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     

Inhibition of serum bactericidal activity by bilirubin

The results presented show that bilirubin inhibited the bactericidal activity of human serum. Addition of serum albumin (which tightly binds bilirubin) to the bactericidal assay prevented the inhibition mediated by bilirubin. These findings suggest that the decreased bactericidal activity of sera from neonates with hyperbilirubinemia may be due to the inhibition of the bactericidal activity by unconjugated bilirubin.     

Technical aspects and clinical correlations of the serum bactericidal test

A review of the studies using 50 % human serum as a diluent for the serum bactericidal test has shown correlations with patient outcome. Human serum used as diluent of the patient's serum appears to be essential because of high protein binding of some antibiotics. An inoculum of 10 ⁵–10⁶ bacteria/ml and a bactericidal criteria of 99.9 % killing are technical aspects that have gained popularity. Careful timing of serum collection for the assay is important. Neither the macrotube nor microtiter techniques are entirely satisfactory. The latter method, however, has the advantage of being more reproducible than the macrotube method, less cumbersome and requiring less serum. Preliminary guidelines for performing and interpreting the test are provided. Future research should be directed toward making the microtiter technique more sensitive for identifying antibiotic tolerance, developing effective methods to eliminate the need for human serum as a diluent and obtaining more clinical correlations.     

Evaluation of post-antibiotic effects of antipseudomonal antibiotics using an automated system

Different methods have been described for determination of the post-antibiotic effects (PAEs) of antibiotics, from the conventional methods to the automatic measurement of bacterial regrowth. The PAEs of ciprofloxacin, ofloxacin and perfloxacin, as compared with those of amikacin, netilmicin and ceftazidime, have now been investigated for three clinical isolates of Pseudomonas species, using automated measurement of the growth with an Anthos microplate reader. It was found that, besides the well-known PAEs of aminoglycosides, quinolone antibiotics also exhibit drug- and concentration-dependent PAEs against clinical isolates of Pseudomonas when used in concentrations of 1/2x, 1 x or 5 x MIC. Of the three quinolones tested, pefloxacin showed the greatest PAE, independently of its MICs against the different strains. Ofloxacin had no or only an insignificant PAE, while ciprofloxacin had a marked PAE for all strains, including the reference strains.     

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.     

Value of the serum bactericidal test in management of patients with bacterial endocarditis

A review is given of the available clinical data on the prognostic value of the serum bactericidal test in the treatment of patients with bacterial endocarditis. It is concluded that the test, even when performed in a standardized manner, does not provide useful information for the majority of patients with bacterial endocarditis. Until further clinical data are available, routine performance of the test in patients with bacterial endocarditis is not recommended.     

Evaluation of the bactericidal effect of the membrane attack complex of serum complement

The bactericidal activity of serum complement and particularly of the membrane attack complex (MAC-C5b-C9) was studied on E. coli C600 with a simple functional test. The test evaluates the in vitro kinetics of the bactericidal effect and the subsequent counting of surviving germs. Homozygous deficiency of a particular membrane attack complex protein was easily detected from a total loss of bactericidal activity. These results were confirmed on Neisseriae meningitidis A and C, but in this case a more complex protocol was required. Deficits of proteins of the membrane attack complex sequence of complement are often found in sera of patients suffering from recurrent Neisseriae infections. This simple test, adapted to family studies, appears, thus, as a valuable basis for a detection of relatives at risk.     

Measurement of functional anti-meningococcal serogroup a activity using strain 3125 as the target strain for serum bactericidal assay

Functional anti-N. meningitidis serogroup A (MenA) activity in human serum is detected by serum bactericidal assay (SBA), using either rabbit (rSBA) or human (hSBA) complement, with F8238 as the recommended MenA SBA target strain. However, the F8238 strain may not be optimal for this purpose because, as we show here, it expresses the L11 immunotype, whereas most MenA invasive strains express the L(3,7)9 or L10 immunotype. Moreover, SBA results may be strain dependent, because immunotypes differ in their sensitivity to complement, emphasizing the need to choose the most appropriate strain. Sera from random subsets of infants, toddlers, children, and adolescents in clinical trials of MenA conjugate vaccines were tested by rSBA using strains 3125 (L10) and F8238 (L11). In unvaccinated subjects from all age groups, the percentages of seropositive samples (rSBA-MenA titer, ≥1:8) was lower using strain 3125 than using strain F8238. However, in toddlers and adolescents immunized with a conjugate MenA vaccine, the percentages of seropositive samples generally were similar using either strain in the rSBA. In two studies, sera also were tested with hSBA. Using hSBA, the differences in the percentages of seroprotective samples (hSBA-MenA titer, ≥1:4) between strains 3125 and F8238 was less apparent, and in contrast with rSBA, the percentage of seroprotective samples from unvaccinated subjects was slightly higher using strain 3125 than using strain F8238. In adults vaccinated with plain MenA polysaccharide, the percentage of seroprotective samples was higher using strain 3125 than with strain F8238, and the vaccine response rates using strain 3125 were better aligned with the demonstrated efficacy of MenA vaccination. In conclusion, SBA results obtained using the MenA L10 3125 strain better reflected vaccine-induced immunity.     

Antimicrobial synergy testing based on antibiotic levels, minimal bactericidal concentration, and serum bactericidal activity

The ratio between the concentrations of different antimicrobial agents varies widely during therapy and often bears no resemblance to the ratios assessed by in vitro methods to evaluate the effectiveness of multiple antimicrobial therapy. The authors examined an alternative method using patient serum that reflects the actual antibiotic levels achieved in the patient. Previous investigators have shown that the ratio of the concentration of free drug (drug-f) to the minimal bactericidal concentration (MBC) in broth approximates the serum bactericidal titer (SBT) when pooled normal human serum is used as the diluent. Theoretically, when two antimicrobial agents are administered, the SBT should be equivalent to (drug-f A/MBC drug A) + (drug-f B/MBC drug B). SBT and drug level determinations were performed on peak and trough serum specimens from ten patients with endocarditis or osteomyelitis who were receiving multiple antimicrobial therapy. The serum dilution synergy method predicted additive interactions twice as often as the checkerboard and kill curve, and predicted synergy less frequently than the kill curve. The checkerboard predicted antagonism four times more often than the other methods and provided equivocal results in four of ten cases. The suggested method may offer an alternative procedure to assess antimicrobial interactions, which is based on antibiotic levels actually achieved in vivo instead of the arbitrary concentrations often used in in vitro tests.     

The effect of cortisone on the bactericidal activity of the serum of infected animals

Summary Serum bacterial activity and that of the inflammatory exudate is decreased toB. pneumoniae in infection of white rats and mice with this culture. Bactericidal activity of rats and mice's serum is also reduced after the administration of cortisone. However, in case of preliminary cortisone administration to infected animals the bactericidal activity of the serum and of the exudate remains high or even rises. The change of the serum bactericidal activity is not of decisive significance in the mechanism of decreased resistance of mice toB. pneumoniae under the effect of cortisone.Presented by Active Member of the AMN SSSR V. V. Zakusov     

Evaluation of serum bactericidal antibody assays for Haemophilus influenzae serotype a

Haemophilus influenzae type a (Hia) is an important pathogen for some American Indian, Alaskan native, and Northern Canada aboriginal populations. Assays to measure serum bactericidal activity (SBA) to Hia have not been developed or validated. Here, we describe two methods for the measurement of SBA: SBA with a viability endpoint (CFU counts) and SBA with a fluorometric endpoint using alamarBlue as the metabolic indicator. Both SBA assays measure Hia-specific functional antibody and correlate with anti-Hia IgG enzyme-linked immunosorbent assay (ELISA) concentration of naturally acquired antibodies.     

Contribution of the complement Membrane Attack Complex to the bactericidal activity of human serum

Direct killing of Gram-negative bacteria by serum is usually attributed to the Membrane Attack Complex (MAC) that is assembled upon activation of the complement system. In serum bactericidal assays, the activity of the MAC is usually blocked by a relatively unspecific method in which certain heat-labile complement components are inactivated at 56 °C. The goal of this study was to re-evaluate MAC-driven lysis towards various Gram-negative bacteria. Instead of using heat-treatment, we included the highly specific C5 cleavage inhibitor OmCI to specifically block the formation of the MAC. Using a C5 conversion analysis tool, we monitored the efficacy of the inhibitor during the incubations. Our findings indicate that ‘serum-sensitive’ bacteria are not necessarily killed by the MAC. Other heat-labile serum factors can contribute to serum bactericidal activity. These unidentified factors are most potent at serum concentrations of 10% and higher. Furthermore, we also find that some bacteria can be killed by the MAC at a slower rate. Our data demonstrate the requirement for the use of specific inhibitors in serum bactericidal assays and revealed that the classification of serum-sensitive and resistant strains needs re-evaluation. Moreover, it is important to determine bacterial viability at multiple time intervals to differentiate serum susceptibility between bacterial species. In conclusion, these data provide new insights into bacterial killing by the humoral immune system and may guide future vaccine development studies for the treatment of pathogenic serum-resistant bacteria.     

Detection of complement-mediated antibody-dependent bactericidal activity in a fluorescence-based serum bactericidal assay for group B Neisseria meningitidis

Serum bactericidal assays (SBAs) for Group B meningococci are considered the methods of choice for the evaluation of functional antimeningococcal antibodies. Many investigators regard SBAs as time- and labor-intensive. Variations in SBA protocols among different laboratories make interpretation of results difficult. Here we describe a fluorescence-based serum bactericidal assay (fSBA) and compare the results obtained with the fSBA to the results obtained with a more conventional SBA. The results generated by both assays were dependent upon the surviving bacteria after incubation, and the assay mixtures contained identical components. Differences between assays lie in how the surviving bacteria are quantified. The fSBA described in the paper uses the fluorescent dye alamarBlue (M. V. Lancaster and R. D. Fields, U.S. patent 5501959, March 1996). The fluorescent signals generated in the fSBA correlate to the oxidative respiration of surviving bacteria. Viable bacteria were detected between 6 and 8 h directly from reaction mixtures in 96-well plates by the fSBA, whereas colonies isolated on semisolid media could be counted after 24 h of incubation. The bactericidal titers generated by both assays were nearly identical. The fSBA described here can be used as an assay for the screening of large quantities of individual sera as complement sources or as a method for the detection of functional antibodies directed against group B Neisseria meningitidis in both human and mouse antisera.     

Effect of increasing doses of amikacin with or without piperacillin in the serum bactericidal test

To determine whether high doses of amikacin would prevent the development of resistance in clinical isolates, the serum bactericidal activity and killing rate of conventional and high doses of amikacin and piperacillin alone and in combination were measured in volunteer sera against a series of ten strains each of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa.Amikacin serum levels were 24.9±6.0 mg/l 1 h after infusion of the 7.5 mg/kg dose and 44.8±5.0 mg/l after the two-fold dose. Median serum bactericidal titers for low dose piperacillin + amikacin were 18–164 and for high-dose piperacillin + amikacin 116–1128. Both were satisfactory, except against piperacillin-resistant Pseudomonas aeruginosa (median bactericidal titers 12), and both combinations had equivalent killing rates.     

A simple rapid technique to measure neutrophil or serum bactericidal activity

A rapid and simple technique to measure both serum and neutrophil bactericidal activity is described. It is based on microtitre equipment and avoids the cumbersome and time consuming serial dilution and plate counting of conventional experiments. A constant concentration of neutrophils and/or serum is incubated with a series of dilutions of bacterial suspensions. Bacterial survival is estimated at various times from their ability to form colonies on agar and the results conveniently expressed as the largest bacterial population that can be eliminated by either neutrophils or serum alone. The technique is as simple and quick as a standard microtitre MBC test commonly used for antibiotics and scoring of the results takes only a few seconds per sample. Data are presented showing the close correlation between results obtained using this technique and those from conventional tests of neutrophil bactericidal activity and phagocytosis-associated chemiluminescence.     

Evaluation of exploration and risk assessment in pre-weaning mice using the novel cage test

Exploration and risk behaviour (risk assessment/risk taking) are critical to enable mice to cope with novel situations and gain control over their environment. Evaluation of those behaviours would therefore be a useful part of early phenotypic characterization of genetically modified mice, allowing early detection of behavioural phenotypes that require special attention and/or are of scientific interest. This study aimed to evaluate exploration and risk behaviour in pre-weaning mice using the novel cage test, which consists in exploration of a novel, clean, Makrolon type III cage. The results of this test were compared with those obtained in more complex and established tests to which the same mice were subjected as adolescents and young adults. Mice of two inbred strains (129S6/Bkl, n=10; C57BL/6Bkl, n=10) and one hybrid (B6CBAF1/Bkl, n=10) were used for validation of the test. The animals were tested in the novel cage (at weaning), the open field test (at 5 weeks), and from 9 weeks of age in three other tests: the elevated plus-maze, the concentric square field and the rat exposure test. The novel cage test effectively detected strain differences in pre-weaning mice as regards exploration and risk behaviour and the results were largely consistent with those obtained in the established tests later in life. In all tests 129S6 displayed a low locomotion and high risk assessment, while C57BL/6 and B6CBAF1 showed high locomotion and exploration. In addition high levels of risk taking were observed in C57BL/6. The novel cage test is rapid, requires no special equipment and is as discriminatory as more complex tests in detecting strain/genotype differences. This suggests that the novel cage test is a valuable tool for evaluation of exploration, risk assessment and risk taking in juvenile mice.     

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.