Effect of methylprednisolone on bacterial clearance and endotoxin liberation during experimental sepsis induced by gram-negative bacteria.

To determine the effect of methylprednisolone administration on the clearance of bacteremia and the release and clearance of endotoxin during antibiotic therapy of gram-negative bacterial sepsis, Escherichia coli K1 sepsis was induced in paired rabbits. Moxalactam and either methylprednisolone or placebo were administered to infected rabbits 1.5 h after intraperitoneal administration of live bacteria. Serial blood samples were obtained for quantitation of bacteremia and endotoxemia, arterial blood gases, and complete blood count. Arterial blood pressure, heart rate, and core body temperature were also monitored. There were no significant differences between the methylprednisolone-treated and placebo-treated groups in either the levels of bacteremia or endotoxemia or in the physiologic, metabolic, or hematologic parameters that were measured. We conclude that methylprednisolone administration has no acute effect on bacterial clearance or on the kinetics of endotoxin release and clearance during antibiotic therapy of gram-negative bacterial sepsis in this experimental model.     

Detection of endotoxin in the plasma of patients with gram-negative bacterial sepsis by the Limulus amoebocyte lysate assay.

A total of 120 Limulus amoebocyte lysate (LAL) determinations were made on plasma obtained from normal, healthy human blood donors. Results demonstrated a mean endotoxin level in blood of 0.02 to 1.57 pg/ml. The amount of Escherichia coli endotoxin added to human plasma samples can be quantitated by both nephelometry and turbidimetry. Endotoxin-spiked samples were shown to be significantly different from unspiked samples. When plasma samples were collected from 45 patients hospitalized at three centers, a strong association was demonstrated between a positive Limulus amoebocyte lysate assay and a septic condition. Sensitivity, specificity, and false-positive and false-negative rates for the Limulus amoebocyte lysate assay as a diagnostic test for gram-negative bacteremia were estimated.     

Concordance of endotoxemia with gram-negative bacteremia in patients with gram-negative sepsis: a meta-analysis.

The Limulus amebocyte lysate (LAL) assay is a sensitive method for detecting endotoxin. Using gram-negative (GN) bacteremia as the basis for comparison, concordance with endotoxemia in 45 studies could be expressed as an odds ratio. Calculation of summary odds ratios by the Mantel-Haenszel-Peto method indicated that the concordance of the results was no higher by the chromogenic LAL assay than by the gelation version, and the sensitivity was improved by only 11% (62 versus 51%). Endotoxemia was detected in 77 (68%) of 114 patients with bacteremia caused by an organism that was not a member of the family Enterobacteriaceae, whereas endotoxemia was detected in only 120 (45%) of 269 patients with bacteremia caused by a member of the family Enterobacteriaceae or an anaerobe (P < 0.001). This difference was also apparent for patients with GN bacteremia for whom a fatal outcome had been recorded. The prevalence of GN bacteremia in the tested population and the type of etiological agent are critical and previously unrecognized variables which affect the interpretation of the LAL test in patients with suspected sepsis.     

Thymocyte selection: not by TCR alone

Summary: Spreading of the immune response is a common theme in organ-specific and systemic autoimmune diseases. We evaluated whether some of the mixed antinuclear antibody patterns characteristic of systemic autoimmunity might be the result of determinant spreading from a single initiating event. Immunisation of healthy mice with individual protein components of the La/Ro ribonudeoprotein (RNP) targeted in systemic lupus erythematosus and primary Sjögren's syndrome induced autoanti-bodies recognising Ro60 (SS-A), Ro52 (SS-A) and La (SS-B) and in some cases the molecular chaperones calreticulin and Grp78. The endogenous antigen(s) driving determinant spreading might be derived from physiological apoptosis which could explain the involvement of some chaperone proteins in the autoimmune response. Diversified anti-La/Ro antibody responses were initiated by challenge with a single subdominant T epitope of La even though some self epitopes of La were efficiently tolerised. The pattern of autoantibody responses in primary Sjögren's syndrome was strongly influenced by HLA class II phenotype which we speculate controls activation of T cells recognising defined peptides from the La/Ro RNP In t his way, HLA class II alleles may be critical in influencing initiation and spreading of systemic autoimmune reactions. Molecular mimicry of such determinants by exogenous agents might readily initiate spreading of an autoimmune response in genetically susceptible hosts.     

Interleukin-6 induced at mucosal surfaces by gram-negative bacterial infection.

Interleukin-6 (IL-6) was produced in response to mucosal and systemic infection of mice with gram-negative bacteria. The IL-6 response was controlled by the lipopolysaccharide gene, Lps; in C3H/HeN mice (Lpsn/Lpsn), the urinary IL-6 levels increased within 30 min after challenge with Escherichia coli, but no response occurred in C3H/HeJ mice (Lpsd/Lpsd). In lipopolysaccharide-responder mice, the levels of local and systemic IL-6 were related to the degree of infection. The urinary response dominated after intravesical challenge, and the serum response dominated after intraperitoneal challenge. The results demonstrate that IL-6 is activated as part of lipopolysaccharide-induced mucosal and systemic responses to gram-negative bacterial infections.     

Rapid diagnosis of gram-negative bacterial meningitis by the Limulus endotoxin assay.

The Limulus amoebocyte lysate endotoxin assay was evaluated as a method for rapid diagnosis of acute bacterial meningitis in a series of 305 patients. The results of Limulus assays on cerebrospinal fluid (CSF) samples from these patients were compared with the results for each patient of routine bacterial cultures and Gram stains. Positive Limulus tests were obtained on initial CSF specimens from 84% of patients with culture-proven bacterial meningitis, including all patients with meningitis due to gram-negative organisms. Initial Gram-stained smears revealed the presence of organisms in 68% of the patients. One patient with pneumococcal meningitis had a weakly positive Limulus assay, whereas patients with meningitis due to other gram-positive organisms, those with aseptic meningitis, or patients without meningitis had negative CSF Limulus tests. The Limulus assay also demonstrated the persistence of endotoxin in the CSF of certain patients during antibiotic therapy, especially patients with Haemophilus influenzae meningitis. The Limulus test proved to be a rapid, reliable indicator of the presence of gram-negative organisms in the CSF of patients suspected of acute bacterial meningitis.     

Activation of glomerular mesangial cells by gram-negative bacterial cell wall components.

D341 Med is a new continuous cell line and transplantable xenograft derived from a cerebellar medulloblastoma. This line grew in vitro in suspension culture with spontaneous macroscopic spheroid formation and demonstrated 20-fold amplification of c-myc. Cultured D341 Med cells injected subcutaneously into athymic mice grew as markedly cellular, highly invasive undifferentiated neoplasms. Intracranial tumors grew as markedly cellular mitotically active neoplasms largely located within the subarachnoid space or lining the ventricular system. Immunocytochemical analysis of the cell line and SQ tumors revealed the high (NFP-H) and middle (NFP-M) molecular weight (Mr) neurofilament proteins (NFPs). Immunoblots demonstrated the presence of molecular species that co-migrated with authentic human NFP-H and NFP-M. This cell line and transplantable xenograft may allow, in conjunction with the authors' other models of human medulloblastoma, analysis of the heterogeneous biologic properties and therapeutic sensitivity of this tumor.     

Rapid detection of gram-negative bacterial peritonitis by the Limulus amoebocyte lysate assay.

The chromogenic Limulus amoebocyte lysate test effectively detected 66 (100%) culture-proven gram-negative peritonitis cases among 185 continuous ambulatory peritoneal dialysis patients with clinical evidence of infectious peritonitis.     

Autotransporter and two-partner secretion: delivery of large-size virulence factors by gram-negative bacterial pathogens

A number of protein secretion mechanisms have been identified in gram-negative pathogens. Many of these secretion systems are dependent upon the Sec translocase for protein export from the cytoplasm into the periplasm and then utilize other mechanisms for transport from the periplasm through the outer membrane. In this article, we review secretion similarities between autotransporter and two-partner secretion systems, and we report similarities between the autotransporter secretion mechanism with that of intimin/invasins. Considering that many secreted proteins are virulence factors, a better understanding of their secretion mechanisms will aid in the development of disease treatments and new bacterial vaccines.     

The T-cell repertoire is not dictated by self antigens alone.

The influence of genetic and environmental factors on the functional cytotoxic T cell (CTL) allorepertoire was studied by comparing the CTL precursor frequencies against the same HLA alloantigens in 116 sibling pairs. A significantly different precursor frequency was found in 68%, 61% and 59% of siblings sharing 0, 1 and 2 HLA haplotypes, respectively. These data show that, although HLA is important in determining the T-cell repertoire, this is hardly reflected in the allorepertoire. Even 50% of the monozygotic twin pairs showed a significant disparity in their CTL allorepertoire, indicating that environmental factors play a role as well. The non-inherited maternal HLA antigens could be identified as one of the environmental factors shaping the CTL allorepertoire.     

Proteolytic activity and fatal gram-negative sepsis in burned mice: effect of exogenous proteinase inhibition.

Circulating proteolytic activity (PA) increases following burn or surgical trauma. Challenging traumatized mice with the yeast Candida albicans further increases PA. Once a PA threshold has been passed, mortality increases as PA increases. The purposes of this study were to determine (i) if gram-negative bacterial challenge affects circulating PA and mortality as Candida challenge does and (ii) if proteinase inhibitor treatment with aprotinin, antithrombin III, and alpha 1-proteinase inhibitor decreases circulating PA and increases the survival of burned mice infected with a bacterium. For all bacteria tested (Proteus mirabilis, Pseudomonas aeruginosa, and Klebsiella pneumoniae), burn plus challenge significantly elevated PA and mortality above levels in mice that were only burned or only challenged. Quantitative culture counts indicated that the mice died of sepsis. Proteinase inhibitor treatment of mice burned and challenged with K. pneumoniae significantly decreased circulating PA, decreased the hepatic microbial load, and increased survival. Hence, in traumatized mice challenged with either C. albicans or gram-negative bacteria, a relationship exists between proteolytic load and subsequent septic death. Parallels between these animal studies and human studies are discussed.     

Patterns of multiple resistance to antibiotics in gram-negative bacteria demonstrated by factor analysis

Principal component analysis was used to demonstrate the main associations between patterns of resistance to antibiotic drugs in 670 gram-negative bacteria consecutively isolated from blood cultures over a period of two years. Six factors were derived, which accounted for 84 % of the total variance of the original matrix. Each factor represented an association between resistance to certain antibiotics as follows: factor 1: aztreonam, third generation cephalosporins and aminoglycosides; factor 2: first and second generation cephalosporins; factor 3: tetracycline and chloramphenicol; factor 4: ampicillin and ureidopenicillins; factor 5: trimethoprim/sulfamethoxazole; factor 6: fluoroquinolones. On two-way analysis of variance the difference in the factor scores was significant between bacteria for all factors except factor 5. The difference in factor scores between community and hospital acquired strains was significant only for factors 1, 2 and 6. Only the score of factor 6 showed a clear trend to increase with time during the two-year study period. Patients who were treated with antibiotics prior to bacteremia had higher scores for all factors, the difference being most marked in patients treated with fluoroquinolones. Factor analysis can be used to describe phenotypic associations between resistance to antibiotics, and the factor score used to compare groups of isolates and to demonstrate temporal and other trends.     

Topical antibiotics for acute bacterial conjunctivitis: a systematic review.

There has been uncertainty about whether antibiotic therapy confers significant benefit in the treatment of acute bacterial conjunctivitis. This study aimed to assess the efficacy of antibiotic therapy in the management of acute bacterial conjunctivitis. Using standard Cochrane search methods, we identified double-blind randomised controlled trials in which any form of antibiotic treatment (topical, systemic or combination) had been compared with placebo in the management of acute bacterial conjunctivitis. Data extraction and analysis followed a pre-defined protocol. Meta-analysis was performed to obtain summary measures of relative risk. Six published trials were identified, of which three fulfilled the eligibility criteria for inclusion in this review. The trials were heterogeneous in terms of their inclusion and exclusion criteria, the nature of the intervention, and the outcome measures assessed. Meta-analysis indicates that acute bacterial conjunctivitis is frequently a self-limiting condition, as clinical remission occurred by days 2 to 5 in 64% (95% confidence interval (CI) = 57-71) of those treated with placebo. Treatment with antibiotics was, however, associated with significantly better rates of clinical remission (days 2 to 5: relative risk (RR) = 1.31, 95% CI = 1.11-1.55), with a suggestion that this benefit was maintained for late clinical remission (days 6 to 10: RR = 1.27, 95% CI = 1.00-1.61). Acute bacterial conjunctivitis is frequently a self-limiting condition but the use of antibiotics is associated with significantly improved rates of early clinical remission, and early and late microbiological remission. Since trials to date have been conducted in selected specialist care patient populations, generalisation of these results to a primary care-based population should be undertaken with a degree of caution.     

Inhibition of bacterial adhesion by subinhibitory concentrations of antibiotics

Background: Urinary Tract Infections (UTIs) due to Escherichia coli is one of the most common diseases encountered in clinical practice. Most common recognised pathogenic factor in E.coli is adhesion. There is accumulating evidence that through subinhibitory concentrations (sub – MICs) of many antibiotics do not kill bacteria, they are able to interfere with some important aspects of bacterial cell function. Materials and Methods: A study was conducted to investigate the effect of sub MICs (1/2–1/8 MIC) of ciprofloxacin, ceftazidime, gentamicin, ampicillin and co - trimoxazole on E. coli adhesiveness to human vaginal epithelial cells using three strains ATCC 25922, MTCC 729 and U 105. Results: The 1/2 MIC of all the antibiotics tested produced the greatest inhibition of bacterial adhesion. Morphological changes were observed with ciprofloxacin, ceftazidime and ampicillin at 1/2 MIC and to a lesser extent at 1/4 and 1/8 MIC. Co-trimoxazole caused the greatest suppression of adhesion at 1/2 MIC of E. coli strain MTCC 729 when compared with the controls, followed by ceftazidime. Conclusion: These results suggest that co - trimoxazole is the most effective antibiotic in the treatment of urinary tract infections caused by uropathogenic E. coli.     

Use of imipenem-cilastatin in neonatal septicemias caused by gram-negative bacilli multiresistant to beta-lactam antibiotics

Seven neonates with septicemia due to Gram negative bacteria resistant to beta-lactam received imipenem-cilastatin therapy. Bacteria isolated were Enterobacter cloacae [3], Enterobacter aerogenes [1], Klebsiella pneumoniae [1], Serratia marcescens [1], Pseudomonas fluorescens [1]. The MICs of imipenem were lower 1 microgram/ml. In 3 children septicemia occurred during previous antimicrobial chemotherapy. 3 IV 60 mg/kg doses of imipenem with amikacin (15 mg kg/d) were administered every day. For five children blood cultures were negative after 48 hours of treatment. E. aerogenes septicemia required pefloxacin because blood cultures remained positive (d5) despite an increased dosage (90 mg/kg/d). All children were cured and imipenem-cilastatin was not responsible for any complication. Those results demonstrate the efficacy of imipenem in the treatment of septicemia in newborns due to multiresistant Gram negative bacteria.