Phosphatidylethanolamine of Helicobacter pylori functions as a steroid-binding lipid in the assimilation of free cholesterol and 3β-hydroxl steroids into the bacterial cell membrane

One of the unique features of Helicobacter pylori is its ability to assimilate free-cholesterol (FC) into its membranes. Via FC assimilation, H. pylori strengthens the membrane lipid barrier and/or evades the host immune system. No previous studies, however, have investigated the FC uptake mechanisms of the H. pylori cell. Phosphatidylethanolamine (PE) is the most prevalent lipid component of bacteria, including H. pylori, but the function of PE remains unclear. We were therefore interested in H. pylori PE (HpPE) and investigated the interaction of its PE with cholesterols. The PE isolated from H. pylori underwent a unique molecular interaction with FC, cholesterol ester (CE), and 2,6-di-O-methyl-β-cyclodextrin (dMβCD), a sterol solubilizer. HpPE interacted not only with the FC molecule, but also with the FC-dMβCD inclusion complex. In contrast, Escherichia coli PE (EcPE), prepared as a reference PE, seemed to bind only FC, and only via a hydrophobic interaction, without binding dMβCD. HpPE was clearly more potent than EcPE in binding FC. Intriguingly, HpPE had a negligible affinity for CE, while EcPE had a high affinity for CE, comparable to its affinity for FC. Further, HpPE interacted with 3β-OH steroids, pregnenolone and dehydroepiandrosterone, in the absence of dMβCD. Gas chromatogram-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) analyses revealed that the fatty acid compositions of HpPE were quite distinct from those of EcPE, and the C(14:0) fatty acid in the HpPE molecule was found to be significant in binding FC selectively. These results indicate that PE is a key candidate of nonesterified steroid-binding lipids in H. pylori.     

Selective and effective bactericidal activity of the cobalt (II) cation against Helicobacter pylori

BACKGROUND: Although the anti-Helicobacter pylori activity of bismuth is well established, the therapeutic potential of other metal ions against the organism is not known. MATERIALS AND METHODS: We measured the minimum inhibitory concentrations of a series of metal ions, including several cobalt (II) compounds against four type strains and seven clinical isolates of H. pylori using three standard broth culture media and a defined medium. Other intestinal bacteria were also investigated for specificity of action. RESULTS: Cobalt chloride had marked activity against H. pylori (minimum inhibitory concentration range was 0.03-1.0 mg/l). The effect was specific because other transition metals had no effect and other intestinal bacteria were not affected by cobalt chloride. Activity was attributable to free cobalt ions as ligands inhibited activity in proportion to their affinity for the ions. Inhibition of cobalt activity was also observed in the presence of nickel, in a dose dependent fashion. However, cobalt activity was not directed towards the nickel-dependent urease enzyme because its effect was similar in wild-type and urease negative mutant strains of H. pylori. Finally, the viability of H. pylori was reduced at the same rate with 2 mg/l cobalt as with 1 mg/l amoxicillin. CONCLUSIONS: Cobalt competes for nickel in its acquisition by H. pylori, but mediates toxicity in a nonurease dependent fashion. As cobalt MIC is similar to some antibiotics and 10 to a hundred times lower than for bismuth, cobalt may represent an effective form of therapy for H. pylori infection.     

Anti-Helicobacter pylori activity of the methanolic extract of Geum iranicum and its main compounds

Geum iranicum Khatamsaz, belonging to the Rosaceae family, is an endemic plant of Iran. The methanol extract of the roots of this plant showed significant activity against one of the clinical isolates of Helicobacter pylori which was resistant to metronidazole. The aim of this study was the isolation and evaluation of the major compounds of G. iranicum effective against H. pylori. The compounds were isolated using various chromatographic methods and identified by spectroscopic data (1H and 13C NMR, HMQC, HMBC, EI-MS). An antimicrobial susceptibility test was performed employing the disk diffusion method against clinical isolates of H. pylori and a micro dilution method against several Gram-positive and Gram-negative bacteria; additionally the inhibition zone diameters (IZD) and minimum inhibitory concentrations (MIC) values were recorded. Nine compounds were isolated: two triterpenoids, uvaol and niga-ichigoside F1, three sterols, beta-sitosterol, beta-sitosteryl acetate, and beta-sitosteryl linoleate, one phenyl propanoid, eugenol, one phenolic glycoside, gein, one flavanol, (+)-catechin, and sucrose. The aqueous fraction, obtained by partitioning the MeOH extract with water and chloroform, was the most effective fraction of the extract against all clinical isolates of H. pylori. Further investigation of the isolated compounds showed that eugenol was effective against H. pylori but gein, diglycosidic eugenol, did not exhibit any activity against H. pylori. The subfraction D4 was the effective fraction which contained tannins. It appeared that tannins were probably the active compounds responsible for the anti-H. pylori activity of G. iranicum. The aqueous fraction showed a moderate inhibitory activity against both Gram-positive and Gram-negative bacteria. The MIC values indicated that Gram-positive bacteria including Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus subtilis are more susceptible than Gram-neagative bacteria including Escherichia coli and Pseudomonas aeruginosa.     

Structural and functional analysis of the riboflavin synthesis genes encoding GTP cyclohydrolase II (ribA), DHBP synthase (ribBA), riboflavin synthase (ribC), and riboflavin deaminase/reductase (ribD) from Helicobacter pylori strain P1

The functions of the riboflavin synthesis gene homologues ribA, ribBA, ribC, and ribD from Helicobacter pylori strain P1 were confirmed by complementation of defined Escherichia coli mutant strains. The H. pylori ribBA gene, which is similar to bifunctional ribBA genes of Gram-positive bacteria, fully complemented the ribB mutation and partially restored growth in a ribC mutant. However, ribBA did not complement the ribA mutation in E. coli, thus explaining the presence of the additional separate copy of the ribA gene in the H. pylori chromosome. In E. coli exclusively ribA conferred hemolytic activity and gave rise to production of molecules with fluorescence characteristics similar to flavins, as observed earlier. The E. coli hemolysin ClyA was not involved in causing the hemolytic phenotype. No riboflavin synthesis genes on plasmids conferred iron uptake functions to a siderophore-deficient mutant of E. coli. Marker exchange mutagenesis of the genes in H. pylori was not successful indicating that riboflavin synthesis is essential for basic metabolic functions of the gastric pathogen.     

Steroid hormones as bactericidal agents to Helicobacter pylori

Helicobacter pylori is a unique bacterial species that assimilates various steroids as membrane lipid components. Our group has recently found, however, that certain steroids may impair the viability of H. pylori. In this study, we go on to reveal that estradiol, androstenedione, and progesterone (PS) all have the potential to inhibit the growth of H. pylori. Of these three steroid hormones, progesterone demonstrated the most effective anti-H. pylori action. 17α-hydroxyprogesterone caproate (17αPSCE), a synthetic progesterone derivative, had a much stronger anti-H. pylori action than progesterone, whereas 17α-hydroxyprogesterone, a natural progesterone derivative, completely failed to inhibit the growth of the organism. Progesterone and 17αPSCE were both found to kill H. pylori through their bacteriolytic action. Among five bacterial species investigated, H. pylori was the only species susceptible to the bactericidal action of progesterone and 17αPSCE. The other four species, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epiderimidis, all resisted this action. Progesterone and free-cholesterol (FC) obstructed each other's effects against the H. pylori cell. Taken in sum, these results suggest that progesterone and FC may bind to the identical region on the H. pylori cell surface. We expect these findings to contribute to the development of a novel anti-H. pylori steroidal agent.     

Unique cholesteryl glucosides in Helicobacter pylori: composition and structural analysis.

A chloroform-methanol-extracted lipid of Helicobacter pylori was studied. Three kinds of glycolipids, accounting for about 25% (wt/wt) of the total lipid, were detected and identified to be cholesteryl glucosides. The structures of two of them were determined to be cholesteryl-alpha-D-glucopyranoside and cholesteryl-6-O-tetrade-canoyl-alpha-D-glucopyranoside, and the plausible structure of the third one was identified as cholesteryl-6-O-phosphatidyl-alpha-D-glucopyranoside. Cholesteryl glucosides are very rare in animals and bacteria. Furthermore, those in H. pylori had an alpha-glycosidic linkage, which is rather unusual for natural glycosides, and a phosphate-linked cholesteryl glycoside like the cholesteryl-6-O-phosphatidyl-alpha-D-glucopyranoside has not been reported previously. As the cholesterol glucosides were detected in strains obtained from diverse geographical locations, the presence of cholesteryl glucosides in H. pylori is a very unique and a characteristic feature of the species. These findings add a new facet to the physiology and biochemistry, especially the cholesterol and glucose metabolism, of H. pylori. Furthermore, the cholesteryl glucosides of H. pylori showed hemolytic activities.     

Intracellular redox status and antibiotic resistance in enterogastric micro-aerophilic bacteria: evidence for the 'scavenging of oxygen' hypothesis

Metronidazole and glutathione reduction activities were measured in situ in the micro-aerophilic bacteria Campylobacter coli and Helicobacter pylori employing 14N- and 1H-nuclear magnetic resonance spectroscopy. The properties of these enzyme activities were investigated in matched pairs of strains with sensitive and resistant phenotypes to the antimicrobial metronidazole. The results indicated that the ability of each type of strain to reduce metronidazole corresponded to its sensitive or resistance phenotype. Higher levels of glutathione reduction and a significantly lower Ki for metronidazole were observed in sensitive strains compared to resistant strains. These findings suggested a relationship between the cellular machinery regulating intracellular redox status in C. coli and H. pylori, and the effects of metronidazole on these bacteria, which supported the 'scavenging of oxygen' hypothesis.     

Interleukin-6 induction by Helicobacter pylori in human macrophages is dependent on phagocytosis

BACKGROUND: The colonization of the gastric mucosa with Helicobacter pylori is accompanied by elevated levels of proinflammatory cytokines, such as interleukin-1 (IL-1), IL-6, and IL-8. The aim of our study was to determine the mechanisms of IL-6 stimulation in phagocytes upon H. pylori infection. MATERIALS AND METHODS: We investigated the secretion of IL-6 by different professional phagocytes from murine and human origin, including granulocyte- and monocyte-like cells and macrophages derived from human peripheral blood monocytes (PBMCs). The influence of viability, phagocytosis, and the impact of different subcellular fractions of H. pylori bacteria were evaluated. RESULTS: IL-6 levels induced by H. pylori were low in cell lines derived from murine and human monocytes and in human granulocyte-like cells. By contrast, macrophages derived from human PBMCs were highly responsive to both H. pylori and Escherichia coli. IL-6 induction was blocked by inhibition of actin-dependent processes prior to infection with H. pylori, but not with E. coli or E. coli lipopolysaccharide (LPS). Using cell fractionation, the most activity was found in the H. pylori membrane. H. pylori LPS exhibited a 10(3)- to 10(4)-fold lower biologic activity than E. coli LPS, suggesting a minor role for toll-like receptor 4 (TLR4)-mediated signalling from the exterior. CONCLUSIONS: From these data, we conclude that macrophages may be a major source of IL-6 in the gastric mucosa upon H. pylori infection. The IL-6 induction by H. pylori in these cells is a multifactorial process, which requires the uptake and presumably degradation of H. pylori bacteria.     

Restriction-modification system differences in Helicobacter pylori are a barrier to interstrain plasmid transfer

Helicobacter pylori cells are naturally competent for the uptake of both plasmid and chromosomal DNA. However, we demonstrate that there are strong barriers to transformation of H. pylori strains by plasmids derived from unrelated strains. We sought to determine the molecular mechanisms underlying these barriers. Transformation efficiency was assessed using pHP1, an Escherichia coli-H. pylori shuttle vector conferring kanamycin resistance. Transformation of 33 H. pylori strains was attempted with pHP1 purified from either E. coli or H. pylori, and was successfully introduced into only 11 strains. Digestion of H. pylori chromosomes with different restriction endonucleases (REs) showed that DNA methylation patterns vary substantially among strains. The strain most easily transformed, JP26, was found to have extremely low endogenous RE activity and to lack a restriction-modification (R-M) system, homologous to MboI, which is highly conserved among H. pylori strains. When we introduced this system to JP26, pHP1 from MboI.M+ JP26, but not from wild-type JP26, transformed MboI R-M+ JP26 and heterologous MboI R-M+ wild-type H. pylori strains. Parallel studies with pHP1 from dam+ and dam- E. coli strains confirmed these findings. These data indicate that the endogenous REs of H. pylori strains represent a critical barrier to interstrain plasmid transfer among H. pylori.     

Natural transformation of an engineered Helicobacter pylori strain deficient in type II restriction endonucleases

Restriction-modification (RM) systems are important for bacteria to limit foreign DNA invasion. The naturally competent bacterium Helicobacter pylori has highly diverse strain-specific type II systems. To evaluate the roles of strain-specific restriction in H. pylori natural transformation, a markerless type II restriction endonuclease-deficient (REd) mutant was constructed. We deleted the genes encoding all four active type II restriction endonucleases in H. pylori strain 26695 using sacB-mediated counterselection. Transformation by donor DNA with exogenous cassettes methylated by Escherichia coli was substantially (1.7 and 2.0 log(10) for cat and aphA, respectively) increased in the REd strain. There also was significantly increased transformation of the REd strain by donor DNA from other H. pylori strains, to an extent corresponding to their shared type II R-M system strain specificity with 26695. Comparison of the REd and wild-type strains indicates that restriction did not affect the length of DNA fragment integration during natural transformation. There also were no differentials in cell growth or susceptibility to DNA damage. In total, the data indicate that the type II REd mutant has enhanced competence with no loss of growth or repair facility compared to the wild type, facilitating H. pylori mutant construction and other genetic engineering.     

The effect of the cag pathogenicity island on binding of Helicobacter pylori to gastric epithelial cells and the subsequent induction of apoptosis

BACKGROUND: Helicobacter pylori infection leads to gastritis, peptic ulcer, and gastric cancer, in part due to epithelial damage following bacteria binding to the epithelium. Infection with cag pathogenicity island (PAI) bearing strains of H. pylori is associated with increased gastric inflammation and a higher incidence of gastroduodenal diseases. It is now known that various effector molecules are injected into host epithelial cells via a type IV secretion apparatus, resulting in cytoskeletal changes and chemokine secretion. Whether binding of bacteria and subsequent apoptosis of gastric epithelial cells are altered by cag PAI status was examined in this study. METHODS: AGS, Kato III, and N87 human gastric epithelial cell lines were incubated with cag PAI-positive or cag PAI-negative strains of H. pylori in the presence or absence of clarithromycin. Binding was evaluated by flow cytometry and scanning electron microscopy. Apoptosis was assessed by detection of DNA degradation and ELISA detection of exposed histone residues. RESULTS: cag PAI-negative strains bound to gastric epithelial cells to the same extent as cag PAI-positive strains. Both cag PAI-positive and cag PAI-negative strains induced apoptosis. However, cag PAI-positive strains induced higher levels of DNA degradation. Incubation with clarithromycin inactivated H. pylori but did not affect binding. However, pretreatment with clarithromycin decreased infection-induced apoptosis. CONCLUSIONS: cag PAI status did not affect binding of bacteria to gastric epithelial cells but cag PAI-positive H. pylori induced apoptosis more rapidly than cag PAI-negative mutant strains, suggesting that H. pylori binding and subsequent apoptosis are differentially regulated with regard to bacterial properties.     

Adhesion of coagulase-negative staphylococci to biomaterials

The adhesion of two Staphylococcus epidermidis strains and one Staphylococcus saprophyticus strain on to poly(tetrafluorethylene-co-hexafluorpropylene) (FEP)-fluorocarbon and cellulose acetate was studied in vitro. Both S. epidermidis strains showed a more hydrophobic character than the encapsulated S. saprophyticus as determined by the bacterial affinity towards xylene. Staphylococcus epidermidis showed a significantly higher adhesion on to the hydrophobic FEP than S. saprophyticus. The adhesion of staphylococci on to the more hydrophilic cellulose acetate was always low. Treatment of S. epidermidis with pepsin or extraction with aqueous phenol yielded cells with a decreased hydrophobicity, which resulted in a decreased adhesion on to FEP. Cells with a decreased hydrophobicity showed a lower rate of reaggregation in suspension. The hydrophobicity and the adhesion on the FEP of S. epidermidis were not affected by exposure to a subminimal inhibitory concentration of penicillin. The strong interaction between S. epidermidis and FEP, which appeared not to be influenced by the age or the metabolic stage of the bacteria, is mainly caused by hydrophobic bonding.     

幽门螺杆菌CagA蛋白N端片段的表达、纯化及其与磷脂酰丝氨酸的亲和力检测

目的:表达和纯化幽门螺杆菌不同菌株的CagA蛋白N端片段,检测其与磷脂酰丝氨酸(PS)的相互作用及亲和力。方法:用PCR方法从幽门螺杆菌3个菌株中扩增出CagA蛋白N端基因,并连接到表达载体pET-28a上;转化大肠杆菌BL21,经IPTG诱导可溶性表达CagA蛋白N端880残基片段;经镍柱亲和纯化后,利用PLOA法检测CagA蛋白与PS的相互作用。结果:构建了3种幽门螺杆菌菌株cagA基因的原核表达质粒pET-28a/cagAJ99、pET-28a/cagA11637及pET-28a/cagASS1,并在大肠杆菌中获得可溶性表达,SDS-PAGE和Western印迹证实得到目标融合蛋白,亲和纯化得到高纯度CagA蛋白。PLOA结果表明,CagA蛋白与PS有明显的相互作用。结论:3种幽门螺杆菌菌株CagA蛋白与PS之间存在相互作用,且不同的CagA与PS有不同的亲和力。     

Cloning and characterization of spermidine synthase and its implication in polyamine biosynthesis in Helicobacter pylori strain 26695

The HP0832 (speE) gene of Helicobacter pylori strain 26695 codes for a putative spermidine synthase, which belongs to the polyamine biosynthetic pathway. Spermidine synthase catalyzes the production of spermidine from putrescine and decarboxylated S-adenosylmethionine (dcSAM), which serves as an aminopropyl donor. The deduced amino acid sequence of the HP0832 gene shares less than 20% sequence identity with most spermidine synthases from mammalian cells, plants and other bacteria. In this study, the HP0832 open reading frame (786 bp) was cloned into the pQE30 vector and overexpressed in Escherichia coli strain SG13009. The resulting N-terminally 6xHis-tagged HP0832 protein (31.9 kDa) was purified by Ni-NTA affinity chromatography at a yield of 15 mg/L of bacteria culture. Spermidine synthase activity of the recombinant protein was confirmed by the appearance of spermidine after incubating the enzyme with putrescine and dcSAM. Substrate specificity studies have shown that spermidine could not replace putrescine as the aminopropyl acceptor. Endogenous spermidine synthase of H. pylori was detected with an antiserum raised against the recombinant HP0832 protein. H. pylori strain 26695 contains putrescine and spermidine at a molar ratio of 1:3, but no detectable spermine or norspermidine was observed, suggesting that the spermidine biosynthetic pathway may provide the main polyamines in H. pylori strain 26695.     

Antibacterial action of a heat-stable form of L-amino acid oxidase isolated from king cobra (Ophiophagus hannah) venom

The major l-amino acid oxidase (LAAO, EC 1.4.3.2) of king cobra (Ophiophagus hannah) venom is known to be an unusual form of snake venom LAAO as it possesses unique structural features and unusual thermal stability. The antibacterial effects of king cobra venom LAAO were tested against several strains of clinical isolates including Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli using broth microdilution assay. For comparison, the antibacterial effects of several antibiotics (cefotaxime, kanamycin, tetracycline, vancomycin and penicillin) were also examined using the same conditions. King cobra venom LAAO was very effective in inhibiting the two Gram-positive bacteria (S. aureus and S. epidermidis) tested, with minimum inhibitory concentration (MIC) of 0.78μg/mL (0.006μM) and 1.56μg/mL (0.012μM) against S. aureus and S. epidermidis, respectively. The MICs are comparable to the MICs of the antibiotics tested, on a weight basis. However, the LAAO was only moderately effective against three Gram-negative bacteria tested (P. aeruginosa, K. pneumoniae and E. coli), with MIC ranges from 25 to 50μg/mL (0.2-0.4μM). Catalase at the concentration of 1mg/mL abolished the antibacterial effect of LAAO, indicating that the antibacterial effect of the enzyme involves generation of hydrogen peroxide. Binding studies indicated that king cobra venom LAAO binds strongly to the Gram-positive S. aureus and S. epidermidis, but less strongly to the Gram-negative E. coli and P. aeruginosa, indicating that specific binding to bacteria is important for the potent antibacterial activity of the enzyme.     

Induction of human beta-defensin-2 mRNA expression by Helicobacter pylori in human gastric cell line MKN45 cells on cag pathogenicity island.

Helicobacter pylori is an etiological agent of gastritis, peptic ulcer, and gastric cancer. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide which belongs to one of the most important host defense systems against bacterial infection in several epithelial tissues. We studied the effect of H. pylori on the expression of hBD-2 mRNA in MKN45 gastric mucosal cells. H. pylori, but not culture filtrate, increased the hBD-2 mRNA level in MKN45 cells; the inductive effect of H. pylori was not detected with Intestine 407 cells. Among H. pylori strains, strain OHPC0002, which lacks a cag Pathogenicity Island (PAI), did not induce hBD-2 mRNA in MKN45 cells. These results suggested that H. pylori cag PAI is critical for the induction of hBD-2 mRNA in MKN45 cells. Exposure of MKN45 cells to Salmonella typhimurium, S. enteritidis, S. typhi, and S. dublin, but not Escherichia coli ML35, also resulted in induction of hBD-2 mRNA.     

Mutagenic activity of certain synthetic steroids: structural requirement for the mutagenic activity in Salmonella and E. coli

Eight steroids, structurally related to cholesterol, were tested for mutagenic activity in the Ames tester strains. All the test compounds were mutagenic without metabolic activation, although metabolic activation markedly enhanced their activity. A significant decrease in the survival of the radiation-sensitive mutants recA and lexA of Escherichia coli was observed as compared to their wild-type counterpart in the presence of the steroids. The role of recA and lexA genes gains further support from the lambda prophage induction in the lysogen as well as with Salmonella strains triggering the error-prone SOS response. Structural features which appear to be essential for mutagenic activity in these strains of the steroids are (1) reactive thio, sulfonyl or sulfinyl groups at the 6 position and (2) a halogen group at the 3 position of the steroidal nucleus. The mutagenicity appears to involve the formation of H2O2 as well as superoxide and hydroxyl radicals.     

Different Penicillin-Binding Protein Profiles in Amoxicillin-Resistant Helicobacter pylori

Background. The β-lactam group of antibiotics kills bacteria by inhibiting the terminal stages of peptidoglycan metabolism. We have recently identified amoxicillin-resistant Helicobacter pylori , none of which expressed β-lactamase. Penicillin-binding proteins (PBPs) represent a group of target enzymes for the β-lactam antibiotic family, and alterations in PBPs have been described in other penicillin-resistant bacteria. The amoxicillin-resistant phenotype characteristically was lost after freezing but could be restored by consecutive transfers into gradient plates.
Materials and Methods. To determine whether amoxicillin resistance in H. pylori was related to alterations in any of the H. pylori PBPs, five H. pylori strains resistant to amoxicillin and three amoxicillin-sensitive strains were tested. PBPs were extracted from bacteria grown to logarithmic phase, labeled in vivo with ³H-benzylpenicillin, and analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Four main PBPs were separated from all amoxicillin-sensitive H. pylori strains.
Results. Only three of the four main PBPs were found in the amoxicillin-resistant H. pylori strains. The differentially detectable PBP (PBP D) had an apparent molecular weight of 30 to 32 kD.
Conclusion. These results suggest that PBP D might play a role in the amoxicillin-resistant phenotype of H. pylori strains lacking β-lactamase activity.     

Surface phagocytosis of Staphylococcus epidermidis and Escherichia coli by human neutrophils: serum requirements for opsonization and chemiluminescence

We examined the serum requirements for surface phagocytosis of Staphylococcus epidermidis and Escherichia coli and for the subsequent chemiluminescent response of human neutrophils. Substantial surface phagocytosis of S. epidermidis occurred in the absence of opsonins, although the presence of 10% pooled or heat-inactivated serum significantly increased phagocytosis. There was no significant difference between these opsonins, indicating that surface phagocytosis of S. epidermidis did not require complement. Unopsonized E. coli were not as readily phagocytized as S. epidermidis (33% versus 57%). In contrast to S. epidermidis optimal phagocytosis of E. coli required complement as 10% heat inactivated donor serum (HHS) was significantly less effective as an opsonin than 10% pooled healthy donor serum (PHS). The time kinetics for phagocytosis of each organism were similar, with most of the phagocytosis occurring in the first 10 min. The chemiluminescent response of neutrophils produced discrepant results. Maximal chemiluminescence was observed when neutrophils were stimulated with bacteria opsonized in PHS. The response to HHS-opsonized bacteria was less, and chemiluminescence to unopsonized bacteria was only marginally higher than the control, even though there was relatively good phagocytosis. These results define the opsonic requirements for surface phagocytosis of S. epidermidis and E. coli and indicate that although complement may not be required for phagocytosis, it is necessary for generation of a maximal oxidative burst, and thus may be essential for efficient intracellular killing.