Endotoxin Release Research Articles

DIFFERENTIAL RELEASE OF ENDOTOXIN FROM PSEUDOMONAS AERUGINOSA TREATED WITH ^|^beta;-LACTAM ANTIBIOTICS AND ITS EFFECT ON THE LETHAL ACTIVITY

The relationship between the level of endotoxin released from Pseudomonas aeruginosa by exposure to beta-lactam antibiotics and the lethal activity against D-galactosamine (D-GalN)-sensitized mice was examined. In vitro treatment of P. aeruginosa with imipenem (IPM) exclusively caused low-level release of free endotoxin, which was not lethal for D-GalN-sensitized mice. Treatment with other beta-lactam antibiotics, such as ceftazidime (CAZ), meropenem (MEPM) and cefozopran (CZOP) caused high-level release of free endotoxin and it exhibited the lethal action against D-GalN-sensitized mice. This study demonstrated close association of free endotoxin released by antibiotic treatment with the lethal activity.

The Effect of Antibiotic Treatment on the Release of Endotoxin during Nontypable Haemophilus influenzae-induced Otitis Media in the Chinchilla

The gram negative bacteria, nontypable Haemophilus influenzae (NTHi) was used to induce otitis media in a total of 18 chinchillas. Three days post-inoculation, three cohorts of 6 chinchillas each were treated daily for four days with either ceftriaxone, chloramphenicol, or diluent without antibiotics. Middle ear fluid (MEF) was obtained daily, assayed for endotoxin content by means of the chromogenic limulus amebocyte lysate assay, and concentration of the NTHi/mL MEF determined by standard plate count. The endotoxin concentration per mL MEF from both the antibiotic treated cohorts decreased during the observation period, but increased in the MEF of the untreated control group. The data indicate that, unlike the dramatic increase in endotoxin concentration, after antibiotic treatment in the cerebrospinal fluid (CSF) during experimental Haemophilus influenzae -induced meningitis, there is no demonstrable sustained release of endotoxin in the middle ear subsequent to antibiotic treatment during experimental otitis media.

Portal Venous Thrombosis in a Backpacker

Effort thrombosis usually afflicts an extremity and is caused by compression. This case report, in contrast, involves superior mesenteric and left portal vein septic thrombosis in a backpacker following prolonged hiking and abdominal straining. The condition may have been caused by localized splanchnic venous ischemia, erosion of the bowel-blood barrier, and release of bacterial endotoxin in this dehydrated and detrained athlete. Diagnosis of this disorder is aided by noting characteristic abdominal pain, fever, nausea, and vomiting, as well as by imaging with MRI, CT, or duplex ultrasonography. Antibiotics and anticoagulants are key to treatment.

Portal Venous Thrombosis in a Backpacker.

In brief Effort thrombosis usually afflicts an extremity and is caused by compression. This case report, in contrast, involves superior mesenteric and left portal vein septic thrombosis in a backpacker following prolonged hiking and abdominal straining. The condition may have been caused by localized splanchnic venous ischemia, erosion of the bowel-blood barrier, and release of bacterial endotoxin in this dehydrated and detrained athlete. Diagnosis of this disorder is aided by noting characteristic abdominal pain, fever, nausea, and vomiting, as well as by imaging with MRI, CT, or duplex ultrasonography. Antibiotics and anticoagulants are key to treatment.

Differential release of smooth-type lipopolysaccharide from Pseudomonas aeruginosa treated with carbapenem antibiotics and its relation to production of tumor necrosis factor alpha and nitric oxide

Endotoxin release from Pseudomonas aeruginosa treated with cell wall-active carbapenem antibiotics and its effect on the production of tumor necrosis factor alpha and nitric oxide were examined. Treatment of bacteria with imipenem induced much lower levels of endotoxin release than treatment with meropenem. The endotoxin released was demonstrated to be of the smooth type and O-specific polysaccharide-rich. The exposure of the filtrates of P. aeruginosa treated with imipenem to physiologically relevant cells caused low-level production of tumor necrosis factor alpha and nitric oxide, while similar treatment with meropenem induced high levels of production.

Potent CD14-mediated signalling of human leukocytes by Escherichia coli can be mediated by interaction of whole bacteria and host cells without extensive prior release of endotoxin.

How invading microorganisms are detected by the host has not been well defined. We have compared the abilities of Escherichia coli and lipopolysaccharides (LPS) purified from these bacteria to prime isolated neutrophils for phorbol myristate acetate-stimulated arachidonate release, to trigger respiratory burst in 1% blood, and to increase steady-state levels of tumor necrosis factor alpha mRNA in whole blood. In all three assays, bacteria were > or = 10-fold more potent than equivalent amounts of LPS and could trigger maximal cellular responses at ratios as low as one bacterium per 20 to 200 leukocytes. Both E. coli and LPS-triggered responses were enhanced by LPS-binding protein and inhibited by an anti-CD14 monoclonal antibody and the bactericidal/permeability-increasing protein (BPI). However, whereas O polysaccharide did not affect the potency of isolated LPS, intact E. coli carrying long-chain LPS (O111:B4) was less potent than rough E. coli (J5). Furthermore, material collected by filtration or centrifugation of bacteria incubated under conditions used to trigger arachidonate release or chemiluminescence was 5- or 30-fold less active, respectively, than whole bacterial suspensions. Extracellular BPI (not bound to bacteria) inhibited bacterial signalling, but BPI bound to bacteria was much more potent. Taken together, these findings indicate that E. coli cells can strongly signal their presence to human leukocytes not only by shedding LPS into surrounding fluids but also by exposing endotoxin at or near their surface during direct interaction with host cells.

Gut is not a source of cytokines in a porcine model of endotoxicosis

We sought to determine whether ischemic gut is a source of endotoxin, tumor necrosis factor (TNF), and interleukin-6 (IL-6) in a porcine model of endotoxicosis. Under general anesthesia pigs underwent neck dissection and laparotomy for placement of catheters in the carotid artery and portal vein and application of an ultrasonic flow probe around the portal vein. Endotoxin, TNF, and IL-6 levels were measured from the carotid artery and the portal vein during a 4 hour period in animals given endotoxin (50 mg/kg; n = 6) and in animals in the control group (n = 6). Gut fluxes of the substances of interest were calculated as the product of concentration and portal venous flow. A tonometer placed in the terminal ileum was used to monitor mucosal pH. Small bowel mucosal pH was significantly depressed in endotoxemic animals (6.8 +/- 0.1) when compared with baseline (7.1 +/- 0.1; p < 0.05) and control levels. In the control group portal venous levels of endotoxin, TNF, and IL-6 did not change significantly from baseline levels (1.5 +/- 0.4 endotoxin units (EU)/ml, 24 +/- 3 pU/ml, and 1.3 +/- 0.4 nU/ml, respectively). In the endotoxemic animals portal venous endotoxin and TNF levels peaked immediately after the endotoxin infusion (2186 +/- 437 EU/ml, and 293 +/- 125 pU/ml, respectively), and portal venous IL-6 levels peaked at 180 minutes (168 +/- 21 nU/ml). At no time did endotoxin, TNF, or IL-6 levels differ between arterial and portal venous blood, and at no time did efflux from the gut significantly exceed gut influx in either the control or endotoxemic animals. Ischemic gut as indicated by decreased mucosal pH is not associated with gut release of endotoxin, IL-6, or TNF in this porcine model of endotoxicosis.

Use of detergent to prevent initial responses to endotoxin in horses

Abstract Objective To determine whether a detergent can prevent most of the early effects of IV infusion with Escherichia coli endotoxin (&lt; 100 ng/kg of body weight) in horses: marked pulmonary hypertension, acute leukopenia, and fever. Animals 8 healthy adult horses (4 male, 4 female), 415 to 615 kg. Design and Procedure Control and detergent experiments were performed in each horse while it was awake but sedated. In control experiments, 10 to 100 ng of E coli endotoxin/kg was given. In detergent experiments, 100 mg of detergent/kg was given 1 hour before injecting endotoxin, similar to the control experiments. Results In control experiments, pulmonary arterial pressure increased transiently over 40 minutes by 33 ± 8 mm of Hg (mean ± SD; P &lt; 0.001), then returned to baseline. Circulating leukocytes decreased to 47 ± 19% (P &lt; 0.02) of baseline by 1 hour after endotoxin, then increased above baseline by 6 hours. Rectal temperature increased by 0.7 ± 0.4 C (P &lt; 0.01). In detergent experiments, the increase in pulmonary arterial pressure was much less than that in the control experiments (8 ± 7 mm of Hg; P &lt; 0.001). Circulating leukocytes did not decrease, and the increase in rectal temperature after endotoxin was blocked. Conclusions This attenuation of the response to endotoxin may occur because the normal steps in the response of pulmonary intravascular macrophages (ie, endocytosis of endotoxin and subsequent release of inflammatory mediators) are altered by the detergent. This low-technology, inexpensive, and safe treatment could be an important new clinical tool for veterinarians in combating endotoxemia. (Am J Vet Res 1996;57:1063–1066)

Antibiotic induced release of endotoxin - clinical data and human studies

In vitro and animal studies, but also clinical data, indicate that treatment with antibiotics may cause systemic or local endotoxin liberation. In several studies this so-called 'endotoxin release' was accompanied by a deterioration of parameters of illness. In addition, in vitro studies have clearly demonstrated that equally effective antibiotics can differ in the amount of endotoxin they release. These differences between antibiotics can be related to different modes of anti-bacterial activities. In a number of animal studies these differences in endotoxin-releasing potential resulted in differences in morbidity and/or mortality. In only one study, antibiotics were compared in this respect in septic patients. The results of that study suggested that differences between antibiotics in endotoxin release may indeed affect the inflammatory response during treatment.

Different endotoxin release and IL-6 plasma levels after antibiotic administration in surgical intensive care patients

Despite the use of broad-spectrum antibiotics, aggressive fluid resuscitation, vasopressor support, the mortality associated with Gram-negative sepsis and septic shock has not decreased significantly in the last two decades. The consequences of host exposure to endotoxin and the relationship of antibiotic administration to endotoxin release have become important areas of intense interest. In vitro studies have demonstrated that there was a difference in endotoxin release between PBP-3 specific antibiotics (β-lactam antibiotics) and PBP-2 specific antibiotics (carbapenems). This is the first clinical report of surgical patients admitted to the surgical and anaesthesiology intensive care unit on the missing endotoxin release after imipenem treatment; however cefotaxime and ceftriaxone showed significantly more positive endotoxin tests in the plasma when compared to imipenem. Ciprofloxacin and vancomycin were intermediate in endotoxin release and tobramycin did not cause endotoxin release. There were also significant differences in endotoxin neutralizing capacity. IL-6 levels were decreased after imipenem faster than after ceftriaxone or cefotaxime; ciprofloxacin seemed to increase IL-6. Endotoxin may be harmful in patients where the immune system has been continuously challenged. Timing, dosage, or combination with other compounds as well as the effect of antibiotics on macrophages need to be tested in larger clinical trials. In this respect a consecutive study was started.

The in vivo significance of antibiotic-induced endotoxin release in experimental Gram-negative sepsis

Considerable evidence now supports the experimental findings that penicillin-binding protein (PBP)-2 specific antimicrobial agents such as imipenem generate less endotoxin than PBP-3 specific agents such as ceftazidime during the process of bacteriolysis of Gram-negative bacteria. To determine if differences in endotoxin release have pathophysiologic significance in vivo, Sprague-Dawley rats were experimentally challenged with intraperitoneal injections of virulent, serum-resistant clinical strains of the following Gram-negative bacilli: Escherichia coli 018:K1, Klebsiella pneumoniae K2, and Pseudomonas aeruginosa 12.4.4 (immuno type 6). After intravenous administration of imipenem (25 mg/kg), ceftazidime (50 mg/kg) or saline control, imipenem and ceftazidime-treated animals had rapid reductions in the quantitative level of bacteremia from all three pathogens. Peritoneal fluid samples revealed spherical forms with imipenem and long, filamentous forms with ceftazidime. Circulating plasma endotoxin levels were consistently higher ( P &lt; 0.05) with ceftazidime than imipenem for 6 h after administration of E. coli or P. aeruginosa intraperitoneal challenge. Endotoxin levels were unchanged to slightly higher with imipenem than ceftazidime following K. pneumoniae intraperitoneal challenge. TNF levels peaked 2 h post-therapy and were consistently higher with ceftazidime-treated animals ( P &lt; 0.05). D-galactosamine-treated animals had LD50 values that were 0.5-2 log higher ( P &lt; 0.001) with imipenem for E. coli and P. aeruginosa but did not differ from ceftazidime in animals challenged with the K. pneumoniae strain. These results indicate that the PBP-2 specific agent imipenem led to significantly less endotoxin release than did ceftazidime with its great affinity to PBP-3. Differential endotoxin release was found after antimicrobial therapy with the E. coli and P. aeruginosa strains but not with the K. pneumoniae strain tested in this study. The clinical relevance of these findings with treatment of systemic Gram-negative infections in humans will require further clinical investigation.

Influence of drug class and dose size on antibiotic-induced endotoxin/IL-6 release and impact on efficacy of anti-endotoxin antibody

There are limited comparative data on antibiotic-mediated endotoxin release and cytokine liberation in septic hosts. To determine the effect of antimicrobial drug class and dose size on the relative concentration kinetics of free endotoxin and interleukin-6 (IL-6), CF-1 mice were made septic following a thermal injury and low-dose (103 CFU) subeschar challenge with Klebsiella pneumoniae serotype K2. Single intraperitoneal (i.p.) doses (in mg/kg) of ceftazidime (TAZ, 25, 200), aztreonam (AZT, 200), piperacillin (PIP, 200), meropenem (MER, 200), imipenem (IMI, 25, 100, 200), ciprofloxacin (CIP, 25) and gentamicin (GEN, 25) were administered at 72 h post burn and infection, when mice were septic with organ dysfunction. AZT, TAZ, MER, PIP (each at 200 mg/kg) and IMI (100 mg/kg) resulted in fold-increases in median endotoxin levels of 15.3, 14.9, 13.1, 8.2 and 12.4, respectively. All were significantly greater than predose baseline values (P &lt; 0.01), however differences among agents did not reach statistical significance. The increases in free endotoxin levels for all of the beta lactams (8.2-15.3-fold) and CIP (7.7-fold) were significantly greater than for GEN (3.9-fold, P &lt; 0.01). The fold-rise in median IL-6 concentrations from baseline for the beta lactams ranged from 3.0-7.7. All of the beta lactams resulted in statistically greater IL-6 release as compared with CIP (1.9-fold, P &lt; 0.01) and GEN (1.4-fold, P &lt; 0.01). The median endotoxin concentrations were significantly higher for the 100 mg/kg (668 EU/ml) and 200 mg/kg (862 EU/ml) doses of IMI compared to the 25 mg/kg dose (378 EU/mL, P &lt; 0.05). There was also a significant increase in endotoxin levels with a dose increase of TAZ from 25 to 200 mg/kg (597 vs 1030 EU/ml, P= 0.017). The addition of antiendotoxin monoclonal antibody (E5, 2 mg/kg i.p. four times daily [qid]) to AZT (75 mg/kg qid) or TAZ (10 mg/kg qid) for 2 days significantly reduced the mortality by ∼20-40% for a 2-3 day period (P &lt; 0.05) compared to AZT or TAZ alone. In contrast, the administration of E5 with either CIP (10 mg/kg) or GEN (10 mg/kg) by the same qid dosing schedule did not improve survival (P&gt; 0.05). In conclusion, data from the present study, generated in a physiologically relevant model of sepsis, found relatively greater release of unbound endotoxin and IL-6 with the beta lactams, intermediate for CIP and lowest for GEN. The size of the dose of two beta lactams was also found to be a potentially important variable, suggesting that dose optimization might be possible to minimize antibiotic-associated endotoxin release. Furthermore, under our experimental conditions, the efficacy of E5 appears to be dependent upon the antibiotic with which it is administered. In order to understand better the potential significance of antibiotic-induced endotoxin release, the pharmacologic variables of the antimicrobial should be systematically evaluated in preclinical and clinical trials.

Contribution of soluble endotoxin released from Gram-negative bacteria by antibiotics to the pathogenesis of experimental sepsis in mice

The results from our laboratory summarized in this review support the importance of endotoxin release from the Gram-negative microbe as a requirement for the full manifestation of the biological activity of the lipopolysaccharide (LPS) macromolecule. They further suggest that LPS is the most important bacterial component released from Gram-negative microbes treated in vitro with cell-wall active antibiotics in terms of biological activity. The use of several experimental mouse models of infection confirm that mice treated with the reversible hepatotoxin, D-galactosamine, are sensitive to both endotoxin lethality and to infection with Gram-negative microbes. Infected LPS-sensitive animals can be protected if they are treated with antibiotics, but the degree of protection (e.g. 5-10-fold) is significantly less than that observed when infection-sensitive but LPS-insensitive mice are similarly treated (500-fold protection). Additional levels of protection in infected, antibiotic-treated mice can be observed with antiendotoxin agents (e.g. polymyxin B-dextran, monoclonal antibody). Finally, antibiotics with equivalent minimal inhibitory concentrations for Escherichia coli, but differing in their mode of action and endotoxin releasing potential in vitro, also differ in their protective efficacy in vivo to reduce mortality in infected animals. Collectively, these data provide evidence to support a role for microbe-derived endotoxin, released from the microbial surface following treatment with cell-wall active antibiotics, in the pathogenesis of experimental Gram-negative sepsis.

The clinical significance of endotoxin released by antibiotics: what is the evidence?

Basic investigations indicate variation among antibiotics in their propensity to release endotoxin from bacterial cells. Evidence for the clinical significance of this phenomenon includes: anecdotal reports; studies evaluating changes in endotoxin levels in response to antibiotics in a variety of infectious diseases; two studies which have prospectively evaluated changes in endotoxin levels in response to different antibiotics and correlated the changes with the patients' clinical course; and one study which evaluated differences in mortality in injured patients based on the endotoxin-releasing potential of antibiotics they received. These studies suggest: (i) different types of antibiotics do induce differing levels of endotoxin release during treatment of bacterial infections in humans; (ii) such antibiotic-associated endotoxin release probably does have a biologic consequence; and (iii) this biologic consequence is possibly of clinical relevance. However, the data are not sufficient to advocate changes in current antibiotic prescribing or dosing decisions. Rather, a variety of in vitro and in vivo experiments as well as clinical studies are advocated.

The influence of antibiotic-induced filament formation on the release of endotoxin from Gram-negative bacteria

The influence of antibiotic-induced filament formation on the release of endotoxin from Gram-negative bacteria

Differences in mode of action of (β-lactam antibiotics influence morphology, LPS release and in vivo antibiotic efficacy

Antibiotic mediated release of endotoxin (lipopolysaccharide, LPS) from Gram-negative bacteria is implicated in septic shock. The β-lactam (cell wall active) class, in particular, has been deemed responsible for release of greater quantities of LPS than other classes of antibiotics. However, it is becoming increasingly recognized that variations in the ability to liberate free LPS exist within the β-lactam subclasses. Until recently, LPS-release studies have primarily included the cephalosporin, monobactam, and penicillin β-lactams, but not the carbapenem subclass. We document here that carbapenems significantly liberate less LPS than other β-lactam subclasses, and that disparity in LPS release also occurs within the carbapenems as well (i.e. imipenem vs meropenem). The propensity to release LPS correlates with the cumulative PBP binding affinity of each β-lactam antibiotic regardless of subclass. A direct correlation has been established between antibiotic concentration, frequency of antibiotic exposure, differential LPS release, PBP specificity (morphology, rate of bacterial lysis or cell mass) and antibiotic efficacy in two mouse models of infection. In the first, differences in the pathogenic (endotoxic) potential of potently biologically active smooth LPS (S-LPS) and poorly biologically active rough chemotype LPS (R-LPS) expressing bacteria underscores the in vivo importance of endotoxin in mediating lethality following antibiotic chemotherapy. Endotoxin is, however, not innately toxic and thus its lethal effects are mediated through LPS stimulation of host cells (i.e. macrophages) to produce deleterious levels of cytokines. In the second experimental model, therefore, the toxic effects of antibiotic-liberated endotoxin have been abrogated by pretreatment of mice with carrageenan (CGN) prior to challenge with endotoxin or Gram-negative bacteria. CGN treatment eliminates or markedly reduces the numbers of LPS-responsive mediator cells thus affecting mortality. In both animal models, an in vivo role for LPS is demonstrated through differential changes in antibiotic efficacy (ED 50) in response to differential effects of LPS via modulation of the endotoxic sensitivity/responsiveness of the host. We conclude that antibiotic class, concentration, dosing intervals (timing) and perhaps, method of treatment (i.e. bolus vs infusion) may each be important in the survival of experimental animals severely infected with Gram-negative bacteria.

A murine model with aspects of clinical relevance for the study of antibiotic-induced endotoxin release in septic hosts

A murine model which utilizes a burn to increase susceptibility to sepsis is presented. The model is inexpensive, highly reproducible, versatile, easy to use, and clinically relevant to many aspects of a Gram-negative sepsis which develops over time from a localized focus of infection. Additionally, studies of LPS-intoxication in mice with the same burn indicate that the animals show increased sensitivity to endotoxin without having to be exposed to exogenous sensitizing chemicals. Hence, this burned mouse model of sepsis might provide a sensitive, clinically relevant model for the study of antibiotic-induced endotoxin release from Gram-negative bacteria in septic hosts.

Assessment of antibiotic-mediated endotoxin release

Assessment of antibiotic-mediated endotoxin release

Do certain antibacterials increase the risk of septic shock?

While antibacterials are known to be effective in treating septicaemia induced by Gram-negative bacteria, treatment with certain antibacterials also appears to increase endotoxin release, which could have negative consequences. In both animal and clinical studies, researchers have shown that the amount of lipopolysaccharide endotoxin released by Gram-negative bacteria is influenced by the choice of antibacterial used. Endotoxins are key factors involved in the development of septic shock syndrome. However, in spite of these findings, researchers have not found a direct link between choice of antibacterial and septic shock outcome. In presentations at the 16th International Symposium on Intensive Care and Emergency Medicine [Brussels, Belgium; March 1996], two researchers described recent investigations in this field.

Elevated serum neopterin level: its relation to endotoxaemia and sepsis in patients with major burns.

The present study was conducted to determine the relationship between levels of neopterin and endotoxin in the circulation, and whether the neopterin level was related to the development of severe sepsis after extensive burns. This prospective study included 35 patients with burn size greater than 30% (30-98%), and 22 healthy volunteers who served as a comparison group. Neopterin levels increased in most patients on day 3 post-burn, but they were not significantly correlated with the extent of the burn surface (P > 0 center dot 05). A high serum neopterin level was found in patients with sepsis (n = 15), and a marked elevation persisted throughout the observation period. The difference between septic and non-septic patients (n = 20) became significant on 14 and 28 days post-burn. Although the presence of early endotoxaemia did not influence the alterations in serum neopterin, patients with endotoxaemia had much higher neopterin values than those who showed no endotoxaemia from the second week onward (P < 0 center dot 05-0 center dot 01). In addition, circulating endotoxin and neopterin levels were positively correlated in patients who developed endotoxaemia on day 14 (r = 0 center dot 368, P < 0 center dot 05) and day 21 (r = 0 center dot 439, P < 0 center dot 01) after major burns. These results suggest that thermal injury can lead to an elevation of serum neopterin independent of the burn surface area. The initial increase in the neopterin level may be a part of the acute-phase response to tissue injury itself, whereas the endotoxin release in the circulation may be responsible for the continuous induction of neopterin during the late stage. In addition, the presence of a constant high neopterin level is associated with a critical event in the development of severe burn sepsis.