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.     

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.     

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.     

Role of neutrophils in experimental septicemia and septic arthritis induced by Staphylococcus aureus.

We have previously described a murine model of hematogenously induced Staphylococcus aureus sepsis and arthritis. In this model, large numbers of granulocytes can be observed both in the circulation and locally in the inflamed synovium within 24 h after bacterial inoculation. To assess the role of neutrophils in this severe infection, mice were given granulocyte-depleting monoclonal antibody RB6-8C5 before being inoculated with S. aureus. All the control mice survived their intravenous injection with 3 x 10(7) CFU of S. aureus, whereas all the mice given RB6-8C5 antibody died of sepsis within 2 to 3 days. Even when the inoculum size was reduced sixfold (i.e., 6 x 10(6) CFU/mouse), 50% of the RB6-8C5-treated animals died within 6 days. The RB6-8C5-treated mice had a significantly higher burden of bacteria in their blood and kidneys 24 and 48 h after bacterial inoculation. In addition, when a suboptimal dose of bacteria was administered, the neutrophil-depleted animals displayed a higher frequency of arthritis than did the controls. The granulocyte-depleted animals exhibited increased levels of the proinflammatory cytokines tumor necrosis factor alpha, interleukin-6, and gamma interferon, reflecting the severity of their disease. This is the first direct demonstration of neutrophils playing a crucial protective role in the early phase of S. aureus infection.     

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.     

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：     

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.     

Rapid detection of bacterial growth in blood cultures by bioluminescent assay of bacterial ATP

A method for rapid detection of bacterial growth in blood cultures by bioluminescent assay of bacterial ATP was developed. Samples from blood cultures were treated with a blood-lysing detergent combined with an ATP-hydrolyzing enzyme to destroy blood cell ATP. Blood cell ATP which was bound to cell debris and escaped the ATPase activity was then separated from the bacteria on Percoll density gradients. Bacterial ATP from the bacterial layer was determined by the firefly bioluminescence system. In simulated blood cultures inoculated with 10 CFU of bacteria per ml of blood, bacterial ATP levels exceeded the detection limit (10(-10) M) after 6 to 10 h of incubation. This ATP level corresponds to approximately 10(4) CFU of bacteria per ml.     

Bioluminescent imaging (BLI) to improve and refine traditional murine models of tumor growth and metastasis

Bioluminescent imaging (BLI) permits sensitive in vivo detection and quantification of cells specifically engineered to emit visible light. Three stable human tumor cell lines engineered to express luciferase were assessed for their tumorigenicity in subcutaneous, intravenous and spontaneous metastasis models. Bioluminescent PC-3M-luc-C6 human prostate cancer cells were implanted subcutaneously into SCID-beige mice and were monitored for tumor growth and response to 5-FU and mitomycin C treatments. Progressive tumor development and inhibition/regression following drug treatment were observed and quantified in vivo using BLI. Imaging data correlated to standard external caliper measurements of tumor volume, but bioluminescent data permitted earlier detection of tumor growth. In a lung colonization model, bioluminescent A549-luc-C8 human lung cancer cells were injected intravenously and lung metastases were monitored in vivo by whole animal imaging. Anesthetized mice were imaged weekly allowing a temporal assessment of in vivo lung tumor growth. This longitudinal study design permitted an accurate, real-time evaluation of tumor burden in the same animals over time. End-point bioluminescence measured in vivo correlated to total lung weight at necropsy. For a spontaneous metastatic tumor model, bioluminescent HT-29-luc-D6 human colon cancer cells implanted subcutaneously produced metastases to lung and lymph nodes in SCID-beige mice. Both primary tumors and micrometastases were detected by BLI in vivo. Ex vivo imaging of excised lung lobes and lymph nodes confirmed the in vivo signals and indicated a slightly higher frequency of metastasis in some mice. Levels of bioluminescence from in vivo and ex vivo images corresponded to the frequency and size of metastatic lesions in lungs and lymph nodes as subsequently confirmed by histology. In summary, BLI provided rapid, non-invasive monitoring of tumor growth and regression in animals. Its application to traditional oncology animal models offers quantitative and sensitive analysis of tumor growth and metastasis. The ability to temporally assess tumor development and responses to drug therapies in vivo also improves upon current standard animal models that are based on single end point data.     

LysGH15, a novel bacteriophage lysin, protects a murine bacteremia model efficiently against lethal methicillin-resistant Staphylococcus aureus infection

Phage-coded lysin is an enzyme that destroys the cell walls of bacteria. Phage lysin could be an alternative to conventional antibiotic therapy against pathogens that are resistant to multiple antibiotics. In this study, a novel staphylococcal phage, GH15, was isolated, and the endogenous lytic enzyme (LysGH15) was expressed and purified. The lysin LysGH15 displayed a broad lytic spectrum; in vitro treatment killed a number of Staphylococcus aureus strains rapidly and completely, including methicillin-resistant S. aureus (MRSA). In animal experiments, a single intraperitoneal injection of LysGH15 (50 μg) administered 1 h after MRSA injections at double the minimum lethal dose was sufficient to protect mice (P < 0.01). Bacteremia in unprotected mice reached colony counts of about 10(7) CFU/ml within 3.5 h after challenge, whereas the mean colony count in lysin-protected mice was less than 10(4) CFU/ml (and ultimately became undetectable). These results indicate that LysGH15 can kill S. aureus in vitro and can protect mice efficiently from bacteremia in vivo. The phage lysin LysGH15 might be an alternative treatment strategy for infections caused by MRSA.     

Rapid antimicrobial susceptibility determination of uropathogens in clinical urine specimens by use of ATP bioluminescence

We describe the first direct testing of the antimicrobial susceptibilities of bacterial pathogens in human clinical fluid samples by the use of ATP bioluminescence. We developed an ATP bioluminescence assay that eliminates somatic sources of ATP to selectively quantify the bacterial load in clinical urine specimens with a sensitivity of <1,000 CFU per milliliter. There was a log-log relationship between light emission and the numbers of CFU in clinical urine specimens. A clinical study was performed to evaluate the accuracy of the ATP bioluminescence assay for determination of the antimicrobial susceptibilities of uropathogens in clinical urine specimens tested in a blinded manner. ATP bioluminescent bacterial density quantitation was used to determine the inoculation volume in growth medium with and without antibiotics. After incubation at 37 degrees C for 120 min, the ATP bioluminescence assay was repeated to evaluate the uropathogen response to antibiotics. The ability of the ATP bioluminescence assay to discriminate between antimicrobial susceptibility and resistance was determined by comparison of the results obtained by the ATP bioluminescence assay with the results obtained by standard clinical microbiology methods. Receiver operator characteristic curves were used to determine the optimal threshold for discriminating between susceptibility and resistance. Susceptibility and resistance were correctly predicted in 87% and 95% of cases, respectively, for an overall unweighted accuracy of 91%, when the results were stratified by antibiotic. For samples in which the pathogen was susceptible, the accuracy improved to 95% when the results for samples with less than a 25-fold increase in the amount of bacterial ATP in the medium without antibiotics were excluded. These data indicate that a rapid bioluminescent antimicrobial susceptibility assay may be useful for the management of urinary tract infections.     

Cutaneous infection in normal and immunocompromised mice.

Since a model of staphylococcal skin infection adequately reflecting human disease was unavailable, a self-limiting animal infection model specific for virulent Staphylococcus species was developed. A virulent strain of S. aureus, NCTC 9789 (ATCC 27700), was used to develop an infection model in adult, male CF-1 mice treated with 0 to 150 mg of cyclophosphamide (CY) per kg 4 days before challenge. Bacteria were inoculated onto the dorsal side of shaved mice at 0 to 10(6) CFU per mouse. Simultaneously, the skin was gently scraped to remove the superficial layers without drawing blood. The wound was occluded with impermeable film secured with surgical tape. At a CY dose of 50 mg/kg and an inoculum of 10(5) CFU, 89% of the mice (96 of 108) developed large abscesses (approximately 15-mm diameter). Mice which were not immunocompromised developed fewer abscesses (20 of 68). Generally, no abscesses formed when the mice were not wounded (1 of 62), occluded (0 of 89), or inoculated (11 of 50). The abscesses developed 24 to 48 h after challenge and persisted for 2 to 3 weeks. The challenge organism was isolated from the abscesses. The rates of abscess formation of three additional S. aureus strains varied widely in normal and CY-treated mice. Three strains of S. epidermidis, one of Micrococcus varians, and one of S. saprophyticus failed to cause abscesses. Bacterial proliferation studies demonstrated that a strain of S. aureus and a strain of S. epidermidis proliferated to the same levels 48 h after challenge. Immunosuppression and wounding had little effect on the levels of proliferation of S. aureus (P greater than 0.2). Without occlusion, however, S. aureus proliferated to significantly lower levels (P less than 0.005). This model may be be useful for screening topical anti-infective agents or studying the mechanisms of bacterial pathogenesis and host response.     

A Staphylococcus aureus capsular polysaccharide (CP) vaccine and CP-specific antibodies protect mice against bacterial challenge.

The efficacy of capsular polysaccharide (CP)-specific antibodies elicited by active immunization with vaccines composed of Staphylococcus aureus types 5 and 8 CP linked to Pseudomonas aeruginosa exoprotein A or with immune immunoglobulin G (I-IgG) obtained from vaccinated plasma donors was tested in lethal and sublethal bacterial mouse challenge models. A dose of 2 x 10(5) CFU of S. aureus type 5 CP per mouse administered intraperitoneally (i.p.) with 5% hog mucin was found to cause 80 to 100% mortality in BALB/c mice within 2 to 5 days. Mice passively immunized i.p. 24 h earlier or subcutaneously 48 h earlier with 0.5 ml of I-IgG showed significantly higher average survival rates than animals receiving standard IgG or saline (P < 0.01) following the bacterial challenge. Animals actively immunized with the monovalent type 5 CP-P. aeruginosa exoprotein A conjugate showed a survival rate of 73% compared with 13% in phosphate-buffered saline-immunized animals. The prechallenge geometric mean titer of type 5 CP antibodies in animals that died was significantly (P < 0.05) lower than that of animals which survived the challenge (95.7 versus 223.6 micrograms/ml, respectively). The IgG was further evaluated in mice challenged i.p. with a sublethal dose of 5 x 10(4) CFU per mouse. Serial blood counts were performed on surviving animals at 6, 12, 24, and 48 h. Surviving animals were sacrificed at 72 h, and bacterial counts were performed on their kidneys, livers, and peritoneal lavage fluids. Animals receiving I-IgG had lower bacterial counts in blood samples and lower bacterial densities in kidneys, livers, and peritoneal lavage samples than mice immunized with standard IgG (P < 0.05). These data suggest that S. aureus type 5 CP antibodies induced by active immunization or administered by passive immunization confer protection against S. aureus infections.     

Establishment of a real-time, quantitative, and reproducible mouse model of Staphylococcus osteomyelitis using bioluminescence imaging

Osteomyelitis remains a serious problem in the orthopedic field. There are only a few animal models in which the quantity and distribution of bacteria can be reproducibly traced. Here, we established a real-time quantitative mouse model of osteomyelitis using bioluminescence imaging (BLI) without sacrificing the animals. A bioluminescent strain of Staphylococcus aureus was inoculated into the femurs of mice. The bacterial photon intensity (PI) was then sequentially measured by BLI. Serological and histological analyses of the mice were performed. The mean PI peaked at 3 days, and stable signals were maintained for over 3 months after inoculation. The serum levels of interleukin-6, interleukin-1β, and C-reactive protein were significantly higher in the infected mice than in the control mice on day 7. The serum monocyte chemotactic protein 1 level was also significantly higher in the infected group at 12 h than in the control group. A significantly higher proportion of granulocytes was detected in the peripheral blood of the infected group after day 7. Additionally, both acute and chronic histological manifestations were observed in the infected group. This model is useful for elucidating the pathophysiology of both acute and chronic osteomyelitis and to assess the effects of novel antibiotics or antibacterial implants.     

Establishment of an experimental model of a Staphylococcus aureus abscess in mice by use of dextran and gelatin microcarriers

Staphylococcus aureus UC 9271 mixed with dextran or gelatin microcarrier beads and injected subcutaneously into mice resulted in the formation of reproducible, sustained abscesses with as few as 2 x 10(3) cfu. Without microcarrier beads, 4 x 10(7) cfu were required to produce an abscess. The abscesses that developed with microcarriers attained a diameter of up to 1.5 cm and persisted for several days before discharging through the skin. The pH of the abscesses fell from 7.1 to 6.6 within 24 h. Histological and microscopic examination of the abscesses revealed an influx of phagocytic cells, mostly polymorphonuclear leucocytes, within 1-2 h after injection. Cell debris accumulated and the abscess became encapsulated 24-48 h after infection. Enzymatic digestion of the abscess contents allowed analysis of the host and bacterial cell populations and treatment with lysostaphin permitted differentiation between phagocytosed and free bacterial populations of S. aureus. Phagocytosed but viable S. aureus comprised c. 50% of the total bacterial population after 24 h; however, by 96 h the phagocytosed population was only 1-5% of the total population, primarily because of an increase in extracellular bacterial numbers. Prevention of abscess formation by antibiotic treatment based upon the minimal inhibitory concentration (MIC) of an antibiotic for S. aureus was not always predictable. Tetracycline did not prevent abscess formation even though it possessed a low MIC for S. aureus; methicillin had a borderline MIC value but was quite active. However, the MIC values were quite predictive of antibiotic cures in a systemic-lethal S. aureus infection in mice.     

Etmopterus spinax,the velvet belly lanternshark,does not use bacterial luminescence

Marine organisms are able to produce light using either their own luminous system, called intrinsic bioluminescence, or symbiotic luminous bacteria, called extrinsic bioluminescence. Among bioluminescent vertebrates, Osteichthyes are known to harbor both types of bioluminescence, while no study has so far addressed the potential use of intrinsic/extrinsic luminescence in elasmobranchs. In sharks, two families are known to emit light: Etmopteridae and Dalatiidae. The deep-sea bioluminescent Etmopteridae, Etmopterus spinax, has received a particular interest over the past fifteen years and its bioluminescence control was investigated in depth. However, the nature of the shark luminous system still remains enigmatic. The present work was undertaken to assess whether the light of this shark species originates from a bioluminescent bacterial symbiosis. Using fluorescent in situ hybridization (FISH) and transmission electron microscopy (TEM) image analyses, this study supports the conclusion that the bioluminescence in the deep-sea lanternshark, Etmopterus spinax, is not of bacterial origin.     

Visualizing fungal infections in living mice using bioluminescent pathogenic Candida albicans strains transformed with the firefly luciferase gene

Animal studies with Candida albicans have provided models for understanding fungal virulence and antifungal drug development. To non-invasively monitor long-term Candida murine infections, clinical isolates were stably transformed with a codon-optimized luciferase gene to constitutively express luciferase. Chronic systemic infections were established in mice with engineered strains, and bioluminescent signals were apparent from kidneys by non-invasive imaging using charged-coupled device cameras. These infections were established in immune-competent mice, and bioluminescence was detectable in animals that showed no physiological consequence of infection, as well as those visually succumbing to the disease. Similarly, bioluminescence was measured from the vaginal tissue of mice infected vaginally. Fungal loads determined by plating vaginal lavages showed a similar pattern to the bioluminescent signals measured, and fungal infection could be detected in animals for over 30 days post infection by both modalities. The effect of the antifungal drug miconazole was tested in this model, and clearance in animals was apparent by both direct imaging and fungal load determination. The use of bioluminescence to monitor these and other models of Candida infections will greatly speed up the analysis of drug development studies, both in ease of visualizing infections and decreasing numbers of animals required to run such studies.     

Lipid X protects mice against fatal Escherichia coli infection.

Lipid X, the major monosaccharide precursor of lipid A, is nontoxic and has previously been shown to protect mice and sheep from the harmful effects of endotoxin. To test the hypothesis that lipid X could be therapeutic against infections with gram-negative organisms, neutropenic ICR mice were infected by intramuscular inoculation of Escherichia coli and subsequently treated with lipid X alone or in combination with the antibiotic ticarcillin. Lipid X slightly prolonged survival; treated mice had a significantly improved rate of survival 18 h after intramuscular inoculation as compared with controls (P less than 0.025). By 24 h, however, this difference disappeared. When lipid X was combined with ticarcillin, survival differences were both significant and prolonged. Treatment of mice with one to two doses of lipid X for a total dose of 1 mg intravenously and with 1,200 mg of ticarcillin per kg every 6 h improved survival over a 48-h treatment period from 5 to 23% (P less than 0.0025). Treatment with lipid X and ticarcillin over a broad range of antibiotic dosages in 362 mice demonstrated improved survival of two- to fourfold (P less than 0.0001 at 24 h after inoculation, P less than or equal to 0.0005 at 48 h, and P less than or equal to 0.0001 at 5 days). Lipid X enabled the dose of ticarcillin necessary to protect 50% of mice from death to be reduced by two- to fivefold. Pretreatment with lipid X was not necessary to improve survival: 16 of 17 (94%) infected and visibly ill animals that received lipid X and ticarcillin 6 h after thigh inoculation survived versus 30 of 44 (68%) control animals treated with ticarcillin alone (P less than 0.0001). Lipid X had no antimicrobial activity in vitro. Lipid X is a novel agent that enhances survival in an animal model of severe infection with gram-negative organisms.     

Lipopolysaccharide triggers invasive streptococcal disease in mice through a tumour necrosis factor-alpha-dependent mechanism

Streptococcus pyogenes sometimes induces invasive streptococcal infection, including streptococcal toxic shock syndrome (STSS). Muscular necrosis is one of the peculiar symptoms of invasive streptococcal infection and STSS. We inoculated S. pyogenes into the muscles of mice. To do so, 5 x 10(8) bacteria in 0.2 ml phosphate-buffered saline were injected into the right hind thigh. None of the mice injected with the bacteria showed muscular necrosis and none died. Tumour necrosis factor-alpha (TNF-alpha) and infiltration of leucocytes were detected in the muscles of infected sites, although the condition of the infected mice did not deteriorate after anti-TNF-alpha monoclonal antibody treatment. The infected mice treated intraperitoneally with Escherichia coli lipopolysaccharide (LPS) showed augmentation of bacterial growth, muscular necrosis and death. TNF-alpha was detected in the sera of the infected mice treated with LPS, but not in the muscles of the infected sites. Infiltration of leucocytes into the infected muscle was not observed in the infected mice treated with LPS. Anti-TNF-alpha monoclonal antibody treatment decreased mortality in the infected mice treated with LPS. Moreover, the infected mice treated with recombinant TNF-alpha showed augmentation of muscular necrosis and death. These results suggest that systemic production of TNF-alpha induced by stimulation with LPS inhibits infiltration of leucocytes into the infected site and exacerbates muscular infection, and that TNF-alpha produced in streptococcal infection is not a defence factor for the host. Invasive streptococcal infection and STSS appear to be induced by both S. pyogenes and the host's immune system.