Respiratory burst facilitates the digestion of Escherichia coli killed by polymorphonuclear leukocytes.

We examined factors that may limit degradation of bacterial protein of Escherichia coli S15 killed by polymorphonuclear leukocytes (PMN). Both human and rabbit PMN degraded up to 40% of [14C]amino acid-labeled protein of ingested and killed E. coli in 2 h as determined by loss of acid-precipitable radioactivity. In contrast, equally bactericidal broken-PMN preparations or isolated granules degraded only about 10% of bacterial protein regardless of pH. To determine whether activation of the respiratory burst contributes to digestion, we compared degradation by intact PMN in room air and under N2. Depletion of O2 by N2 flushing had no effect on the bactericidal activity of either human or rabbit PMN but reduced degradation by approximately 50%. Protein degradation during phagocytosis was also reduced in the presence of cyanide or azide, inhibitors of myeloperoxidase (MPO). PMN of two patients with chronic granulomatous disease ingested and killed E. coli S15 as well as did normal PMN but degraded bacterial protein as did normal PMN incubated under N2. The low degradative activity of PMN disrupted by sonication could be raised to nearly the level of intact PMN incubated in room air by preincubation of the PMN with 10(-7) M formyl-methionyl-leucyl-phenylalanine (fMLP) before sonication and by pretreatment of E. coli with MPO. Depletion of O2 or chloride during these preincubations with formyl-methionyl-leucyl-phenylalanine respectively, virtually abolished and markedly diminished stimulation of bacterial protein degradation. We conclude that enhanced MPO-mediated O2 metabolism of intact PMN plays a role in the digestion of killed E. coli.     

Influence of modified natural and synthetic surfactant preparations on bacterial killing by polymorphonuclear leucocytes

In addition to its biophysical functions, surfactant plays an important role in pulmonary host defense. In this investigation we studied the influence of various commercially available surfactants on the phagocytosis of bacteria that are common pathogens in the neonatal period. Group B streptococci (GBS), Escherichia coli and Staphylococcus aureus were cultured with isolated human polymorphonuclear leucocytes (PMN) and non-specific serum in the presence or absence of different modified natural (Curosurf, Alveofact, Survanta) or totally synthetic, protein-free surfactant preparations (Exosurf, Pumactant). Prior to and after 30 and 60 min of incubation with PMN at different surfactant concentrations (1, 10 or 20 mg/ml), the number of viable bacteria was determined by colony counting. Killing of S. aureus by PMN was not influenced by any of the surfactants. Alveofact and Curosurf had no significant negative impact on phagocytosis. At 20 mg/ml, Curosurf even reduced the number of viable E. coli. Survanta at 10 and 20 mg/ml and Exosurf at all concentrations impaired the killing of non-encapsulated GBS and E. coli. Pumactant at 1-20 mg/ml interfered with the phagocytosis of E. coli. In further experiments we demonstrated that Curosurf did not interfere with the phagocytosis of an encapsulated GBS-strain opsonised by a specific antiserum either. In additional experiments we analysed the influence of the different surfactants on the release of reactive oxygen metabolite by PMN and found that the changes in nitroblue tetrazolium reduction did not necessarily correlate with the findings of the studies on killing. In conclusion, we found that killing by PMN was influenced by the bacterial species and the composition and concentration of the different surfactant preparations. The strongest impairment in phagocytic function of PMN was observed with the protein-free synthetic surfactant Exosurf, a phospholipid preparation that contains the alcohols hexadecanol and tyloxapol as spreading agents.     

Relationship between extracellular stimulation of intracellular killing and oxygen-dependent microbicidal systems of monocytes.

Human monocytes require serum components immunoglobulin G, C3/C3b, and B/Bb to exert optimal microbicidal action against ingested microorganisms. The present study was performed to find out whether these factors act by enhancing oxygen-dependent antimicrobial mechanisms. Serum enhanced oxygen consumption and superoxide production by monocytes before phagocytosis, but did not further increase these processes in monocytes that had recently ingested bacteria. Furthermore, serum did not boost iodination during intracellular killing by monocytes. Phorbol myristate acetate, N-formyl-methyonyl-leucyl-phenylalanine, concanavalin A, and concanavalin A-Sephadex all stimulated the conversion of O2 to H2O2 by monocytes, but only concanavalin A augmented intracellular killing. Reactive oxygen intermediates generated by cell-free enzymes (xanthine oxidase or glucose oxidase) in concentrations comparable to those accumulating extracellularly during incubation of monocytes containing bacteria with phorbol myristate acetate did not promote intracellular killing. The presence of catalase during phagocytosis inhibited killing, but had no effect on killing in the postphagocytic state. Monocytes deprived of glucose for 24 h showed markedly impaired O2 consumption, O2- generation, and bacterial killing; all of these effects were rapidly reversed by restoration of glucose. It is concluded that both an intact respiratory burst and extracellular serum factors are necessary for optimal killing of intracellular Staphylococcus aureus by human monocytes. Serum does not appear to act by enhancing the respiratory burst, but rather to have a separate, synergistic role, the biochemical basis of which is unknown.     

Resistance of transgenic mice expressing human group II phospholipase A2 to Escherichia coli infection

Group II phospholipase A2 (PLA2) is a newly recognized antibacterial acute-phase protein. Recently we observed that transgenic mice expressing group II PLA2 (PLA2(+) mice) were able to resist experimental Staphylococcus aureus infection by killing the bacteria, as indicated by improved survival and by the small numbers of live bacteria in their tissues (V. J. O. Laine, D. S. Grass, and T. J. Nevalainen, J. Immunol. 162:7402-7408, 1999). To establish the role of group II PLA2 in Escherichia coli infection, the host responses of PLA2(+) mice and their PLA2-deficient C57BL/6J littermates (PLA2(-) mice) were studied after intraperitoneal administration of E. coli. The levels of group II PLA2 in sera of PLA2(+) mice increased after the administration of E. coli, and the concentration of group II PLA2 correlated significantly with the catalytic activity of PLA2 in serum. PLA2(+) mice showed lower rates of mortality and less bacterial growth in peritoneal lavage fluid, blood, and spleen and liver tissues than PLA2(-) mice. Unlike the observations with staphylococcal infection, serum and peritoneal lavage fluid did not inhibit the growth of E. coli in vitro. The results indicate that expression of the group II PLA2 transgene improves the host defense of mice against E. coli infection.     

Pre-opsonisation of Escherichia coli induces resistance to neutrophil killing in serum and urine: relationship to growth phase.

Tests of phagocytosis and killing by polymorphonuclear neutrophil leucocytes (PMNL) are usually done with pre-opsonised organisms. Phagocytosis of 11 strains of Escherichia coli, pre-opsonised, and in the stationary phase, resulted in the killing of only one strain although all the organisms were phagocytosed. However, when the same strains were added unopsonised to a PMNL-serum mixture, eight were killed after phagocytosis. With two of these strains, the amount of killing was inversely proportional to the time of pre-oposonisation. E. coli incubated for 30 min in dilute peptone water in Hanks's Balanced Salts Solution before phagocytosis also became resistant to killing; bacterial division did not occur during this period. Experiments with bacteria in urine confirmed these findings and showed that E. coli exposed to serum or urine before phagocytosis became resistant to killing by PMNL. E. coli rapidly changes its sensitivity to phagolysosome killing during transition from stationary to lag phase in a nutrient medium. This resistance is retained through the exponential phase but is lost during the stationary phase. The killing of Pseudomonas, Enterobacter, and Acinetobacter by PMNL was unaffected by varying the method of opsonisation or the phase of growth. If this phenomenon occurs in vivo it may affect the outcome of infections caused by strains of E. coli that survive killing by PMNL.     

Role of bacterial phospholipases in serum-mediated killing of Escherichia coli.

The importance of bacterial phospholipases during serum-mediated killing of Escherichia coli was examined by using wild-type DR+ DS+ and an isogenic phospholipase-deficient mutant DR- DS-. No difference in serum sensitivity was observed when the parental DR+ DS+ and mutant DR- DS- strains were exposed to various concentrations of normal guinea pig serum. Examination of the free fatty acid (FFA) and lipid composition during serum-mediated killing of the two E. coli strains indicated that FFA release occurred only in the parental DR+ DS+ strain. No FFA release or lipid degradation was detected in the mutant DR- DS- strain during serum killing. The addition of heat-inactivated E. coli antiserum (rabbit) to normal guinea pig serum caused FFA release in both E. coli strains. This FFA release was found to be independent of serum-mediated killing and due to a highly active and heat-resistant rabbit serum phospholipase that hydrolyzed the bacterial lipids after serum killing. The data presented indicate that serum-mediated killing of E. coli is independent of FFA release and that activation of bacterial phospholipases and the resulting release of FFA are only a result rather than a cause of serum-mediated cell death.     

Reduction in superoxide anion secretion and bactericidal activity of neutrophils from aged rats: reversal by the combination of gamma interferon and growth hormone.

Polymorphonuclear neutrophils (PMN) from bone marrow of 24-month-old rats kill Escherichia coli less efficiently than PMN from 3-month-old rats. Secretion of O2- and killing of E. coli by PMN from both young and old rats can be significantly augmented by preincubation with either 250 U of gamma interferon (IFN-gamma) or 250 ng of growth hormone (GH) per ml. This priming is specific, because neutralizing monoclonal antibodies against either IFN-gamma or GH completely abrogate the enhanced O2- secretion by PMN from young rats. However, in contrast to PMN from young rats, PMN from aged rats are not primed to kill E. coli by 10-fold-lower concentrations of either IFN-gamma (25 U/ml) or GH (25 ng/ml). To explore the mechanism for the reduction in bacterial killing by PMN from old rats, a syngeneic GH-secreting pituitary cell line (GH3) was implanted in vivo. PMN from GH3-treated aged rats, but not control aged rats, could now be primed in vitro for O2- secretion by IFN-gamma (25 U/ml). Although PMN from aged rats do not respond to the lower doses of either IFN-gamma or GH, the combination of both reagents totally restores the ability of PMN to secrete O2- and to kill E. coli. This synergistic priming is observed with PMN from aged rats, but not with those from young rats, and can be detected when both reagents are added simultaneously or when they are added in either sequence. Furthermore, addition of a monoclonal antibody against either IFN-gamma or GH abrogates the synergism of these two molecules. Collectively, these data identify an important alteration in myeloid cells from aged rodents by showing that their PMN are intrinsically unable to respond to low concentrations of IFN-gamma by secreting O2- and killing bacteria. The results also define a previously unrecognized synergism in PMN from aged animals by showing that GH synergizes with IFN-gamma both in vivo and in vitro to restore these suppressed responses.     

Interactions of Neisseria gonorrhoeae with adherent polymorphonuclear leukocytes

Neisseria gonorrhoeae causes severe exudative urethritis. The exudates from infected individuals contain large numbers of polymorphonuclear leukocytes (PMN) with ingested gonococci. The fate of N. gonorrhoeae within PMN has been a topic of debate for years. In this study, we examined the interactions of N. gonorrhoeae with PMN adherent to surfaces as a system that better models events during clinical disease. Using chemiluminescence to measure reactive oxygen species (ROS), we found that N. gonorrhoeae stimulated PMN to produce a respiratory burst. Different kinetics were seen when PMN were stimulated with opsonized zymosan particles. In addition, ROS were produced predominantly inside the PMN in response to gonococci. Laser scanning confocal microscopy and transmission electron microscopy showed that N. gonorrhoeae rapidly associated with PMN under these experimental conditions and was internalized. Some gonococci were cleared in the first 30 to 60 min after phagocytosis, but a majority of the population persisted for 6 h after phagocytosis. Quantification of viable organisms showed that a significant portion of the population resisted killing. The viability of this subpopulation remained unchanged for 2 h after phagocytosis. A significant increase of viable gonococci from 1 to 6 h was also observed, suggesting intracellular replication. Four different N. gonorrhoeae strains demonstrated the same capacity to resist PMN-mediated killing, whereas Escherichia coli was rapidly killed by PMN under the same conditions. Taken together, these findings suggest that a subpopulation of N. gonorrhoeae resists killing and replicates within PMN phagosomes in spite of NADPH oxidase activation.     

Toll-like receptor prestimulation increases phagocytosis of Escherichia coli DH5alpha and Escherichia coli K1 strains by murine microglial cells

Meningitis and meningoencephalitis caused by Escherichia coli are associated with high rates of mortality. When an infection occurs, Toll-like receptors (TLRs) expressed by microglial cells can recognize pathogen-associated molecular patterns and activate multiple steps in the inflammatory response that coordinate the brain's local defense, such as phagocytosis of invading pathogens. An upregulation of the phagocytic ability of reactive microglia could improve the host defense in immunocompromised patients against pathogens such as E. coli. Here, murine microglial cultures were stimulated with the TLR agonists Pam(3)CSK(4) (TLR1/TLR2), lipopolysaccharide (TLR4), and CpG oligodeoxynucleotide (TLR9) for 24 h. Upon stimulation, levels of tumor necrosis factor alpha and the neutrophil chemoattractant CXCL1 were increased, indicating microglial activation. Phagocytic activity was studied after adding either E. coli DH5alpha or E. coli K1 strains. After 60 and 90 min of bacterial exposure, the number of ingested bacteria was significantly higher in cells prestimulated with TLR agonists than in unstimulated controls (P < 0.01). Addition of cytochalasin D, an inhibitor of actin polymerization, blocked >90% of phagocytosis. We also analyzed the ability of microglia to kill the ingested E. coli strains. Intracellularly surviving bacteria were quantified at different time points (90, 150, 240, and 360 min) after 90 min of phagocytosis. The number of bacteria killed intracellularly after 6 h was higher in cells primed with the different TLR agonists than in unstimulated microglia. Our data suggest that microglial stimulation by the TLR system can increase bacterial phagocytosis and killing. This approach could improve central nervous system resistance to infections in immunocompromised patients.     

Quantitative granulocyte chemiluminescence in the rapid detection of impaired opsonization of Escherichia coli.

Previous work has demonstrated that 40% of clinically isolated Escherichia coli are resistant to in vitro killing by normal human polymorphonuclear granulocytes (PMN) due to ineffective opsonophagocytosis. Using these E. coli isolates, we have demonstrated the usefulness of measuring the chemiluminescence (CL) of granulocytes undergoing phagocytosis in detecting this impaired opsonization. CL correlated well with several other methods to assess PMN function including in vitro killing assays, postphagocytic O2 consumption, and morphological phagocytic indexes. Of the available methods to assess granulocyte function, CL is the most rapid and simple and would appear to be a potentially useful screening method to determine possible opsonic deficiencies of human PMNs and serum. Impaired opsonization to these resistant E. coli isolates was demonstrable in several different donors and could be reversed by type-specific rabbit antibody to the particular resistant isolate. Bacterial levels of superoxide dismutase and catalase, enzymes which have been implicated as possible virulence factors in some microorganisms, did not correlate with resistance in these E. coli isolates. However, heat denaturation of these isolates reversed this resistance and would suggest heat-labile antigenic determinants present in E. coli as possible resistance factors.     

Interaction of Escherichia coli with polymorphonuclear leukocytes in pathogenesis of urinary tract infection in mice.

Two type 1 fimbria-producing strains of Escherichia coli, 31-B and K12W1-3, and two type 1 fimbriae-defective mutants derived from 31-B, BH5 and BH9, were compared for their capacity to induce vesical infection in mice undergoing water diuresis and to interact in vitro with murine peritoneal exudate polymorphonuclear leukocytes (PMN). Strains 31-B and BH5 caused rapid bacterial multiplication in the bladder wall after being inoculated intrabladderly; their log-phase cells grown at 37 degrees C, in striking contrast to their stationary-phase or 17 degrees C-grown cells, resisted phagocytic killing by PMN in the presence of normal murine serum. Strains K12W1-3 and BH9 failed to cause vesical infection, and their cells were always susceptible to the opsonophagocytic killing by PMN irrespective of the growth conditions. Nevertheless, the log-phase cells of the three isogenic strains, 31-B, BH5, and BH9, grown at 37 degrees C gave almost the same chemiluminescent response patterns during incubation with PMN in normal serum. The phagocytic resistance in strains 31-B and BH5 was eliminated by briefly treating bacterial cells with EDTA. These results suggest that the two virulent strains may express an antiphagocytic activity during their growth in the bladder and continue to stimulate the oxidative metabolic burst of PMN without being ingested and killed, and that the antiphagocytic activity may be related to a bacterial surface component(s) that is removed by EDTA.     

Effect of temperature on bacterial killing by serum and by polymorphonuclear leukocytes.

Bacterial killing by serum alone and by polymorphonuclear )PMN) leukocytes was studied at 37 degrees C and compared with killing at 39 and 41 degrees C. The test organisms for serum killing were Staphylococcus aureus 502A (serum resistant) and Escherichia coli O14 (serum sensitive). The organisms used in PMN killing tests were Streptococcus pneumoniae type 29 and E. coli O86.S aureus was not killed by serum alone at any temperature. Changes in temperature did not affect the rate of serum killing of E. coli O14 for the first 60 min, but by 90 and 120 min there was a discrepancy with continued killing at 37 degrees C, but no further killing at 39 and 41 degrees C. PMN phagocytic killing of the pneumococcus was enhanced at 39 degrees C compared with 37 degrees C, and phagocytic killing of E. coli O86 was decreased at 41 degrees C when compared with 37 degrees C. Therefore, it appears that under certain circumstances fever may aid the host PMNs in destroying organisms, whereas under other circumstances it may interfere with such destruction.     

Electron microscopic study of phagocytosis of Escherichia coli by human polymorphonuclear leukocytes.

The fate of Escherichia coli strains within the polymorphonuclear leukocytes was studied by determining the killing of bacteria, measuring the release of degradation products, and examining the phagocytic bacteria by electron microscopy. When sufficiently opsonized, both unencapsulated and encapsulated E. coli strains were rapidly phagocytized by polymorphonuclear leukocytes. Once phagocytized, the two unencapsulated E. coli strains (K-12 and O111) were rapidly killed (99% of the bacteria were killed during the first 5 min of phagocytosis) and extensively degraded (about 40% of the radiolabeled material was released from bacteria after 15 min of phagocytosis). Electron micrographs taken after 15 min of phagocytosis revealed extensive structural changes in most of the internalized bacteria. In contrast to the rapid killing and extensive breakdown of these strains, encapsulated E. coli O78:K80 was more resistant to killing and withstood degradation by polymorphonuclear leukocytes (only 5% of the radioactivity was released from the radiolabeled bacteria after 1 h of phagocytosis). Electron micrographs of thin sections taken after 1 h of phagocytosis revealed virtually no structural changes. Most of the internalized bacteria were still surrounded by thick capsular material.     

Lactoferrin release and interleukin-1, interleukin-6, and tumor necrosis factor production by human polymorphonuclear cells stimulated by various lipopolysaccharides: relationship to growth inhibition of Candida albicans.

Lipopolysaccharides (LPSs) from Escherichia coli, Serratia marcescens, and Salmonella typhimurium, at doses from 1 to 100 ng/ml, strongly enhanced growth inhibition of Candida albicans by human polymorphonuclear leukocytes (PMN) in vitro. Flow cytometry analysis demonstrated that LPS markedly augmented phagocytosis of Candida cells by increasing the number of yeasts ingested per neutrophil as well as the number of neutrophils capable of ingesting fungal cells. LPS activation caused augmented release of lactoferrin, an iron-binding protein which itself could inhibit the growth of C. albicans in vitro. Antibodies against lactoferrin effectively and specifically reduced the anti-C. albicans activity of both LPS-stimulated and unstimulated PMN. Northern (RNA blot) analysis showed enhanced production of mRNAs for interleukin-1 beta, tumor necrosis factor alpha, and interleukin-6 and in neutrophils within 1 h of stimulation with LPS. The cytokines were also detected in the supernatant of the activated PMN, and their synthesis was prevented by pretreatment of LPS-stimulated PMN with protein synthesis inhibitors, such as emetine and cycloheximide. These inhibitors, however, did not block either lactoferrin release or the anti-Candida activity of LPS-stimulated PMN. These results demonstrate the ability of various bacterial LPSs to augment neutrophil function against C. albicans and suggest that the release of a candidastatic, iron-binding protein, lactoferrin, may contribute to the antifungal effect of PMN. Moreover, the ability to produce cytokines upon stimulation by ubiquitous microbial products such as the endotoxins points to an extraphagocytic, immunomodulatory role of PMN during infection.     

Leptin improves pulmonary bacterial clearance and survival in ob/ob mice during pneumococcal pneumonia

The adipocyte-derived hormone leptin is an important regulator of appetite and energy expenditure and is now appreciated for its ability to control innate and adaptive immune responses. We have reported previously that the leptin-deficient ob/ob mouse exhibited increased susceptibility to the Gram-negative bacterium Klebsiella pneumoniae. In this report we assessed the impact of chronic leptin deficiency, using ob/ob mice, on pneumococcal pneumonia and examined whether restoring circulating leptin to physiological levels in vivo could improve host defences against this pathogen. We observed that ob/ob mice, compared with wild-type (WT) animals, exhibited enhanced lethality and reduced pulmonary bacterial clearance following Streptococcus pneumoniae challenge. These impairments in host defence in ob/ob mice were associated with elevated levels of lung tumour necrosis factor (TNF)-alpha, macrophage inflammatory peptide (MIP)-2 [correction added after online publication 28 September 2007: definition of MIP corrected], prostaglandin E(2) (PGE(2)), lung neutrophil polymorphonuclear leukocyte (PMN) counts, defective alveolar macrophage (AM) phagocytosis and PMN killing of S. pneumoniae in vitro. Exogenous leptin administration to ob/ob mice in vivo improved survival and greatly improved pulmonary bacterial clearance, reduced bacteraemia, reconstituted AM phagocytosis and PMN H(2)O(2) production and killing of S. pneumoniae in vitro. Our results demonstrate, for the first time, that leptin improves pulmonary bacterial clearance and survival in ob/ob mice during pneumococcal pneumonia. Further investigations are warranted to determine whether there is a potential therapeutic role for this adipokine in immunocompromised patients.     

Ascorbate and phagocyte function.

Scorbutic guinea pig neutrophils (PMN) were found to produce H2O2 and kill Staphylococcus aureus as well as control PMN, suggesting that ascorbate does not contribute significantly to phagocyte H2O2 production or bacterial killing. Total and reduced ascorbate contents of human PMN was observed to fall upon phagocytosis, whereas dehydroascorbate increased to a lesser extent. These observations are consistent with the view that ascorbate constitutes a functional part of the PMN's redox-active components and may thus function to protect cell constituents from denaturation by the oxidants produced during phagocytosis.     

Effect of pH and osmolality on in vitro phagocytosis and killing by neutrophils in urine.

Phagocytosis and intracellular killing of two strains of Escherichia coli and a Staphylococcus saprophyticus by polymorphonuclear neutrophils (PMN) in pooled sterile urine at three osmolalities (800, 485, and 200 mosM/kg of H2O) between pHs 5 and 8 was investigated. Urine at 800 mosM virtually abolished phagocytosis of both E. coli strains, regardless of pH, and reduced the phagocytosis of S. saprophyticus to 30%; no killing of any organisms took place at this osmolality. On the other hand, phagocytosis was a good in urine as in Hanks balanced salt solution at both 485 and 200 mosM between pHs 6 and 8. Phagocytosis of all three strains was virtually abolished at pH 5. Killing of the strains by PMN was optimal between pHs 6.5 and 7.5 in urine at 485 mosM (being at least 90% of the control values in Hanks balanced salt solution), whereas at 200 mosM killing was reduced to 50 to 70% of these values. Reduced killing of all three strains occurred at pH 8, whereas at pH 6 only S. saprophyticus was killed. Thus, the bactericidal activity of PMN in urine was more sensitive than phagocytic function to alterations in pH. The dominant modulating factor affecting PMN function in urine of 500 mosM or less was pH, but osmolality had a greater influence at 800 mosM. Thus, raising the pH of urine and reducing the osmolality may increase the ability of natural defense mechanisms to eliminate infecting organisms.     

Alteration of bacteria induced by subinhibitory concentrations of cefixime: consequences on bactericidal activity of human polynuclear neutrophils]

Subinhibitory concentrations of most parenteral cephalosporins have been reported to alter bacterial infectivity and, in particular, to increase the susceptibility of altered bacteria to the killing effects of polymorphonuclear neutrophils (PMN). Few data on this issue are available for oral cephalosporins. This study investigated the effects of sub-MIC concentrations of the new oral cephalosporin cefixime on two bacterial targets, i.e., S. aureus 209P (MIC 20 mg/l) and E. coli K12 (MIC 0.15 mg/l). After overnight incubation (18 hours) with 10 or 5 mg/l cefixime, susceptibility of S. aureus to the killing effects of PMNs was increased two-fold as compared with control organisms and susceptibility to the O2-independent PMN bactericidal system (PMN extract) was also increased. In contrast, the susceptibility of E. coli to PMN and to cell-free bactericidal systems was identical for cefixime-exposed strains (0.1 and 0.05 mg/l) and for unexposed controls. However, cefixime-exposed E. coli were filamentous, suggesting that bactericidal efficacy in terms of the bacterial mass eliminated was enhanced in exposed strains. These data show that low levels of cefixime are capable of producing major alterations in susceptible and resistant bacteria and of increasing their susceptibility to PMN. These effects may be relevant in vivo, in particular when low concentrations of antibiotics persist over long periods in infected sites.     

Bactericidal action of eosinophils from normal human blood.

The ability of normal human eosinophils to ingest and kill Staphylococcus aureus and Escherichia coli was investigated and compared with the reactions shown by neutrophils from the same donors. The rate of phagocytosis of S. aureus by eosinophils was 50% of that shown by neutrophils. Unlike neutrophils, eosinophils were not able to kill ingested S. aureus at low bacterium/phagocyte ratios. The degree of S. aureus killing increased with increasing ratios, being equal to that of neutrophils when bacterium/phagocyte ratios of about 15 were used. This was probably due to a better triggering of the eosinophil oxidase system at high bacterium/phagocyte ratios. The early kinetics of the association of bacteria with eosinophils, the perforation of the bacterial envelope and the inactivation of bacterial proteins, was monitored in the ML-35 mutant strain of E. coli. The association of E. coli with eosinophils was 70% of that with neutrophils. Eosinophils had only 25% of the capacity of neutrophils to perforate the E. coli envelope. E. coli loses its colony-forming ability when the bacterial envelope has been perforated, indicating that eosinophils also kill E. coli more slowly than do neutrophils. This was confirmed with a plating assay for colony formation. The perforation of E. coli is independent of peroxidase-mediated reactions. Hence, the defective bactericidal action of eosinophils is probably not related to the differences between myeloperoxidase and eosinophil peroxidase. On the other hand, the inactivation of bacterial proteins is peroxidase dependent and was also seen to occur to a lesser extent in eosinophils compared with neutrophils. We conclude that eosinophils ingest E. coli but only slowly perforate (kill) these bacteria and barely inactivate the bacterial enzymes. In contrast, neutrophils quickly ingest and perforate (kill) E. coli and quickly inactivate the bacterial enzymes.     

Wall teichoic acid deficiency in Staphylococcus aureus confers selective resistance to mammalian group IIA phospholipase A(2) and human beta-defensin 3

Wall teichoic acids (WTAs) and membrane lipoteichoic acids (LTAs) are the major polyanionic polymers in the envelope of Staphylococcus aureus. WTAs in S. aureus play an important role in bacteriophage attachment and bacterial adherence to certain host cells, suggesting that WTAs are exposed on the cell surface and could also provide necessary binding sites for cationic antimicrobial peptides and proteins (CAMPs). Highly cationic mammalian group IIA phospholipase A(2) (gIIA PLA(2)) kills S. aureus at nanomolar concentrations by an action(s) that depends on initial electrostatic interactions, cell wall penetration, membrane phospholipid (PL) degradation, and activation of autolysins. A tagO mutant of S. aureus that lacks WTA is up to 100-fold more resistant to PL degradation and killing by gIIA PLA(2) and CAMP human beta-defensin 3 (HBD-3) but has the sensitivity of the wild type (wt) to other CAMPs, such as Magainin II amide, hNP1-3, LL-37, and lactoferrin. In contrast, there is little or no difference in either gIIA PLA(2) activity toward cell wall-depleted protoplasts of the wt and tagO strains of S. aureus or in binding of gIIA PLA(2) to wt and tagO strains. Scanning and transmission electron microscopy reveal increased surface protrusions in the S. aureus tagO mutant that might account for reduced activity of bound gIIA PLA(2) and HBD-3 toward the tagO mutant. In summary, the absence of WTA in S. aureus causes a selective increase in bacterial resistance to gIIA PLA(2) and HBD-3, the former apparently by reducing access and/or activity of bound antibacterial enzyme to the bacterial membrane.