The microbial factor in the chemiluminescence of the peripheral blood neutrophils of patients with suppurative surgical infection

The luminol-dependent chemiluminescence of neutrophils in the peripheral blood of 30 healthy adults and 39 patients with the local and generalized forms of purulent infection was studied. Nonstimulated chemiluminescence and the index of chemiluminescence stimulation in the presence of opsonized Staphylococcus aureus added in vitro were determined. The former characteristic was found to be directly and the latter one, inversely related to the concentration of S. aureus, Escherichia coli and Candida albicans, but not E. epidermidis, Pseudomonas aeruginosa or Citrobacter, in the primary focus. At the microbial concentration exceeding 10(4) cells/g of tissue, the former characteristic was essentially higher than the level of chemiluminescence in healthy persons. With the improvement of the general state of the patients and in the absence of microorganisms in the wound as the result of complex treatment this characteristic decreased to values comparable with the reaction of neutrophils in healthy persons.     

Oxygen-dependent killing of Staphylococcus aureus by human neutrophils

Luminol-dependent chemiluminescence was used as a monitor of reactive oxidant generation during phagocytosis of Staphylococcus aureus by human neutrophils. Reactive oxidants play a crucial role in the killing of this organism because: (a) S. aureus was killed most rapidly when the rate of increase of chemiluminescence was greatest; (b) neutrophils which had been activated to generate reactive oxidants by re-aeration of anaerobic suspensions killed this bacterium more efficiently than control suspensions; and (c) neutrophils from a patient with chronic granulomatous disease could neither generate reactive oxidants nor kill S. aureus.     

Neutrophil-stimulating activity of staphylococci based on reactive chemiluminescence data

The neutrophil-stimulating properties of 38 S. aureus strains and 32 S. epidermidis strains were studied in the reaction of luminol-mediated chemiluminescence. All S. aureus strains and 29 S. epidermidis strains were found to possess neutrophil-stimulating activity, the mean activity index for S. aureus being significantly higher. The stimulating activity of the strains varied within a wide range (the variation coefficient was 120.0 +/- 21.9%) and did not correlate with the content of protein A in bacterial cells and the degree of their hydrophoby. The opsonization of staphylococci with normal human serum enhanced the neutrophil reaction 1.5- to 100-fold and simultaneously leveled out the chemiluminescence indices in experiments with different strains (the variation coefficient was 8.0 +/- 1.5%). The nature of the neutrophil-stimulating effect of staphylococci and its relationship to the exploratory reactions of phagocytes are discussed.     

Role of myeloperoxidase in the killing of Staphylococcus aureus by human neutrophils: studies with the myeloperoxidase inhibitor salicylhydroxamic acid

We have used salicylhydroxamic acid (SHAM) to inhibit intraphagosomal myeloperoxidase activity in order to evaluate the role of this enzyme in the killing of Staphylococcus aureus by human neutrophils. 50 microM-SHAM reduced the luminol-dependent chemiluminescence response stimulated during phagocytosis of unopsonized latex beads and opsonized S. aureus by over 80% and 60%, respectively. When opsonized S. aureus were incubated with neutrophils, 45% were killed within 15 min incubation and 60% by 1 h. However, in neutrophil suspensions incubated with 50 microM-SHAM, only 13% were killed by 15 min whilst 71% still remained viable after 1 h. This inhibitor had no effect upon the number of bacteria phagocytosed or upon degranulation. In a cell-free system, 2.5 microM-H2O2 alone killed 55% of the bacteria, whereas in the presence of myeloperoxidase (i.e. 10 mU myeloperoxidase and 2.5 microM-H2O2) virtually all of the bacteria were killed: the addition of 50 microM-SHAM abolished this myeloperoxidase-enhanced killing but did not affect the H2O2-dependent killing. We therefore conclude that in normal neutrophils whilst H2O2 is required for killing of this pathogen, both myeloperoxidase-dependent and -independent pathways exist.     

Effect of Lactic Acid Bacteria on Growth of Staphylococcus aureus

Cultures of lactic acid bacteria, mostly from foods, were tested for their effect on the growth of Staphylococcus aureus in Trypticase Soy Broth (BBL). Some of the effectors, e.g., Streptococcus faecalis, S. faecium, Lactobacillus lactis, L. brevis, and Leuconostoc mesenteroides, stimulated growth of S. aureus during early hours of growth, especially at higher temperatures of incubation, but most cultures were inhibitory, and some (S. faecium and L. mesenteroides) were even killing by the time of attainment of the maximal phase of growth of the Staphylococcus. Low-temperature meat lactobacilli and Leuconostoc dextranicum inhibited S. aureus at 10, 15, 20, and 25 C throughout its growth. Streptococcus faecalis var. liquefaciens inhibited at these temperatures and at 30 and 37 C, as well. When the ratio of effectors to staphylococci in the inoculum was 100:1, the three enterococci, the meat Lactobacillus, and L. dextranicum prevented the attainment of 5 x 10(6) staphylococci per milliliter at 15 C, and all but the meat Lactobacillus did so at 22 C. A ratio of 1:1 accomplished similar results at 15 C, except that S. aureus was only delayed for 12 hr by S. faecalis. A ratio of 1:100 usually was ineffective. In general, the more effector bacteria there were in the inoculum, the greater was the overall inhibition (or stimulation) of S. aureus. Inhibition was most effective at 10 or 15 C, less so at 20 or 25 C, and least at 30 or 37 C, whereas stimulation during early growth was greater at the higher temperatures. Results with different strains of the effectors and with two strains of S. aureus were similar, for the most part.     

Differences in the effect of arachidonic acid on polymorphonuclear and mononuclear leukocyte function

Incubation of human polymorphonuclear leukocytes with arachidonic acid resulted in a stimulation of the oxidative metabolism of the cells. Upon stimulation with 80 microM arachidonic acid, neutrophils (5 X 10(6) cells/ml) produced superoxide (53 +/- 8 nmol/5 X 10(6) cells per 15 min), generated chemiluminescence (1211 100 +/- 157 000 cpm) and consumed oxygen (20 +/- 1 nmol/10(6) cells per 5 min). The stimulation of the cell metabolism could be reduced 40-60% by prior incubation of the cells with 10 microM indomethacin. Incubating polymorphonuclear leukocytes with arachidonic acid also resulted in a diminished chemotaxis towards an attractant, a decreased uptake of opsonized staphylococci and aggregation of the cells. This may be due to inhibitory products of arachidonic acid metabolism and toxic oxygen species produced during stimulated oxidative metabolism. The effects of arachidonic acid are specific for neutrophils, as mononuclear phagocytes only produced 17 +/- 8 nmol superoxide/5 X 10(6) cells per 15 min and generated 27 000 +/- 15 000 cpm chemiluminescence when stimulated with 80 microM arachidonic acid. When monocytes and neutrophils were stimulated with particles such as opsonized staphylococci, the amount of superoxide produced, oxygen consumed and chemiluminescence generated were similar. The phagocytic activity of the monocytes was also not affected by prior incubation with arachidonic acid. We conclude that in contrast to monocytes, neutrophil metabolism can be stimulated with arachidonic acid and this stimulation resulted in a decreased phagocytic activity of these cells.     

The chemoluminescence of the primary focus of a suppurative infection and of isolated neutrophils exposed to dimephosphon]

The influence of dimephosphone at concentrations of 0.001 M-0.75 M on the chemiluminescence of tissues at the focus of purulent infection in the ear of a guinea pig, on the survival rate of the experimental animals injected with the lethal dose of Staphylococcus aureus, as well as on the spontaneous and stimulated chemiluminescence of blood neutrophils in patients with wound infection, was studied. The study showed that different concentrations of dimephosphone oppositely influenced the intensity of the chemiluminescence of neutrophil suspensions and tissues at the focus of infection: low concentrations were found to produce stimulating action and high concentrations, suppressive action. At the highest concentration used in this study (0.75 M) dimephosphone prevented the death of the animals receiving lethal doses of S. aureus.     

Effect of stobadine on opsonized zymosan stimulated generation of reactive oxygen species in human blood cells

To predict more precisely the effect of stobadine, a pyridoindole antioxidant agent, in the whole organism, we studied its effect on opsonized zymosan-stimulated free radical generation in whole blood, on superoxide generation in the mixture of PMNL : platelets (1:50), as well as on superoxide generation and myeloperoxidase release in isolated PMNL. Without stimulation, stobadine had no effect on reactive oxygen species (ROS) generation and myeloperoxidase release. Stobadine in a concentration of 10 or 100 micromol/l significantly decreased luminol-enhanced chemiluminescence in opsonized zymosan-stimulated whole blood. In concentrations of 10 and 100 micromol/l, it reduced myeloperoxidase release from isolated neutrophils. Stobadine significantly decreased superoxide generation in isolated neutrophils in 100 micromol/l concentration. Its effect was much less pronounced in the mixture of neutrophils and platelets in the ratio close to physiological conditions (1:50). Our results suggest that stobadine might exert a beneficial effect in diseases or states where superfluous ROS generation could be deleterious.     

Simultaneous measurement of phagocytosis and phagosomal pH by flow cytometry: role of polymorphonuclear neutrophilic leukocyte granules in phagosome acidification

Human polymorphonuclear neutrophilic leukocytes (PMNLs) phagocytosed fluorescein-isothiocyanate (FITC)-labelled Staphylococcus aureus. Free bacteria, phagocytes, and nonphagocytes were discriminated and quantified by flow cytometry (FCM). The relative fluorescence of phagocyte-associated and free bacteria (Nf:N) was calculated by dividing the mean phagocyte fluorescence by that of the free bacteria and the number of phagocytosed bacteria. Bactericidal capacity and chemiluminescence were measured by standard methods. The red-to-green fluorescence ratio of acridine orange-stained PMNLs (R/G) was measured by FCM. Degradation of bacteria was monitored by the reduction in FITC and ethidiumbromide fluorescence of bacteria liberated from the phagocytes. Bacterial FITC fluorescence was pH dependent. Nf:N was 0.5 to 0.7. Using a standard curve for the interrelationship between bacterial fluorescence and pH, phagosomal pH was 5.0-5.5. Phagocytes, kept at 4 degrees C for 24 h had Nf:N approximately 1, did not degrade bacteria, but killed them and emitted chemiluminescence. NH4Cl increased phagocyte fluorescence by 27% and decreased R/G by 50%. Cyanide and azide did not affect Nf:N. Nf:N of phagocytes from a patient with chronic granulomatous disease was 32% below, and R/G was 32% higher than the controls. Acidification of the phagosomes seems to be related to discharge of PMNL granule contents and independent of the respiratory burst.     

Relationship between the susceptibility of various bacteria to active oxygen species and to intracellular killing by macrophages

The susceptibilities of six micro-organisms to active oxygen species generated in the xanthine oxidase-mediated bactericidal system were as follows: Escherichia coli 81 greater than or equal to Listeria monocytogenes EGD greater than or equal to Salmonella typhimurium HKB-1 greater than or equal to Staphylococcus aureus Smith much greater than Mycobacterium tuberculosis H37Rv approximately equal to Candida albicans NIH A207 (the last two organisms were essentially resistant to this system). The H2O2-Fe-mediated halogenation system exhibited a higher microbicidal activity. When the micro-organisms were compared for their sensitivity to bactericidal activity of resident mouse peritoneal macrophages (M phi s), C. albicans, Staph. aureus and E. coli were killed rapidly, whereas M. tuberculosis, L. monocytogenes and S. typhimurium were more resistant. In tests for the ability to trigger an oxidative burst in mouse peritoneal M phi s (as measured by chemiluminescence), Staph. aureus showed the highest activity followed by the other organisms in the following order: C. albicans greater than E. coli greater than L. monocytogenes congruent to M. tuberculosis. S. typhimurium exhibited no triggering activity. The high susceptibility of Staph. aureus and E. coli to M phi bactericidal activity, and the partial resistance of L. monocytogenes and M. tuberculosis, correlated with their susceptibility to active oxygen and the H2O2-Fe-mediated halogenation reaction.     

Use of resistant mutants to study the interaction of triton X-100 with Staphylococcus aureus.

Staphylococcus aureus mutants resistant to the nonionic detergent Triton X-100, isolated from the wild-type strain H and the autolysin-deficient strain RUS3, could grow and divide in broth containing 5% (vol/vol) Triton X-100, while growth of the parental strains was markedly inhibited above the critical micellar concentration (0.02%) of the detergent. Growth-inhibitory concentrations of Triton X-100 killed wild-type cells without demonstrable cellular lysis. Triton X-100 stimulated autolysin activity of S. aureus cells under nongrowing conditions, and this lytic response was markedly reduced in energy-poisoned cells. In contrast, the detergent had no effect on the activity of autolysins in cell-free systems, and growth in the presence of Triton X-100 did not alter either the cellular autolysin activity or the susceptibility of cell walls to exogenous lytic enzymes. Treatment with either Triton X-100 or penicillin G in the growth medium stimulated release of predominantly acylated intracellular lipoteichoic acid and sensitized staphylococci to Triton X-100-induced autolysis. There was no significant difference in the cell wall and membrane compositions or Triton X-100 binding between the parental strains and the resistant mutants. The resistant mutant TXR1, derived from S. aureus H, had a higher level of L-alpha-glycerophosphate dehydrogenase activity, and its oxygen uptake was more resistant to inhibition by a submicellar concentration (0.008%) of Triton X-100. Growth in the presence of subinhibitory concentrations of Triton X-100 rendered S. aureus H cells phenotypically resistant to the detergent and greatly stimulated the level of oxygen uptake. Membranes isolated from such cells exhibited enhanced activity of the respiratory enzymes succinic dehydrogenase and L-alpha-glycerophosphate dehydrogenase.     

Staphylococcal opsonization and anti-Staphylococcus aureus IgG subclass antibodies in patients with severe or recurrent S. aureus infections

Abstract Opsonization of Staphylococcus aureus (Oxford strain) and specific IgG subclass antibodies against formalised staphylococci were meausred in plamas from 27 patients with significant S. aureus infections and 35 healty adults and 15 children. There were no statistically significant differences in the IgG2 and IgG4 levels between two groups and IgG3 was not detected, but the median plasma IgG1 level was significantly higher in patients with staphylococcal infections ( P < 0.00003). The concentration of IgG2 anti- S. aureus antibodies was 25–47 times greater than that of IgG1. If plasmas were decomplemented, the raised IgG1 levels were associated with increased opsonophagocytosis by normal neutrophils ( P < 0.0002).     

Responses of baboons to traditionally pyrogenic agents

It is not clear whether baboons develop fever in response to endotoxin or other pyrogens. We injected various pyrogens intravenously in 12 unrestrained baboons (Papio ursinus) and measured their body temperature using intra-abdominal radiotelemeters. Serum iron concentration was also measured. The baboons developed fever after injection of killed Staphylococcus aureus (5 X 10(7) organisms/kg). No significant fever was measured after injection of lipopolysaccharide (Salmonella typhosa) (0.1, 8, 40, and 100 micrograms/kg), bovine serum albumin (4 mg/kg), killed Salmonella minnesota (5 X 10(7) organisms/kg), and killed Salmonella typhi (5 X 10(7) organisms/kg). A significant decrease in serum iron concentration was found only after injection of S. aureus and lipopolysaccharide, 100 micrograms/kg. The phagocytic synthesis of interleukin-1 following pyrogen stimulation in baboons and some other primates appears to differ from that in man and in nonprimates.     

Gamma interferon enhances the killing of Staphylococcus aureus by human neutrophils

The effect of purified human interferon-gamma on the responsiveness of human neutrophils was investigated. Pre-incubation of neutrophils with 100 U interferon ml-1 for 10 min at 37 degrees C resulted in a 2.5-fold increase in N-formylmethionyl-leucyl-phenylalanine-stimulated reactive oxygen metabolite generation (as assayed by luminol-dependent chemiluminescence). Pre-treatment of neutrophils with interferon also potentiated their ability to kill Staphylococcus aureus, and thus it is proposed that this lymphokine may also enhance neutrophil function in vivo under certain pathological conditions.     

Reactivity of neutrophils in systems with peptidoglycans of different species of Staphylococcus

The work presents the results of the comparative study of the luminol-dependent chemiluminescence of human neutrophils stimulated with peptidoglycans of 14 staphylococcal species. The intensity of chemiluminescence greatly varied, which was not linked with specific antigenic and structural features of peptidoglycans. The maximal chemiluminescence was induced by S. aureus and S. cohnii peptidoglycans and the minimal chemiluminescence, by S. lentus and S. epidermidis peptidoglycans. The data obtained in experiments on cross restimulation with homologous and heterologous peptidoglycans suggest that the mechanisms of neutrophil activation are similar in character, differing from the mechanism of latex-induced chemiluminescence. These data were analyzed from the viewpoint of the heterogeneity of bacterial peptidoglycans and its effect on their relationship with neutrophils.     

Luminol-dependent chemiluminescence in antibody-sensitized neutrophils stimulated with protein A-bearing staphylococci

When mouse polymorphonuclear leukocytes (PMNs) sensitized with rabbit antibody to mouse Ehrlich ascites tumor cells were stimulated by Staphylococcus aureus Cowan I cells, a conspicuous luminol-dependent chemiluminescence was observed in the absence of opsonin. The profile of the chemiluminescence (CL) response evoked by staphylococcal cells from antibody-sensitized PMNs had two peaks. An initial peak, observed within 1 min after stimulation, was sharp and high and a second peak, observed about 5 min after stimulation, was low and extended. The CL response of antibody-sensitized PMNs stimulated by S. aureus Cowan I cells was dose-dependently blocked by preincubation with soluble SpA. Cells of a mutant derived from S. aureus Cowan I strain with trace amounts of cell-bound SpA failed to stimulate the antibody-sensitized PMNs to generate the CL response. The antibody-sensitized PMNs were found to phagocytize SpA-bearing S. aureus cells even in the absence of opsonic serum. These results suggest that the observation presented here might provide a useful tool for the investigation of CL response of PMNs.     

A potential new pathway for Staphylococcus aureus dissemination: the silent survival of S. aureus phagocytosed by human monocyte-derived macrophages

Although considered to be an extracellular pathogen, Staphylococcus aureus is able to invade a variety of mammalian, non-professional phagocytes and can also survive engulfment by professional phagocytes such as neutrophils and monocytes. In both of these cell types S. aureus promptly escapes from the endosomes/phagosomes and proliferates within the cytoplasm, which quickly leads to host cell death. In this report we show that S. aureus interacted with human monocyte-derived macrophages in a very different way to those of other mammalian cells. Upon phagocytosis by macrophages, S. aureus persisted intracellularly in vacuoles for 3-4 days before escaping into the cytoplasm and causing host cell lysis. Until the point of host cell lysis the infected macrophages showed no signs of apoptosis or necrosis and were functional. They were able to eliminate intracellular staphylococci if prestimulated with interferon-gamma at concentrations equivalent to human therapeutic doses. S. aureus survival was dependent on the alternative sigma factor B as well as the global regulator agr, but not SarA. Furthermore, isogenic mutants deficient in alpha-toxin, the metalloprotease aureolysin, protein A, and sortase A were efficiently killed by macrophages upon phagocytosis, although with different kinetics. In particular alpha-toxin was a key effector molecule that was essential for S. aureus intracellular survival in macrophages. Together, our data indicate that the ability of S. aureus to survive phagocytosis by macrophages is determined by multiple virulence factors in a way that differs considerably from its interactions with other cell types. S. aureus persists inside macrophages for several days without affecting the viability of these mobile cells which may serve as vehicles for the dissemination of infection.     

Staphylococcal opsonization and anti-Staphylococcus aureus IgG subclass antibodies in patients with severe or recurrent S. aureus infections

Opsonization of Staphylococcus aureus (Oxford strain) and specific IgG subclass antibodies against formalised staphylococci were measured in plasmas from 27 patients with significant S. aureus infections and 35 healthy adults and 15 children. There were no statistically significant differences in the IgG2 and IgG4 levels between two groups and IgG3 was not detected, but the median plasma IgG1 level was significantly higher in patients with staphylococcal infections (P less than 0.00003). The concentration of IgG2 anti-S. aureus antibodies was 25-47 times greater than that of IgG1. If plasmas were decomplemented, the raised IgG1 levels were associated with increased opsonophagocytosis by normal neutrophils (P less than 0.0002).     

Therapeutic effect of some antibiotics on experimental staphylococcal infection and its correlation with in vitro activity of antibiotics in sub-inhibitory concentration against Staphylococcus aureus strains

The aim of the study was to demonstrate of whether the therapeutic effects of antibiotics depend on their in vitro activity in sub-inhibitory concentrations against staphylococci. Cloxacillin, gentamicin and lincomycin were used in the study. Groups of S. aureus strains, containing 6 strains with similar MIC values each but different sensitivity to sub-inhibitory antibiotic concentrations (sub-MIC) were selected (a total of 36 trains): i. strains increasing their sensitivity to phagocytosis and bactericidal activity of rabbit leukocytes after incubation with an antibiotic in 0.1 MIC concentration, ii. strains with sensitivity to the above factors unaffected by incubation with an antibiotic in 0.5 MIC concentration. The doses of staphylococci causing death of 90-100% of Swiss albino mice 10 days after i.p. infection were determined. The injected doses (LD 90-100) and various doses of antibiotics were used to determine ED50 values as well as the survival rate of the mice with experimental staphylococcal infections after treatment with these antibiotics. It was demonstrated that effective doses (ED 50) of the antiboitics were significantly lower when the antibiotics were administered once to mice infected with strains S. aureus sensitive to sub-MIC concentrations of the investigated antibiotics than for mice infected with strains resistant to their sub-MIC concentrations. Similar correlations were observed in mice which were given the antibiotics several times (for 7 days): the percentage of the surviving mice was higher in the group infected with sub-MIC sensitive strains. The therapeutic effect of cloxacillin, gentamicin and lincomycin demonstrated a significant correlation with the S. aureus strains used to induce the infections and their sensitivity, or lack of sensitivity in vitro, to phagocytosis and bactericdal activity of leukocytes in the presence of antibiotics in sub-MIC concentrations.     

Luminol-dependent chemiluminescence of the primary focus at different infecting doses of Staphylococcus aureus

Guinea pigs were infected intramuscularly with different doses of S. aureus. At different periods after infection the chemiluminescence (CL) of the primary focus in the animals infected with 10(10) colony-forming units (CFU) of S. aureus (abscess formation on day 3, mortality rate equal to 70%) was poor and increased only on days 6-9; in animals injected with 10(9) CFU (abscess formation on day 6, mortality rate equal to 16%) the CL increased on day 3 and lasted till day 20; in the animals infected with 10(8) CFU (the appearance of infiltration, no mortality) the reaction increased on day 1, then decreased; and in those infected with 10(2) CFU (no local manifestations of the process) the reaction was low at all periods. CL peak was recorded early mainly due to the neutrophil reaction, while a later CL peak (in cases of the favorable outcome of the infection) was explained by the macrophagal reaction. Thus, for the favorable outcome of the infection (in the presence of inflammation) the early increase of the CL of neutrophils migrating to the focus is characteristic, while the resolution of the local process is manifested by CL decrease and the prevalence of the macrophagal component of the reaction.