in vitro bladder model system. The in vitro bladder model was infected with a range of urease positive and negative bacterial strains. Developed sensors enabled catheter blockage to be predicted ~17-24 h in advance of its occurrence. Signaling only occurred following infection with urease positive bacteria and only when catheter blockage followed. In summary, sensors were developed that could predict urinary catheter blockage in in vitro infection models. Translation of these sensors to a clinical environment will allow the timely and appropriate management of catheter blockage in long-term catheterized patients.     

Biofilm-Infected Intracerebroventricular Shunts Elicit Inflammation within the Central Nervous System

Central nervous system catheter infections are a serious complication in the treatment of hydrocephalus. These infections are commonly caused by Staphylococcus epidermidis and Staphylococcus aureus, both known to form biofilms on the catheter surface. Our objective was to generate a novel murine model of central nervous system catheter-associated biofilm infection using a clinical S. aureus isolate and characterize the nature of the inflammatory response during biofilm growth. Silicone catheters were precoated with S. aureus to facilitate bacterial attachment, whereupon infected or sterile catheters were stereotactically inserted into the lateral ventricle of the brain in C57BL/6 mice and evaluated at regular intervals through day 21 postinsertion. Animals tolerated the procedure well, with no clinical signs of illness or bacterial growth seen in the control group. Bacterial titers associated with central nervous system catheters were significantly elevated compared to those from the surrounding parenchyma, consistent with biofilm formation and minimal planktonic spread of infection. Catheter-associated bacterial burdens progressively increased, with maximal colonization achieved at day 7 postinfection. Analysis of inflammatory infiltrates by fluorescence-activated cell sorting (FACS) revealed significant macrophage and neutrophil influx, which peaked at days 3 and 5 to 7, respectively. In contrast, there were no detectable immune infiltrates associated with tissues surrounding sterile catheters. Biofilm infection led to significant increases in chemokine (CXCL1 and CCL2) and proinflammatory cytokine (interleukin 17 [IL-17]) expression in tissues surrounding infected central nervous system catheters. Based on these results, we propose this approach is a valid animal model for further investigations of catheter-associated central nervous system shunt infections.     

Application of orange essential oil as an antistaphylococcal agent in a dressing model

ABSTRACT: BACKGROUND: Staphylococcus aureus is the pathogen most often and prevalently involved in skin and soft tissue infections. In recent decades outbreaks of methicillin-resistant S. aureus (MRSA) have created major problems for skin therapy, and burn and wound care units. Topical antimicrobials are most important component of wound infection therapy. Alternative therapies are being sought for treatment of MRSA and one area of interest is the use of essential oils. With the increasing interest in the use and application of natural products, we screened the potential application of terpeneless cold pressed Valencia orange oil (CPV) for topical therapy against MRSA using an in vitro dressing model and skin keratinocyte cell culture model. METHODS: The inhibitory effect of CPV was determined by disc diffusion vapor assay for MRSA and vancomycin intermediate-resistant S. aureus (VISA) strains. Antistaphylococcal effect of CPV in an in vitro dressing model was tested on S. aureus inoculated tryptic soya agar plate. Bactericidal effect of CPV on MRSA and VISA infected keratinocyte cells was examined by enumeration of extra- and intra-cellular bacterial cells at different treatment time points. Cytotoxic effects on human skin cells was tested by adding CPV to the keratinocyte (HEK001) cells grown in serum free KSFM media, and observed by phase-contrast microscope. RESULTS: CPV vapour effectively inhibited the MRSA and VISA strains in both disc diffusion vapour assay and in vitro dressing model. Compared to untreated control addition of 0.1% CPV to MRSA infected keratinocyte decreased the viable MRSA cells by 2 log CFU/mL in 1 h and in VISA strain 3 log10 CFU/mL reduction was observed in 1 h. After 3 h viable S. aureus cells were not detected in the 0.2% CPV treatment. Bactericidal concentration of CPV did not show any cytotoxic effect on the human skin keratinocyte cells in vitro. CONCLUSIONS: At lower concentration addition of CPV to keratinocytes infected with MRSA and VISA rapidly killed the bacterial cells without causing any toxic effect to the keratinocytes. Therefore, the results of this study warrant further in vivo study to evaluate the potential of CPV as a topical antistaphylococcal agent.     

Use of in vivo-generated biofilms from hemodialysis catheters to test the efficacy of a novel antimicrobial catheter lock for biofilm eradication in vitro

This biofilm study was conducted to assess the in vitro activity of tetrasodium EDTA on catheters that had been routinely removed from hemodialysis patients at Leeds Teaching Hospitals Trust due to maturation of fistula. Catheters were screened by culture of through-catheter flush, and isolates were identified by standard methodologies; 20 isolates were found to be biofilm positive. Initial biofilm cell count levels averaged above 10(5) CFU/1-cm catheter section. Bacteria identified in the biofilms were gram-negative (1 isolate), gram-positive (11 isolates), or mixed species (8 isolates). After a 24-h lock, 40 mg of tetrasodium EDTA per ml was effective at eradicating the total biofilm viable count in almost all cases. The efficacy of tetrasodium EDTA as a catheter lock potentially shows that this agent could substantially reduce catheter-related infections and be used to treat patients with limited access.     

Scanning electron microscopy of bacteria adherent to intravascular catheters

Scanning electron microscopy was used to assess the morphological features of coagulase-negative staphylococci adherent to polyvinylchloride intravascular catheter specimens. Clinical specimens were obtained by using patient catheters from which coagulase-negative staphylococci (greater than or equal to 15 colonies per catheter) grew on semiquantitative blood agar roll cultures. In vitro specimens were prepared by a previously published technique in which sterile polyvinylchloride catheters were immersed in 10(6) CFU of coagulase-negative staphylococci per ml suspended in phosphate-buffered saline. Unused sterile polyvinylchloride catheters were also examined. Scanning electron microscopy of unused sterile polyvinylchloride catheters demonstrated multiple linear surface irregularities. Scanning electron microscopy of infected patient catheters showed a diffuse amorphous material covering the entire surface and the presence of bacteria which appeared anchored to that surface by several different means. These included a slime layer, "foot" processes, and lodgement in surface irregularities. Scanning electron microscopy of in vitro specimens demonstrated no background surface coating, but it did show attachment of cocci to the surface by the same mechanisms as described for clinical specimens. These observations of similar means of attachment in clinical and in vitro specimens suggest that intrinsic catheter surface properties, bacterial surface features, and perhaps coating with host substances may all play a role in bacterial attachment to intravascular catheters. More sophisticated analysis of these interactions may clarify mechanisms of pathogenesis.     

Detection of viable but non-culturable staphylococci in biofilms from central venous catheters negative on standard microbiological assays

Viable bacteria were sought in 44 Maki-negative biofilms from central venous catheters (CVCs) using epifluorescence microscopy after live/dead staining. Thirty (77%) samples contained viable but non-culturable (VBNC) cells; the majority were positive on real-time PCR specific for Staphylococcus epidermidis (one also for Staphylococcus aureus). Viable cells were significantly (p <0.01) associated with CVCs from febrile patients, three of whom showed S. epidermidis-positive blood cultures, suggesting that CVC-associated biofilms can be reservoirs for staphylococci in the VBNC state. The possible role of VBNC staphylococci in persistent infections related to medical devices requires further investigation.     

Use of chitosan bandage to prevent fatal infections developing from highly contaminated wounds in mice

HemCon bandage is an engineered chitosan acetate preparation used as a hemostatic control dressing, and its chemical structure suggests that it should also be antimicrobial. We tested its ability to rapidly kill bacteria in vitro and in mouse models of infected wounds. We used the Gram-negative species Pseudomonas aeruginosa and Proteus mirabilis and the Gram-positive Staphylococcus aureus that had all been stably transduced with the entire bacterial lux operon to allow in vivo bioluminescence imaging. An excisional wound in Balb/c mice was inoculated with 50-250 million cells followed after 30 min by application of HemCon bandage, alginate sponge bandage, silver sulfadiazine cream or no treatment. HemCon was more adhesive to the wound and conformed well to the injury compared to alginate. Animal survival was followed over 15 days with observations of bioluminescence emission and animal activity daily. Chitosan acetate treated mice infected with P. aeruginosa and P. mirabilis all survived while those receiving no treatment, alginate and silver sulfadiazine demonstrated 25-100% mortality. Chitosan acetate was much more effective than other treatments in rapidly reducing bioluminescence in the wound consistent with its rapid bactericidal activity in vitro as well as its light-scattering properties. S. aureus formed only non-lethal localized infections after temporary immunosuppression of the mice but HemCon was again more effective in reducing bioluminescence. The data suggest that chitosan acetate rapidly kills bacteria in the wound before systemic invasion can take place, and is superior to alginate bandage and silver sulfadiazine that may both encourage bacterial growth in the short term.     

Antibodies to capsular polysaccharides are not protective against experimental Staphylococcus aureus endocarditis.

The protective efficacy of antibodies to the Staphylococcus aureus capsular polysaccharide was examined in a rat model of catheter-induced endocarditis. Capsular antibodies were induced either by active immunization with killed S. aureus or by passive immunization with hyperimmune rabbit antiserum to S. aureus. Control rats were injected with phosphate-buffered saline or passively immunized with normal rabbit serum or rabbit antiserum to a nonencapsulated strain. Animals with indwelling catheters were challenged intravenously with 5 x 10(4) to 4 x 10(6) CFU of the homologous S. aureus strain (capsular serotype 5 strain Reynolds or serotype 1 strain SA1 mucoid). Both immunized and control rats developed S. aureus endocarditis. The numbers of S. aureus cells recovered from the blood and aortic valve vegetations of immunized rats were similar to those of control rats, indicating that capsule-specific antibodies were not protective. To determine whether the presence of an indwelling catheter interfered with antibody-mediated protection against S. aureus endocarditis, catheters were removed 2 h after insertion in additional groups of rats. An inoculum of 10(8) CFU of strain Reynolds was needed to provoke endocarditis in rats catheterized for 2 h, compared with 5 x 10(4) CFU for rats with indwelling catheters. Passively transferred capsular antibodies were not protective since both immunized and nonimmunized animals developed endocarditis, and quantitative cultures of blood and valvular vegetations revealed no differences between immunized and control animals. The findings of this study indicate that antibodies to the capsular polysaccharide are not protective in the rat model of experimental S. aureus endocarditis.     

Replication dynamics of Mycobacterium tuberculosis in chronically infected mice

The dynamics of host-pathogen interactions have important implications for the design of new antimicrobial agents to treat chronic infections such as tuberculosis (TB), which is notoriously refractory to conventional drug therapy. In the mouse model of TB, an acute phase of exponential bacterial growth in the lungs is followed by a chronic phase characterized by relatively stable numbers of bacteria. This equilibrium could be static, with little ongoing replication, or dynamic, with continuous bacterial multiplication balanced by bacterial killing. A static model predicts a close correspondence between "viable counts" (live bacteria) and "total counts" (live plus dead bacteria) in the lungs over time. A dynamic model predicts the divergence of total counts and viable counts over time due to the accumulation of dead bacteria. Here, viable counts are defined as bacterial CFU enumerated by plating lung homogenates; total counts are defined as bacterial chromosome equivalents (CEQ) enumerated by using quantitative real-time PCR. We show that the viable and total bacterial counts in the lungs of chronically infected mice do not diverge over time. Rapid degradation of dead bacteria is unlikely to account for the stability of bacterial CEQ numbers in the lungs over time, because treatment of mice with isoniazid for 8 weeks led to a marked reduction in the number of CFU without reducing the number of CEQ. These observations support the hypothesis that the stable number of bacterial CFU in the lungs during chronic infection represents a static equilibrium between host and pathogen.     

脊柱化脓性感染模型犬感染的判断与植入物治疗

背景：应用生物发光基因标记细菌建立动物感染模型,模拟脊柱感染的局部环境,揭示脊柱感染发生的病理生理机制。 目的：探讨前路一期清创自体髂骨植骨钛板内固定治疗脊柱化脓性感染的可行性及安全性。 方法：取中国家犬24只,用生物发光基因标记金葡菌Xen29建立犬脊柱化脓性感染模型。应用X射线、CT、MRI等检查对模型进行系统的动态影像学观察。建模4周后所有犬行前路一期清创自体髂骨植骨钛板内固定。内固定中、内固定后连续4周应用抗生素抗感染治疗。分别于内固定后4,8,12,24周处死犬,将钛板及钛板比邻的骨组织取出,进行传统的细菌培养、普通细菌16S rRNA 基因及金葡菌特异性Nuc基因进行PCR检测、生物发光成像(BLI)检测。 结果与结论：家犬内固定后观察切口愈合良好,没有窦道形成及脓性物质渗出。标本大体观察及MRI检查结果均未见脓肿形成、椎骨骨髓炎等感染复发征象。用传统的细菌培养、普通细菌16S rRNA 基因PCR检测作为判断感染与否的标准,感染率分别为41.7%(10/24)、75%(18/24),表明用PCR检测细菌16S rRNA 基因的检测方法判断感染的敏感性要明显高于传统细菌培养方法(P < 0.05)。金黄色葡萄球菌特异性Nuc基因的PCR检测验证有金黄色葡萄球菌存在(1/24)。而利用BLI技术对假体周围细菌进行特异性基因检测,并未检出基因标记的细菌Xen29(0/24)。证实体内假体上细菌附着是一种相对的普遍现象,并且内固定后取出的假体上分离出来的细菌与内固定前脊柱所感染的细菌种类并非同源。提示前路一期清创自体髂骨植骨钛板内固定治疗脊柱化脓性感染是安全有效的。内固定的使用不会造成感染复发或持续性慢性感染。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

Contribution of Quorum Sensing to the Virulence of Pseudomonas aeruginosa in Burn Wound Infections

The Pseudomonas aeruginosa quorum-sensing systems, las and rhl, control the production of numerous virulence factors. In this study, we have used the burned-mouse model to examine the contribution of quorum-sensing systems to the pathogenesis of P. aeruginosa infections in burn wounds. Different quorum-sensing mutants of P. aeruginosa PAO1 that were defective in the lasR, lasI, or rhlI gene or both the lasI and rhlI genes were utilized. The following parameters of the P. aeruginosa infection were examined: (i) lethality to the burned mouse, (ii) dissemination of the P. aeruginosa strain within the body of the infected mouse (by determining the numbers of CFU of P. aeruginosa within the liver and spleen), and (iii) spread of the P. aeruginosa strain within the burned skin (by determining the numbers of CFU of P. aeruginosa at the inoculation site and at a site about 15 mm from the inoculation site [distant site]). In comparison with that of PAO1, the in vivo virulence of lasI, lasR, and rhlI mutants was significantly reduced. However, the most significant reduction in in vivo virulence was seen with the lasI rhlI mutant. The numbers of CFU that were recovered from the livers, spleens, and skin of mice infected with different mutants were significantly lower than those of PAO1. At 8 and 16 h post burn infection, comparable numbers of CFU of PAO1 and lasI and rhlI mutants were obtained from both the inoculation and distant sites of the burned skin of infected mice. In contrast, CFU of the lasR mutant and the lasI rhlI double mutant were recovered only from the inoculation site of infected mice at 8 and 16 h post burn infection. The ability of a plasmid carrying either the lasI or rhlI gene or the lasI and rhlI genes to complement the defect of the lasI rhlI double mutant was also examined. The presence of any of these plasmids within the lasI rhlI double mutant significantly enhanced its in vivo virulence, as well as its ability to spread within the burned skin. These results suggest that the quorum-sensing systems play an important role in the horizontal spread of P. aeruginosa within burned skin and in the dissemination of P. aeruginosa within the bodies of burned-and-infected mice and contributed to the overall virulence of P. aeruginosa in this animal model.     

Pseudomonas aeruginosa invades corneal epithelial cells during experimental infection.

Pseudomonas aeruginosa is considered an extracellular pathogen. Using assays to determine intracellular survival in the presence of gentamicin, we have demonstrated that some strains of P. aeruginosa are able to invade corneal cells during experimental bacterial keratitis in mice. Although intracellular bacteria were detectable 15 min after inoculation, the number of intracellular bacteria increased in a time-dependent manner over a 24-h period. Levels of invasion were similar when bacteria were grown as a biofilm on solid medium and when they were grown in suspension. Intracellular bacteria survived in vitro for at least 24 h, although only minimal bacterial multiplication within cells was observed. P. aeruginosa PAK and Escherichia coli HB101 did not cause disease in this model and were not isolated from corneas after 24 h even when an inoculum of 10(8) CFU was applied. Transmission electron microscopy of corneal epithelium from eyes infected for 8 h revealed that intracellular bacteria were present within membrane-bound vacuoles, which suggests that bacterial entry was an endocytic process. At 24 h, the observation of many bacteria free in the cytoplasm indicated that P. aeruginosa was able to escape the endocytic vacuole. The ability of some P. aeruginosa strains to invade corneal epithelial cells may contribute to the pathogenesis or to the progression of disease, since intracellular bacteria can evade host immune effectors and antibiotics commonly used to treat infection.