On-line determination of serum bactericidal activity using recombinant luminescent bacteria

Intensity of luminescence quenching in recombinant strains of Escherichia coli with cloned lux-operones by human blood serum is directly proportional to the degree of bactericidal effect assessed by nephelometric and bacteriological methods. This correlation was most characteristic of E. coli with luminescence genes from Photobacterium leiognathi, which substantiates its use in the development of the kinetic bioluminescent method to determine of serum bactericidal activity. The possibility of using this method for evaluation of activity of classic and alternative pathways of compliment activation was demonstrated by using zymosan or EGTA-Mg²⁺-treated sera and C1-C5-deficient sera. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 8, pp. 200–204, August, 2006     

Determination of serum bactericidal activity with the aid of luminous bacteria

Nonmarine luminous bacteria belonging to the genus Vibrio cholerae were extremely sensitive to the bactericidal activity of human serum. Luminous bacteria incubated in a medium containing serum showed a decrease in their in vivo luminescence that was directly proportional to the decrease in the viable count and was a function of the serum concentration. Both immunoglobulins and the complement system were required to exert the serum bactericidal activity. Serum lacking immunoglobulins or certain complement components, especially C3, did not affect the luminescence. The bactericidal effect of the serum on luminous bacteria was diminished by the presence of lipopolysaccharide or by pretreatment of the serum with different species of killed bacteria. As found in other systems, the bacteriolytic activity of serum was only augmented by lysozyme, but was not lysozyme dependent; although the luminous bacteria were converted into spheroplasts in serum containing 0.5 M sucrose, their in vivo luminescence was almost not affected. This system could easily distinguish between the C classical pathway and the properdin pathway. Ethylene glycol-bis (beta-aminoethyl ether)-N,N'-tetraacetic acid, which inhibits only the classical complement pathway, did not inhibit the decrease in luminescence as did EDTA. Thus, it was possible to distinguish between deficiencies in complement components participating in both pathways and complement components that were involved only in the classical pathway. This system could also be used as a substitute to the hemolytic system in complement fixation tests.     

Effects of chitosan on the biological properties of gram-negative bacteria endotoxins

The destructive action of Gram-negative bacteria lipopolysaccharides and their complexes with chitosan, a natural polycation, was comparedin vivo andin vitro. Lipopolysaccharides in complex with chitosan modified the biological properties of endotoxin, among other things, reducing its toxic and aggregation effects. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 8, pp. 169–172, August, 1995 Presented by V. I. Pokrovskii, Member of the Russian Academy of Medical Sciences     

Properties of equine anti-lipopolysaccharide hyperimmune plasma: binding to lipopolysaccharide and bactericidal activity against gram-negative bacteria

Anti-lipopolysaccharide equine hyperimmune plasma (anti-LPS), which has been used successfully to treat LPS (endotoxin)-mediated disorders, has been further characterised. IgG present in anti-LPS had the highest affinity for LPS prepared from Salmonella typhimurium, S. typhi, S. abortus equi and Shigella flexneri and intermediate affinity for Escherichia coli O55:B5, E. coli O127:B8 and S. enteritidis. Anti-LPS destroyed by means of complement activation a wide range of gram-negative bacteria, including various species and strains of Klebsiella, Enterobacter, E. coli, Sh. flexneri, Providencia, Salmonella and Pseudomonas. Control plasmas or saline had little or no effect. Maximum killing occurred within seconds to minutes. Electronmicroscopy showed that anti-LPS treatment of K. pneumoniae caused extensive cell wall and cytoplastic membrane disruption, followed by the appearance of spheroplasts and cell ghosts. Antibodies were required in 100,000-fold excess to inhibit the limulus amoebocyte lysate reaction with LPS from E. coli. Anti-LPS thus contains IgG that binds to a wide range of LPS, and can destroy a wide range of gram-negative bacteria by means of complement activation.     

Pharmacodynamic effects of meropenem on gram-negative bacteria

The in vitro initial killing and post-antibiotic effect (PAE) of meropenem on five gramnegative reference strains were evaluated by bioluminescence assay of bacterial adenosine triphosphate (ATP) and viable count. Morphology studies were performed in parallel. Meropenem showed concentration-dependent long (2–5 h) PAEs onEnterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa andSerratia marcescens when assayed by bioluminescence and induced spheroplasts at almost all concentrations. The bioluminescence PAEs reached a maximum response at 4 × MIC. These PAEs of meropenem onEscherichia coli, Klebsiella pneumoniae andSerratia marcescens were longer than corresponding PAEs of imipenem shown in previous studies. The higher affinity of meropenem than imipenem for PBP 3 might explain the longer PAEs obtained with meropenem. However, there was only a very short PAE, no PAE or even a negative PAE when viable count was used as the initial value for the PAE calculation. A strong initial decrease in viability but a less pronounced change in intracellular ATP was registered. Since this initial change in cell numbers is the initial value for the PAE calculation, the length of PAE was highly method dependent. In summary, a strong initial killing and no PAE were shown using viable count as the initial value for the PAE calculation, but a weak initial killing and long PAEs were shown when bioluminescence was used throughout the experiments.     

Emergence of resistance in gram-negative bacteria during therapy with expanded-spectrum cephalosporins

To assess the clinical importance of emergence of beta-lactam resistance caused by stable derepression of chromosomal beta-lactamases, sequential cultures from patients treated with expanded-spectrum cephalosporins were monitored for the persistence of bacteria possessing these enzymes. Antibiotic susceptibilities and beta-lactamase production before and after cefoxitin induction were determined in sequential isolates of individual bacterial strains. Of 49 strains isolated from 44 patients, 25 strains (51%) were eradicated by cephalosporin therapy, 17 strains (35%) persisted with unchanged susceptibility in sequential cultures, and 7 strains (14%) from 7 patients developed multiple beta-lactam resistance during cephalosporin therapy. In 6 of the 7 strains, resistance was associated with stable derepression of beta-lactamases. In the patient group whose strains developed resistance, subsequent use of non-beta-lactam antibiotics was more frequent and mortality was higher.     

In vitro sensitivity of oral, gram-negative, facultative bacteria to the bactericidal activity of human neutrophil defensins.

Neutrophils play a major role in defending the periodontium against infection by oral, gram-negative, facultative bacteria, such as Actinobacillus actinomycetemcomitans, Eikenella corrodens, and Capnocytophaga spp. We examined the sensitivity of these bacteria to a mixture of low-molecular-weight peptides and highly purified individual defensin peptides (HNP-1, HNP-2, and HNP-3) isolated from human neutrophils. Whereas the Capnocytophaga spp. strains were killed significantly by the mixed human neutrophil peptides, the A. actinomycetemcomitans and E. corrodens strains were resistant. Killing was attributable to the defensins. The bactericidal activities of purified defensins HNP-1 and HNP-2 were equal, and both of these activities were greater than HNP-3 activity against strains of Capnocytophaga sputigena and Capnocytophaga gingivalis. The strain of Capnocytophaga ochracea was more sensitive to defensin-mediated bactericidal activity than either C. sputigena or C. gingivalis was. The three human defensins were equipotent in killing C. ochracea. C. ochracea was killed under aerobic and anaerobic conditions and over a broad pH range. Killing was most effective under hypotonic conditions but also occurred at physiologic salt concentrations. We concluded that Capnocytophaga spp. are sensitive to oxygen-independent killing by human defensins. Additional studies will be required to identify other components that may equip human neutrophils to kill A. actinomycetemcomitans, E. corrodens, and other oral gram-negative bacteria.     

Role of rabbit lysozyme in in vitro serum and plasma serum bactericidal reactions against Bacillus subtilis.

The antibacterial activity of purified rabbit lysozyme was kinetically investigated at concentrations comparable to those in normal rabbit serum and plasma serum. The bactericidal capability, lysozyme content, and electrophoretic composition of "purified beta-lysin," fractionated from normal rabbit serum, were also examined. In contrast to the extensive antibacterial activity of dilute normal rabbit serum observed in vitro, rabbit lysozyme was only weakly bactericidal for Bacillus subtilis. Inhibition of lysozyme enzymatic and bactericidal activities in normal rabbit serum by antilysozyme immunoglobulin G slightly reduced the initial rate of killing. The addition of neutralizing antibody or histamine (another lysozyme inhibitor) to partially purified bactericidal serum fractions had no effect on killing kinetics. Increasing the ionic strength of reaction mixtures containing normal serum or partially purified bactericidal fractions to levels which completely inhibited lysozyme activity resulted in stimulation of their respective killing kinetics. The addition of inhibitors to normal rabbit plasma serum completely eliminated its bactericidal activity. With regard to the killing of B. subtilis by rabbit and human blood fractions, these analyses clearly demonstrated that (i) although lysozyme is not a significant antibacterial component of normal rabbit serum, it represents the principal factor in normal rabbit plasma serum; (ii) different primary bactericidal mechanisms which are not detectable by singlepoint analyses operate in the sera of different species; and (iii) purified beta-lysin isolated from normal rabbit serum by the classical procedure is a heterogenous mixture of components.     

Effects of anti-inflammatory agents on mucosal inflammation induced by infection with gram-negative bacteria.

Gram-negative bacterial infections of the urinary tract elicit a mucosal inflammatory response. Interleukin-6 is secreted into the urine, and polymorphonuclear leukocytes (PMNL) are recruited. In the present study we examined the effect of anti-inflammatory agents on these parameters and on bacterial clearance from the kidneys. Dexamethasone reduced interleukin-6 secretion, the PMNL response, and bacterial clearance. Diclofenac abolished the urinary interleukin-6 response but reduced the PMNL response and bacterial clearance only at the highest concentrations. Indomethacin drastically decreased bacterial clearance without the corresponding effect on interleukin-6 production or the PMNL response. The results demonstrate that the inhibition of inflammation impairs bacterial clearance from the kidneys. This is, however, not a direct function of inhibited interleukin-6 production or PMNL recruitment.     

Immunosuppressive effects of pathogenic gram-negative bacteria

The review deals with the Immunosuppressive effects of virulent gram-negative bacteria (Salmonella, Shigela, Pseudomonas aeruginosa), with the importance of these effects for the bacteria to survive in the infected body. The above bacteria affect the immunity system in a different way, yet have common features. They are characterized by the occurrence of endotoxin shock, by the suppression of the phagocytic system and cell-mediated immunity. A significant role in suppressing a cellular immune response is played by the lipopolysaccharide of virulent bacteria that greatly differs from that of nonvirulent strains. The immunosuppressive activity of the bacteria and their lipopolysaccharide is closely related to their virulent properties.     

Differential toxicity of inhaled gram-negative bacteria

The toxicity by inhalation of various gram-negative bacteria, isolated from settings associated with inhalation disease, was studied by a variety of means. These microorganisms were not equally toxic. Citrobacter freundii aerosol challenges of rabbits provoked significant (up to fivefold) increases in plasma haptoglobin 24 to 48 h after inhalation. Other strains tested failed to provoke such statistically consistent increases. Measurements of C-reactive protein in these same animals did not lead to as reliable results, due to the variability of the responses. Mice responded differently to inhalation in that haptoglobin responses were either unaffected or depressed. When a strain of mice was used that exhibits more severe inflammatory responses to endotoxin (C3H/HeJ), C. freundii and Escherichia coli aerosols provoked significant haptoglobin increases. Free lung cell analyses demonstrated that the macrophage and neutrophil responses differed depending on the strain of bacteria used. Again, C. freundii induced the greatest responses. When murine B lymphocytes were stimulated with lipopolysaccharide preparations from different gram-negative bacteria, distinctly different dose-response curves were obtained. The types of responses obtained indicate that (i) brief inhalation of bacterial aerosols previously thought to be innocuous may lead to pulmonary inflammation, and (ii) that these bacteria differ in their toxicity, with C. freundii being the most toxic organism of the five studied.     

Receptors for human plasminogen on gram-negative bacteria.

A total of 188 strains representing 11 species of gram-negative bacteria were examined for the ability to interact with human plasminogen. Highly purified human plasminogen was labeled with 125I, and its uptake by different bacterial strains was measured. All 14 strains of Haemophilus influenzae and all 13 strains of Branhamella catarrhalis tested were positive with respect to plasminogen uptake. Also, eight species belonging to the family Enterobacteriaceae were tested, and of those, Proteus mirabilis demonstrated the most substantial uptake, with 28 of 39 strains taking up more than 10% of the plasminogen. Ten strains of Pseudomonas aeruginosa were also tested, of which seven showed uptake values higher than 10%. With H. influenzae and B. catarrhalis strains, Scatchard analysis indicated a two-phase receptor interaction, one more-avid receptor with a Kd of 6 to 8 nM and 2,000 to 2,500 sites per bacterium and a second receptor with a Kd of 50 to 80 nM and 9,000 sites per bacterium. With Pseudomonas aeruginosa strains, a single receptor interaction was detected with a Kd of 60 nM and the number of sites was estimated as 8,000 per bacterium. Scatchard analysis with strains of P. mirabilis indicated binding of a less-specific nature. However, plasminogen uptake by this species could be reduced by 50% by the addition of 2 mM unlabeled plasminogen. This estimate of Kd, as well as uptake studies with plasminogen fragments, suggests different properties of this receptor. With all receptor types, the addition of plasmin-aprotinin complex inhibited plasminogen uptake, which demonstrates that both forms of the molecule react with the same receptors. Plasminogen uptake could be eliminated by the addition of lysine or epsilon-aminocaproic acid, which suggests that the lysine-binding sites of the plasminogen molecule are involved in the receptor-ligand interaction.     

Role of lipopolysaccharide in induction of Ia expression during infection with gram-negative bacteria

Lipopolysaccharide (LPS), a major component of the outer membrane of gram-negative bacteria, is known to be a potent modulator of many host immune functions, including the expression of products of the class II major histocompatibility locus (Ia molecules) by macrophages. LPS-mediated Ia induction is controlled by the lps gene. We sought to determine the role of LPS in the induction of Ia expression during infection with gram-negative bacteria. To address this question, we tested a simple prediction: if LPS is the primary determinant of Ia induction during gram-negative infection, then the Ia response to intraperitoneal injection of these organisms should be under the control of the lps gene. We found that while both LPS-responder and LPS-low-responder mice showed strong Ia responses to injection of either a gram-positive bacterium (Listeria monocytogenes) or concanavalin A, only the LPS-responder mice responded strongly to gram-negative organisms or to LPS alone. We interpret these results as strong evidence for the role of LPS as the primary determinant of Ia induction by gram-negative bacteria.     

Effects of antibiotic resistance plasmids on the bactericidal activity of normal rabbit serum.

The susceptibility of human salivary proteins to degradation by Candida albicans was studied. The organism was cultivated in either whole-salivary supernatant or parotid fluid, both of which were supplemented with glucose (0.1%). The culture pH's were at, or above, neutrality. After growth, the culture supernatant solutions were examined by polyacrylamide gel electrophoresis for alterations in their profiles of salivary proteins. No evidence of proteolysis of whole-saliva or parotid fluid proteins was found. Salivary proteins, however, are susceptible to degradation by preparations of C. albicans protease. Candida protease was incubated with parotid fluid adjusted to several pH values. After incubation the reaction mixtures were subjected to polyacrylamide gel electrophoresis. Extensive degradation of parotid proteins was found at pH 4, very slight proteolysis at pH 5, and no degradation at pH 6 or 7. No selectivity in proteolysis of the several parotid proteins was noted. These results indicate that C. albicans protease is strictly dependent upon low (ca. 4) pH for activity on salivary proteins. Furthermore, it is suggested that due to the pH requirements of the enzyme, it is unlikely to be of major significance to the pathogenesis of Candida-induced oral inflammatory lesions.     

Influence of extracellular bactericidal agents on bacteria within macrophages

We employed gentamicin-sensitive and -resistant derivatives of Escherichia coli in a macrophage phagocytosis assay that compared lambda bacteriophage and gentamicin as extracellular bactericidal agents. Colony counts and direct microscopic examination of phagocytized E. coli supported the conclusion that gentamicin entered macrophages, even at low concentrations, and contributed to their bactericidal activity. Also, two E. coli strains differing in the ability to express the adhesin of type 1 pili (FimH) were distinguishably different in intracellular survival when lambda was used as the extracellular killing agent but were indistinguishable when gentamicin was employed.     

Electrophoretic behavior of beta-lactamases in gram-negative bacteria

The ongoing discoveries of new beta-lactamases, mainly penicillinases, in Gram-negative bacteria has emphasized the problem of their precise identification, and thus their phylogeny. Crude extracts, prepared by sonication, of 14 plasmid beta-lactamases, types TEM, carbenicillinases (CARB or PSE) and oxacillinases (OXA) were analysed by a simple, rapid (3.5 to 4 hours) method of electrophoresis on polyacrylamide (7%) agarose (1.4%) gels, using Tris-glycine buffer at pH 8.7. Preliminary serial dilutions were made to determine enzymic activity levels. Enzymes were then characterized by their relative electrophoretic mobilities. These mobilities had coefficients of variability between 2% and 10%, ranged from 5 to 61, and were correlated with their isoelectric points (pI). Thus, the lower the pI is, the greater the mobility is. Despite the high resolving power of the polyacrylamide-agarose gel system, enzymes with similar pI's and of similar types (PSE-1 and CARB-3, or OXA-1 and OXA-4) or different types (SHV-1 and OXA-6) could not be distinguished on the basis of their mobilities. However, this technique provides for rapid and easy identification of the major penicillinases in Gram-negative bacteria. A combination of polyacrylamide-agarose gel electrophoresis and pH gradient electrophoresis (titration curve) could provide a powerful approach to the study of the molecular structure of these enzymes.     

Comparison of the effects of different lipopolysaccharides on the serum bactericidal reactions of two strains of Escherichia coli.

The serum killing of Escherichia coli ML308 225 and PB94 was inhibited by lipopolysaccharide extracted from either organism, but not by lipopolysaccharide from three pathogenic Enterobacteriaceae.