Comparison of the bactericidal activity of different vertebrate sera

Schwab, G. E. (University of Adelaide, Adelaide, South Australia), and P. R. Reeves. Comparison of the bactericidal activity of different vertebrate sera. J. Bacteriol. 91:106-112. 1966.-The bactericidal activity for gram-negative bacteria of normal sera from eight species of vertebrates was investigated to compare complement-mediated killing in sera from animals representing various classes of vertebrates. Although all the sera were bactericidal, there was considerable variation in the range of bacterial species killed and in the bactericidal titers. The temperature dependence of the bactericidal and hemolytic complement activities was also studied. The curves relating activity to temperature were similar in shape for sera from a homeotherm and poikilotherms, but those for the homeotherm reached their maximum at temperatures of 5 to 10 C higher than the poikilotherms. The role of lysozyme and complement in killing rough gram-negative bacteria was examined, and the results suggest that, as for smooth organisms, killing is due to antibody plus complement. These natural antibodies, like those for smooth strains, were shown to be specific.     

Titration of Bactericidal Activity against Smooth and Rough Strains of Salmonella which Are Resistant in Isotonic Medium

Bactericidal factors in antisera against various chemotype strains of Salmonella were assayed by means of the spot method. Certain smooth and rough strains were resistant to the killing effect of immune mouse sera when the activity was assayed in an isotonic salt medium and guinea pig complement was used. However, the sensitivity was found to be increased in various degrees by assaying it in a medium containing hypotonic concentrations of NaCl or tris(hydroxymethyl)-aminomethane-HCl (Tris-HCl). Keeping the resistant bacteria in hypotonic solutions before or after treatment with complement increased the titer of antiserum, indicating that the hypotonic solution increases the sensitivity of bacterial cells to the antiserum and/or complement. The optimal salt concentration for the assay of serum-sensitive and -resistant smooth strains was 0.02 M NaCl. With Ra through Rd chemotype strains, 0.1 m NaCl before complement treatment and 0.05 m NaCl after the treatment was the best. Isotonic medium was necessary for the titration of the Re chemotype strain. Specificity of the killing effect which was assayed by the hypotonic spot method was shown by adsorption studies on the immune sera. Use of C4-deficient guinea pig complement resulted in low titers against certain strains of bacteria and high titers were restored by the addition of the C4 component of complement. These results indicate that serologically specific bactericidal factors including antibody can be assayed by the spot method using hypotonic NaCl or Tris-HCl.     

Bactericidal activity of normal human serum to gram-negative rods with sialic acid containing lipopolysaccharide

The complement plays the most important role in eliminating bacterial invasion of the host, by facilitating phagocytosis of potential pathogens and by participating in the direct essential role in protecting gram-negative bacteria against bactericidal activity of serum. Sialic acids which are important constitutes of animal tissue glycoconjugates are also present in antigens of some bacterial strains. The susceptibility of gram-negative strains with sialic acid--containing lipopolysaccharides to bactericidal action sera was examined.     

Binding of complement inhibitor C4b-binding protein contributes to serum resistance of Porphyromonas gingivalis

The periodontal pathogen Porphyromonas gingivalis is highly resistant to the bactericidal activity of human complement, which is present in the gingival crevicular fluid at 70% of serum concentration. All thirteen clinical and laboratory P. gingivalis strains tested were able to capture the human complement inhibitor C4b-binding protein (C4BP), which may contribute to their serum resistance. Accordingly, in serum deficient of C4BP, it was found that significantly more terminal complement component C9 was deposited on P. gingivalis. Moreover, using purified proteins and various isogenic mutants, we found that the cysteine protease high molecular weight arginine-gingipain A (HRgpA) is a crucial C4BP ligand on the bacterial surface. Binding of C4BP to P. gingivalis appears to be localized to two binding sites: on the complement control protein 1 domain and complement control protein 6 and 7 domains of the alpha-chains. Furthermore, the bacterial binding of C4BP was found to increase with time of culture and a particularly strong binding was observed for large aggregates of bacteria that formed during culture on solid blood agar medium. Taken together, gingipains appear to be a very significant virulence factor not only destroying complement due to proteolytic degradation as we have shown previously, but was also inhibiting complement activation due to their ability to bind the complement inhibitor C4BP.     

Sialic Acid-Containing Lipopolysaccharides of Salmonella O48 Strains—Potential Role in Camouflage and Susceptibility to the Bactericidal Effect of Normal Human Serum

Sialic acid (N-acetylneuraminic acid, NeuAc) plays an essential role in protecting gram-negative bacteria against the bactericidal activity of serum and may contribute to the pathogenicity of bacteria by mimicking epitopes that resemble host tissue components (molecular mimicry). The role of sialic acid (NeuAc)-containing lipopolysaccharides (LPS) of Salmonella O48 strains in the complement activation of normal human serum (NHS) was investigated. NeuAc-containing lipooligosaccharides cause a downregulation of complement activation and may serve to camouflage the bacterial surface from the immunological response of the host. Serotype O48 Salmonella strains have the O-antigen structure containing NeuAc while its serovars differ in outer membrane protein composition. In this study, the mechanisms of complement activation responsible for killing Salmonella O48 serum-sensitive rods by NHS were established. Four of such mechanisms involving pathways, which are important in the bactericidal mechanism of complement activation, were distinguished: only the classical/lectin pathways, independent activation of the classical/lectin or alternative pathway, parallel activation of the classical/lectin and alternative pathways, and only the alternative pathway important in the bactericidal action of human serum. To further study the role of NeuAc, its content in bacterial cells was determined by gas-liquid chromatography-mass spectrometry in relation to 3-deoxy-D-manno-2-octulosonic acid (Kdo), an inherent constituent of LPS. The results indicate that neither the presence of sialic acid in LPS nor the length of the O-specific part of LPS containing NeuAc plays a decisive role in determining bacterial resistance to the bactericidal activity of complement and that the presence of sialic acid in the structure of LPS is not sufficient to block the activation of the alternative pathway of complement. We observed that for three strains with a very high NeuAc/Kdo ratio the alternative pathways were decisive in the bactericidal action of human serum. The results indicated that those strains are not capable of inhibiting the alternative pathway very effectively. As the pathogenicity of most Salmonella serotypes remains undefined, research into the interactions between these bacterial cells and host organisms is indispensable.     

In vitro effect of subinhibitory concentrations of ceftazidime and meropenem on the serum sensitivity of Pseudomonas aeruginosa strains

The aim of this study was to determine the effect of subminimal inhibitory concentrations (subMICs) of ceftazidime, meropenem and gentamicin on the in vitro serum sensitivity of Pseudomonas aeruginosa strains isolated from a variety of isolation sites at two medical wards and an intensive care unit in a government university hospital in Croatia. A total of 20 serum-resistant P aeruginosa strains isolated from different clinical specimens were selected. Bacteria were exposed to 1/2, 1/4, 1/8, 1/16, and 1/32 x MIC of each antibiotic tested. Sensitivity of P. aeruginosa strains to bactericidal activity of normal human serum before and after bacterial exposure to subMICs was determined. Significant difference in serum sensitivity of the strains was observed after the bacteria were exposed to subMICs of ceftazidime and meropenem (p < 0.01), while the exposure to subMICs of gentamicin did not affect significantly the resistance of tested strains to the serum bactericidal activity. Comparing the number of serum-resistant strains before and after exposure to subMICs of antibiotics, statistically significant differences were determined (p < 0.01) after exposure of the strains to 1/2, 1/4, 1/8 and 1/16 x MIC of meropenem, and after exposure to 1/2, 1/4 and 1/8 x MIC of ceftazidime. SubMICs of ceftazidime and meropenem affected not only the resistance to serum bactericidal activity of bacteria, but also their morphology. The alterations in bacterial morphology caused by subMICs of ceftazidime and meropenem could be connected with consecutive bacterial serum sensitivity.     

The sensitivity of smooth and rough mutants ofSalmonella typhimurium to bactericidal and bacteriolytic action of serum,lysozyme and to phagocytosis

The sensitivity of smooth and rough mutants ofSalmonella typhimurium belonging to different chemotypes to bactericidal and bacteriolytic action of antibodies, complement and lysozyme was investigated. By the action of specific antibodies and complement the majority of strains was killed and in the presence of sufficient amount of lysozyme even lysed. Sera of newborn piglet which do not contain antibodies however, were able to kill regularly only strains of lower chemotypes (Rc and Rb). Smooth strains were usually resistant to the action of piglet serum whereas strains of Ra chemotype were killed only by sera of some piglets. Using the liver clearance method a significant correlation between the lipopolysaccharide chemotype of the particular strain and the degree of phagocytosis was found.     

Biphasic effect of gingipains from Porphyromonas gingivalis on the human complement system

Periodontitis is an inflammatory disease of the supporting structures of the teeth and is caused by, among other agents, Porphyromonas gingivalis. P. gingivalis is very resistant to killing by human complement, which is present in a gingival fluid at 70% of the serum concentration. We found that the incubation of human serum with purified cysteine proteases of P. gingivalis (gingipains) or P. gingivalis wild-type strains W83 and W50 resulted in a drastic decrease of the bactericidal activity of the serum. In contrast, serum treated with P. gingivalis mutants lacking gingipains (particularly strains without HRgpA) maintained significant bactericidal activity. To understand in detail the mechanism by which gingipains destroy the serum bactericidal activity, we investigated the effects of gingipains on the human complement system. We found that all three proteases degraded multiple complement components, with arginine-specific gingipains (HRgpA and RgpB) being more efficient than lysine-specific gingipain (Kgp). Interestingly, all three proteases at certain concentrations were able to activate the C1 complex in serum, which resulted in the deposition of C1q on inert surfaces and on bacteria themselves. It is therefore plausible that P. gingivalis activates complement when present at low numbers, resulting in a local inflammatory reaction and providing the bacteria with a colonization opportunity and nutrients. At later stages of infection the concentration of proteases is high enough to destroy complement factors and thus render the bacteria resistant to the bactericidal activity of complement.     

The interaction of complement components with Aeromonas species

The interaction of seven serum-sensitive Aeromonas strains with the complement system was investigated using a 2-h quantitative assay. Of the strains tested, four isolates activated both the alternative and classical pathways, two activated only the alternative pathway, and one strain was sensitive to the bactericidal action of complement through the classical pathway only. Two of the four Aeromonas caviae strains were such efficient activators of the complement system that when challenged with human sera deficient in normal concentrations of C3 and C4, they were still subject to complement-mediated bacterial lysis. This phenomenon, in conjunction with previous studies on complement activation by Aeromonas spp., may help account for the decreased incidence observed of systemic disease caused by Aeromonas caviae.     

A rapid microtiter plate serum bactericidal assay method for determining serum complement-mediated killing of Mannheimia haemolytica

In this study, we describe a rapid microtiter serum bactericidal assay (RMSBA) that can be used to measure the functionality of immune sera. It quantifies bactericidal activity of immune sera in the presence of complement against a homologous bacterium, M. haemolytica in this case. There is high correlation between data from RMSBA and standard complement-mediated bacterial killing assay (r=0.756; p<0.0001). The RMSBA activity of sera can be generated in less than 5 h instead of overnight incubation. RMSBA costs substantially less in terms of time, labor, and resources and is highly reproducible.     

Complex surveillance of Streptococcus pyogenes. IV. The phenomenon of interference of human serum under the conditions of the bactericidal test.

The phenomenon of interference of human sera under the conditions of the bactericidal test was described. In the presence of these sera, secondarily non-resistant strains of Streptococcus pyogenes were capable of vigorous growth and anti M 12 bactericidal activity was impaired. The factor was found in the albumin fraction and has probably the character of a blocking cytophilic lymphokin. It can markedly distort the interpretation of the bactericidal test.     

A comparative study of the bactericidal activity of horseshoe crab (Limulus polyphemus) hemolymph and vertebrate serum

This paper describes a partially heat-labile, naturally occurring bactericidal factor in cell-free hemolymph preparations obtained from Limulus polyphemus. This bactericidal activity has been shown to be directed against two Gram-negative bacteria, Escherichia coli and Klebsiella pneumoniae, whereas it had no effect on the Gram-positive bacteria tested, Micrococcus lysodeikticus and Staphylococcus aureus. Maximal bactericidal activity of this factor was observed at 30°C and pH 6.0. Since complement and antibody are required for antimicrobial activity in vertebrate sera, the activity of this factor in the presence of various complement inhibitors was assayed. The bactericidal activity of Limulus hemolymph is abolished by treatment with endotoxin; however, other anticomplementary substances were without effect. Limulus amebocyte lysate is known to contain protein which may be precipitated by endotoxin; it is possible that the reduction of bactericidal activity produced by endotoxin treatment may be caused by the denaturation of a bactericidal protein moiety produced by the hemocytes.     

Evaluation of efficacy of disinfectants against Salmonella from the feed industry

Aims:  To evaluate disinfectants against Salmonella under conditions relevant for the feed industry.
Materials and Results:  A survey on the use of disinfectants in feed industry showed that a range of different types was used. Nine disinfectants, reflecting the most commonly used active ingredients, were tested for bactericidal activity on Salmonella isolated from the feed industry. All disinfectants were efficient against Salmonella in suspension. The bactericidal effect varied considerably between different types of active compounds on bacteria dried on surfaces or grown as biofilm. Tenside-based disinfectants and hypochlorite were found to have low bactericidal activity and the efficiency was significantly reduced when the ratio of amount disinfectant per cell decreased. It was shown that concentrations of 70–80% ethanol were effective against Salmonella. Among the disinfectants tested a product containing 70% ethanol was most efficient followed by Virkon S.
Conclusions:  Many disinfectants had low bactericidal activity against Salmonella at surfaces while Virkon S and a product containing 70% ethanol were most effective. Another advantage of ethanol-based disinfectants is evaporation of ethanol, resulting in low residual water after use.
Significance and Impact of the Study:  Use of the disinfectants found to be efficient against surface associated Salmonella , may assist the industry in combating Salmonella .     

Impaired bactericidal activity of serum of a child suffering from focal proliferative glomerulonephritis.

The serum of a child with focal proliferative glomerulonephritis was found to exhibit a weaker bactericidal activity against Pseudomonas aeruginosa, Salmonella typhimurium, Salmonella enteritidis and Escherichia coli strains as compared with sera of the child's parents. The child's serum showed a low haemolytical activity of complement as well as a low C3 concentration. The authors believe that the abnormal complement concentration could cause the impaired bactericidal activity of the patient serum.     

The interaction of Escherichia coli with normal human serum: the kinetics of serum-mediated lipopolysaccharide release and its dissociation from bacterial killing

We have examined the killing of E. coli and kinetics of lipopolysaccharide (LPS) release after the exposure of the bacteria to normal human serum (NHS) and sera deficient in complement components, or with inactivated complement components. LPS of the galactose epimerase-deficient strain E. coli J5 were specifically radiolabeled by growing the bacteria in a medium containing [3H]galactose. Exposure of the washed bacteria to NHS resulted in a significant reduction (greater than 99%) in viability within 15 min and the concomitant release of radiolabeled LPS. However, maximal release of LPS was consistently 30% of the total radiolabel incorporated into the LPS molecules. The amount of tritium-labeled LPS released was shown to be directly proportional to the concentration of bacteria exposed to NHS, suggesting that release of LPS was not limited by the availability of some critical serum component(s). The consumption of complement in NHS by incubation with E. coli was demonstrated by decreased alternative and classical pathway-specific hemolytic activity. The use of Factor D-depleted and VEM-treated human sera demonstrated that, with these bacteria, both the alternative and classical pathways of complement contribute to bacterial killing and release of LPS. It is noteworthy that, in VEM-treated and Factor D-depleted sera, the rate of killing and the kinetics of LPS release were somewhat slower as compared to control serum. Bacterial killing in C7-depleted and C9-deficient human sera was minimal. Neither killing nor LPS release occurred in heat-inactivated (56 degrees C, 30 min) human serum. The amount of [3H]LPS released by C9-deficient serum was qualitatively similar to the amount released by the action of NHS. Tritium-labeled LPS was not released in C7-depleted serum. These data indicate that bacterial killing can be dissociated from LPS release, and suggest that, whereas LPS release may be necessary for the bactericidal effects of serum complement, it is probably not sufficient to effect killing. Furthermore, a significant fraction of LPS can be removed from the outer membrane of the bacteria without an apparent affect on viability.     

Bactericidal activity of human sera with a physiologic or genetic defect of the complement system

The serum of patient suffering genetically conditioned C2 defect showed a weak bactericidal activity against Pseudomonas aeruginosa strains. Out of 23 tested strains one was susceptible comparing with 16 ones sensitive to normal human serum. The normal cord serum exerted a bactericidal effect on 8 strains. All sera were found to be active against Salmonella strains tested.     

Variability of the lytic susceptibility of Neisseria gonorrhoeae to human sera.

The bactericidal activity of human sera for Neisseria gonorhoeae was studied. Sera were obtained from a group of patients with gonococcal infections who had acute urethritis, acute pelvic inflammatory disease, disseminated gonococcal infection, or who were asymptomatic carriers. The homologous and heterologous strains were tested with these sera. The development of serum bactericidal antibodies was not a universal event. With few exceptions, the susceptibility of a particular strain to human antibody and complement appeared to be largely independent of the particular person from whom the serum was obtained and was due instead to antigenic properties intrinsic to each individual strain. Lipopolysaccharide appeared to be the predominant antigen against which bactericidal antibodies were directed. The principal bactericidal antibody class was IgM. Blocking antibodies were not found to account for the lack of lytic activity. A correlation of bactericidal antibodies with protection from developing gonococcal infection could not be demonstrated in three pateints.     

The method for estimation of opsonic activity in sera of infants

Three methods for the estimation of opsonic activity in the sera of newborn children were tested. Two of them, based on the phagocytosis of opsonised bacteria labelled with radioactive phosphorus³²P as measured byin vivo blood clearance or uptake of bacteria in perfused isolated liver, were found to be unsuitable for long term dynamic study mainly because they do not permit the testing of series of samples. The third method (using isolated phagocytic cellsin vitro) permits the differentiation of the opsonic effect of complement and antibody and, furthermore, the firmness of the bond between microbes and phagocytes (which reflects the degree of opsonization) can be established. It was found that a 2-mercaptoethanol-resistant antibody, probably of the IgG type, was responsible for the opsonic activity of children's sera toEscherichia coli 083. Homologous antibody (toEscherichia coli 083) could be differentiated from beterologous antibody (toEscherichia coli 086) using the opsonic test only at low dilutions of sera. The combination of newborn piglet complement and antibody of children's sera yielded higher values of opsonic activity than either component separately.     

Humoral immune parameters of cultured Atlantic halibut (Hippoglossus hippoglossus L.)

Several humoral immune factors were studied in a group of cultured halibut (Hippoglossus hippoglossus L.). The serum protein and IgM concentration was comparable to levels seen in other teleost species. A strong antibody activity against TNP-BSA was observed but not against other antigens tested. Lysozyme and anti-protease activity was detected and showed variable heat sensitivity. Unlike the anti-protease activity, the lysozyme activity of the sera was not sensitive to storage at -20 degrees C. No spontaneous haemolytic activity was observed and the sera had no bactericidal effect on any of the bacterial strains tested. Iron binding capacity of the sera was high. Individual variation was considerable in all the factors tested.     

Bactericidal activity of hemolymph from the horseshoe crab, Limulus polyphemus

Bactericidal activity was found in Limulus serum, with great individual variation in titers toward different bacteria and also among individual horseshoe crabs toward the same bacterial species. These titers varied between monthly determinations of activity. There were crabs with zero activity toward each bacterial species tested. Although environmental factors are likely influences on the bactericidal activity of Limulus serum, the marked variability within similarly treated groups indicates large individual differences in the horseshoe crab population. The highest titers were recorded against those Gram-negative bacilli found normally in the environment. Lower titers were found against those species found normally in warmblooded animals and present in water as contaminants. The serum bactericidal factor is probably released from the circulating amoebocytes during clotting since there was no activity in the “plasma” portion of the blood. Exposure to heat (56°C, 30 min) destroyed the bactericidal activity.