Bacteria In Serum Research Articles

Soluble MAC is primarily released from MAC-resistant bacteria that potently convert complement component C5.

The membrane attack complex (MAC or C5b-9) is an important effector of the immune system to kill invading microbes. MAC formation is initiated when complement enzymes on the bacterial surface convert complement component C5 into C5b. Although the MAC is a membrane-inserted complex, soluble forms of MAC (sMAC), or terminal complement complex (TCC), are often detected in sera of patients suffering from infections. Consequently, sMAC has been proposed as a biomarker, but it remains unclear when and how it is formed during infections. Here, we studied mechanisms of MAC formation on different Gram-negative and Gram-positive bacteria and found that sMAC is primarily formed in human serum by bacteria resistant to MAC-dependent killing. Surprisingly, C5 was converted into C5b more potently by MAC-resistant compared to MAC-sensitive Escherichia coli strains. In addition, we found that MAC precursors are released from the surface of MAC-resistant bacteria during MAC assembly. Although release of MAC precursors from bacteria induced lysis of bystander human erythrocytes, serum regulators vitronectin (Vn) and clusterin (Clu) can prevent this. Combining size exclusion chromatography with mass spectrometry profiling, we show that sMAC released from bacteria in serum is a heterogeneous mixture of complexes composed of C5b-8, up to three copies of C9 and multiple copies of Vn and Clu. Altogether, our data provide molecular insight into how sMAC is generated during bacterial infections. This fundamental knowledge could form the basis for exploring the use of sMAC as biomarker.

Study on the Prevalence of Periodontopathogenic Bacteria in Serum and Subgingival Bacterial Plaque in Patients with Rheumatoid Arthritis

The purpose of this study was to detect bacterial periodontal DNA from subgingival dental plaque and serum in patients affected by rheumatoid arthritis and periodontitis. The study group included 19 patients with periodontitis and refractory rheumatoid arthritis. The patients were clinically examined and diagnosed and the bacterial DNA was detected in the subgingival bacterial plate and serum by PCR. Severe chronic periodontitis was the most commonly diagnosed (42.2%). The DNA of periodontopathogenic bacteria was detected 100% in subgingival plate samples, and in serum samples it was identified in 84.2% of cases. The most commonly found species in subgingival plate samples were P. intermedia (100%), T. denticola (84.2%) and P. gingivalis (78.9%). In serum samples, the most frequently detected species were P. intermedia (89.4% and 73.6% respectively) and P. gingivalis (57.8% and 42.1%, respectively). A. actinomycetemcomitans and P. gingivalis did not show statistically significant differences between samples. This finding suggests that it could be an association because the same bacteria species detected in the serum were present in bacterial plaque samples. Patients with rheumatoid arthritis contain levels of oral pathogens in the serum and subgingival plaque that are common to red complex organisms, namely Porphyromonas gingivalis, Tannerella forsythia and Prevotella intermedia.

The relationship of the lipoprotein SsaB, manganese and superoxide dismutase in Streptococcus sanguinis virulence for endocarditis.

Streptococcus sanguinis colonizes teeth and is an important cause of infective endocarditis. Our prior work showed that the lipoprotein SsaB is critical for S. sanguinis virulence for endocarditis and belongs to the LraI family of conserved metal transporters. In this study, we demonstrated that an ssaB mutant accumulates less manganese and iron than its parent. A mutant lacking the manganese-dependent superoxide dismutase, SodA, was significantly less virulent than wild-type in a rabbit model of endocarditis, but significantly more virulent than the ssaB mutant. Neither the ssaB nor the sodA mutation affected sensitivity to phagocytic killing or efficiency of heart valve colonization. Animal virulence results for all strains could be reproduced by growing bacteria in serum under physiological levels of O(2). SodA activity was reduced, but not eliminated in the ssaB mutant in serum and in rabbits. Growth of the ssaB mutant in serum was restored upon addition of Mn(2+) or removal of O(2). Antioxidant supplementation experiments suggested that superoxide and hydroxyl radicals were together responsible for the ssaB mutant's growth defect. We conclude that manganese accumulation mediated by the SsaB transport system imparts virulence by enabling cell growth in oxygen through SodA-dependent and independent mechanisms.

Distinct localization of the complement C5b-9 complex on Gram-positive bacteria

The plasma proteins of the complement system fulfil important immune defence functions, including opsonization of bacteria for phagocytosis, generation of chemo-attractants and direct bacterial killing via the Membrane Attack Complex (MAC or C5b-9). The MAC is comprised of C5b, C6, C7, C8, and multiple copies of C9 that generate lytic pores in cellular membranes. Gram-positive bacteria are protected from MAC-dependent lysis by their thick peptidoglycan layer. Paradoxically, several Gram-positive pathogens secrete small proteins that inhibit C5b-9 formation. In this study, we found that complement activation on Gram-positive bacteria in serum results in specific surface deposition of C5b-9 complexes. Immunoblotting revealed that C9 occurs in both monomeric and polymeric (SDS-stable) forms, indicating the presence of ring-structured C5b-9. Surprisingly, confocal microscopy demonstrated that C5b-9 deposition occurs at specialized regions on the bacterial cell. On Streptococcus pyogenes, C5b-9 deposits near the division septum whereas on Bacillus subtilis the complex is located at the poles. This is in contrast to C3b deposition, which occurs randomly on the bacterial surface. Altogether, these results show a previously unrecognized interaction between the C5b-9 complex and Gram-positive bacteria, which might ultimately lead to a new model of MAC assembly and functioning.

FH-dependent complement evasion by disease-causing meningococcal strains with absent fHbp genes or frameshift mutations

Meningococci bind human fH to down-regulate complement, which enhances survival of the bacteria in serum. A major fH ligand is the vaccine candidate, factor H-binding protein (fHbp). Although fHbp has been considered an essential meningococcal virulence factor, rarely, invasive isolates with absent fHbp genes or frameshift mutations have been identified. In previous studies fH binding to these isolates was not detected. The aim of the present study was to investigate fH binding and complement evasion by invasive meningococcal serogroup B clinical isolates with absent fHbp genes or frameshift mutations. Four of the seven isolates tested bound human fH by flow cytometry and survived in IgG-depleted human serum. In all four, fH binding was decreased after inactivating the gene encoding NspA. Binding of fH to fHbp and NspA is specific for human fH. To investigate fH-dependent evasion of host defenses, human fH transgenic infant rats, or control littermates negative for human fH, were challenged IP with 103–104CFU of two of the isolates with no detectable fH binding by flow cytometry. At 6h, bacteremia caused by both strains was higher in human fH transgenic rats than in control rats (P<0.002). In conclusion, six of the seven isolates had evidence of fH binding and/or human fH-dependent complement evasion in transgenic rats. In four, NspA was as an alternative fH ligand. fHbp vaccination may select for mutants that do not require fHbp for complement evasion. Inclusion of additional target antigens in vaccines containing fHbp may delay emergence of these mutants.

Peripheral Arterial Disease Associated With Caries and Periodontal Disease

Peripheral arterial disease (PAD) is an important cardiovascular disorder of the peripheral arteries. Chronic infections, such as periodontitis, may play an important role in the etiology and pathophysiology of PAD and other cardiovascular conditions. Recently, Streptococcus mutans has been found with high frequency in atheromatous plaques. The aim of this study is to evaluate the possible clinical and microbiologic association between PAD and periodontitis and dental caries. Thirty patients with PAD and 30 control individuals were selected. PAD and its severity were established by the use of the ankle-brachial index (ABI). Clinical attachment loss (AL); probing depth; decayed, missing, and filled teeth (DMFT) index; and C-reactive protein (CRP) levels were evaluated. The presence of bacterial DNA from Streptococcus mutans, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, Treponema denticola, and Aggregatibacter actinomycetemcomitans was identified by polymerase chain reaction in subgingival biofilm and serum. Patients with ≥30% AL ≥ 4 mm had six-fold increased risk of having PAD (odds ratio = 8.18; 95% confidence interval = 1.21 to 35.23; P = 0.031). There was statistical difference in the CRP (P = 0.0413) and DMFT index (P = 0.0002), with elevated number of missing teeth (P = 0.0459) in the PAD group compared with the control group. There were no significant differences in the frequency of bacteria in serum and subgingival plaque. There was a positive relationship between periodontitis based on AL and PAD determined by the ABI (odds ratio = 8.18).

Antibodies to some enteropathogenic bacteria in serum of apparently healthy individuals in Ugobowo, Benin City, Nigeria

Antibodies to some enterobacteriaceae such as Escherichia coll, Proteus mirabilis, Klebsiella and Shigella spp. were assayed for in the sera of 50 apparently healthy individuals aged between 20-50 years at Ugobowo, Benin city, Nigeria. Sera were obtained by standard method and stored at -4°C for 24-48 hr. Antigens were prepared from bacteria isolates and were used for tile/passive haemagglutination. Results showed that 74, 66, 60 and 50% of the study subjects had antibodies to E. coli, Proteus, Ktebsiella and Shigella spp. respectively. Antibody to E. coli was highest. The highest antibody titre recorded was 1 in 8 for E. coli. Thirty-eight sera had cross reactivity to all four isolates. This study has the advantage of documenting a specific antibody response at a time when the pathogen may no longer be present or may not be isolated for other reasons such as the action of a chemotherapeutic agent.Keywords: Antibodies, antigens, bacteria, haemagglutination, Nigeria

Antiserum against Escherichia coli J5 contains antibodies reactive with outer membrane proteins of heterologous gram-negative bacteria.

The binding of IgG in antiserum to Escherichia coli J5 to the surface of Enterobacteriaceae and to cell wall fragments released from serum-exposed bacteria was studied in a search for potentially protective epitopes other than lipopolysaccharide (LPS). IgG titers to multiple heterologous gram-negative smooth bacteria increased following incubation of the bacteria in serum and decreased following absorption with serum-exposed heterologous bacteria. IgG eluted from absorbing bacteria bound to at least three conserved bacterial outer membrane proteins (OMPs), but not LPS, as assessed by immunoblotting. The same OMPs were present in LPS-containing macromolecular cell wall fragments released by incubation of heterologous gram-negative bacteria in human serum. Part of the protection offered by J5 antiserum could be from binding of IgG to conserved OMPs at the bacterial surface or to OMPs in cell-wall fragments released from dying bacteria.

Identification of phosphatidylinositol mannoside as a mycobacterial adhesin mediating both direct and opsonic binding to nonphagocytic mammalian cells.

The molecular basis for the binding of Mycobacterium tuberculosis to nonphagocytic cells, which are readily infected in vitro, and the in vivo significance of this interaction are incompletely understood. Of six cell types tested, we found that only two, Chinese hamster ovary (CHO) fibroblasts and primary porcine aortic endothelial cells, were able to bind M. tuberculosis H37Rv efficiently in vitro. Binding to both CHO and endothelial cells was markedly (three- to fivefold) enhanced by 10 to 20% human or bovine serum, suggesting that the bacteria were coated by a serum opsonin. Preincubation with individual candidate opsonins revealed that recombinant human mannose-binding protein (rMBP), fibronectin, and transferrin were each able to enhance binding threefold. Preincubation of bacteria in serum depleted of mannan-binding lectins or in genetic MBP-deficient serum resulted in enhancements that were only approximately 60 and 58%, respectively, of that produced by preincubation in control serum. In contrast, serum depleted of fibronectin or transferrin retained its opsonizing capacity, suggesting that the latter two are not significant opsonins in whole serum. Binding of M. tuberculosis and Mycobacterium smegmatis to both CHO and endothelial cells in the presence or absence of serum was blocked (60 to 70%) by a monoclonal antibody, MAb 1D1, selected for recognition of intact bacilli. The 1D1 antigen was purified from mycobacterial cell walls and chemically identified as a polar phosphatidylinositol mannoside (PIM). Latex beads coated with purified 1D1 antigen bound to CHO cells, which was enhanced threefold by serum and abolished by periodate treatment, suggesting a requirement for the PIM mannoses in opsonic adhesion. This was likely mediated, at least in part, by serum MBP, as rMBP bound strongly to 1D1 antigen in both thin-layer chromatography overlay and plate binding assays, the latter in a mannan-inhibitable manner. This is the first demonstration that mycobacterial PIMs can function as adhesins for binding to nonphagocytic cells, both directly and after opsonization with serum proteins, including MBP.

Protein degradation by Prevotella intermedia and Actinomyces meyeri supports the growth of non-protein-cleaving oral bacteria in serum.

The proteolytic activities of oral bacteria are thought to play an important role in the aetiology of dental abscesses. Bacteria-derived proteases may contribute to tissue destruction, and are likely to impair host defence by degrading immunoglobulins and complement. Degraded periodontal tissue and tissue fluid are likely to constitute essential sources of nutrients in the abscess. Tissue fluid, which is derived from serum, is rich in protein and poor in carbohydrate, suggesting that breakdown of protein and fermentation of amino acids is a crucial step to generate energy for growth of the microflora. The number of oral bacterial species that perform hydrolytic cleavage of protein into polypeptides, the first step in protein degradation, is relatively small compared to the large majority of peptidase-producing species. In this study, we therefore investigated the growth-promoting effect of proteinase-producing species like Prevotella intermedia and Actinomyces meyeri on the growth of some non-proteinase producing bacteria in mixed cultures. We used serum as a substitute for the supposed natural substrate of the abscess microflora. The breakdown of serum proteins was investigated using capillary electrophoresis. Poor growth was found in mono- and mixed cultures of non-proteinase producing species Eubacterium lentum, Fusobacterium nucleatum. Peptostreptococcus micros, and Streptococcus intermedius. The presence of P. intermedia in mixed cultures strongly enhanced growth of these 4 species, according to the hypothesis that the growth of the mixed cultures was peptide-limited. The enhanced growth of P. intermedia in pronase-digested serum indicated peptide-limited growth of this organism in serum, despite its production of proteinase. We found that growth of monocultures of Actinomyces meyeri was poor. In contrast, A. meyeri grew well in mixed cultures and its presence stimulated growth of F. nucleatum and P. micros, suggesting a synergistic relationship. The growth of mono- and mixed cultures was investigated using one representative strain of each species. Thus, there is a small risk of having selected unique strains. Proteinase inhibitors reduced the growth of Porphyromonas gingivalis, Prevotella nigrescens, and P. intermedia in trypticase peptone-yeast extract medium with, and without, IgG. Our study indicated that proteinase-producing organisms play a key role in mixed cultures of oral bacteria in human serum by providing polypeptides for growth. This may explain their association with dental abscesses.

Normal human serum depleted of Clq, factor D and properdin: its use in studies of complement activation

Normal human sera were depleted of C1q, factor D (D) and properdin (P) by a simple and reproducible procedure providing reagents for analysis of complement-dependent functions. Classical pathway activity was restored with purified C1q, and alternative pathway activity with purified D and P. Since both activation pathways were abolished, antibodies and other components could be removed without loss of complement activity during immunoabsorption procedures. Synergism between the two pathways during haemolysis of rabbit erythrocytes was clearly demonstrated, and was also found on analysis of C3 cleavage in serum incubated with other alternative pathway activators such as zymosan and inulin. Experiments with a Neisseria meningitidis serogroup W-135 strain isolated from a patient with inherited P deficiency showed that both pathways were capable of supporting antibody-dependent killing of the bacteria in serum. The alternative pathway was possibly more efficient than the classical pathway in the assay system. In C1q,D,P-depleted serum with high concentrations of anticapsular IgG antibodies, the addition of D alone resulted in efficient alternative pathway-mediated killing. The alternative pathway was equally efficient in a C1q,D,P-depleted serum with low concentrations of anticapsular antibody, but in this case the reaction required both D and P.

Degradation of Escherichia coli chromosomal and plasmid DNA in serum.

Incubation of serum-sensitive [3H]thymidine labelled Escherichia coli PC2166 (RSF1030) and E. coli AM1281 (pBR322) harbouring small plasmids (mol. wt 5.5 X 10(6) and 2.6 X 10(6] in serum resulted in killing of 99.9% of the bacteria within 15 min and in the release of 85% of the radioactivity into the medium after 1 h incubation. The fate of chromosomal and plasmid DNA during incubation of the bacteria in serum was analysed by measurement of the amount of DNA-associated radioactivity, by TCA precipitation, by agarose gel electrophoresis and by the capacity of DNA to transform competent acceptor bacteria. Chromosomal DNA and high molecular weight plasmid DNA were rapidly degraded after 1 h incubation of bacteria in serum. However, low molecular weight plasmid DNA was virtually unaffected and remained physicochemically as well as biologically intact during up to 4 h of incubation of bacteria in serum.

Evidence for antibacterial activity of endodontic gutta-percha cones

In the presence of endodontic gutta-percha cones, several species of bacteria were killed in vitro. Silver points, when compared with gutta-percha cones, were not or were considerably less effective against the target organism, Staphylococcus aureus, in our assay system. Growth of bacteria in serum could be abolished by the mere presence of gutta-percha, especially when time was allowed for previous contact between the serum and the cones. It is concluded that gutta-percha cones possess a slowly acting, relatively weak, but, in our opinion, significant inherent antimicrobial property.

Generation of antistreptolysin O activity in contaminated sera.

THE ability to lyse erythrocytes of reduced streptolysin O, produced by strains of Lancefield's group A streptococci apparently results from attachment to membrane cholesterol. Cholesterol in aqueous emulsion will inhibit streptolysin activity at concentrations of about 1.0 µg ml−1 (ref. 1). Sera contaminated with bacteria can occasionally give false positive tests for anti-streptolysin antibody but the underlying mechanism has never been defined. We have excluded the possibility that bacterial contamination leads to release of free cholesterol from either ester-bound or protein-bound cholesterol by showing that (1) cholesterol added to contaminated sera showing positive antistreptolysin activity is bound to the same extent as in normal serum, and (2) that ultrafiltrates of such contaminated sera do not contain free cholesterol. Therefore it seemed likely that bacteria in serum might release proteolytic enzymes which would cleave the streptolysin molecule. But the studies reported here indicate that the system is more complex.

Fate of staphylococci in fresh rabbit blood and serum.

SummaryA method is presented to enable measurement of the initial bactericidal activity in freshly-drawn blood. Although Staphylococcus epidermidis was quickly killed by rabbit serum, whole blood did not become bactericidal until it clotted. The β-lysin which is presumably responsible for the death of gram-positive bacteria in serum could not be released from platelets into the circulating blood by injection of endotoxin into sensitized rabbits. No inhibitor for β-lysin was found in unclotted blood.

The growth and death of wound bacteria in serum, exudate and slough.

Six strains of Str. pyogenes and four strains of Staph. aureus were found to grow well from large or small inocula in fresh human serum; four strains of E. coli failed to grow from large inocula and were killed when the inocula were small. Ps. pyocyanea, Proteus and Str. viridans sometimes grew and sometimes failed to grow in fresh serum.In serum which had been treated with trypsin or heated at 56° C. for 1 hr. the Gram-negative bacilli grew readily, and growth was still better in serum which had been heated and then trypsinized. Heating at 56° C. and treatment with proteolytic enzymes were shown to destroy complement and to inactivate the bactericidal activity of fresh serum against a Shigella highly sensitive to the properdin system. It is suggested that these changes might be enough to explain the conversion of serum by proteolytic enzymes into a suitable medium for the growth of coliform bacilli. This change might be brought about by bacterial or leucocytic enzymes. Fibrinolytic serum from a cadaver was found to retain at least a part of its complement and bactericidal activity.Strains of E. coli, Ps. pyocyanea, Proteus, Staph. aureus and Str. pyogenes were tested for growth in burn slough suspension and blister fluid. In slough suspension all strains grew well, but the Gram-negative bacilli grew rather more vigorously than the Gram-positive cocci. In blister fluid E. coli was killed and other strains grew.These findings are discussed in relation to wound sepsis.