NorA efflux pump mediates Staphylococcus aureus response to Pseudomonas aeruginosa pyocyanin toxicity.

Endogenous transporters protect Staphylococcus aureus against antibiotics and also contribute to bacterial defense from environmental toxins. We evaluated the effect of overexpression of four efflux pumps, NorA, NorB, NorC, and Tet38, on S. aureus survival following exposure to pyocyanin (PYO) of Pseudomonas aeruginosa, using a well diffusion assay. We measured the PYO-created inhibition zone and found that only an overexpression of NorA reduced S. aureus susceptibility to pyocyanin killing. The MICPYO of the NorA overexpressor increased threefold compared to that of wild-type RN6390 and was reduced 2.5-fold with reserpine, suggesting that increased NorA efflux caused PYO resistance. The PYO-created inhibition zone of a ΔnorA mutant was consistently larger than that of a plasmid-borne NorA overexpressor. PYO also produced a modest increase in norA expression (1.8-fold at 0.25 µg/mL PYO) that gradually decreased with increasing PYO concentrations. Well diffusion assays carried out using P. aeruginosa showed that ΔnorA mutant was less susceptible to killing by PYO-deficient mutants PA14phzM and PA14phzS than to killing by PA14. NorA overexpression led to reduced killing by all tested P. aeruginosa. We evaluated the NorA-PYO interaction using a collection of 22 clinical isolates from adult and pediatric cystic fibrosis (CF) patients, which included both S. aureus (CF-SA) and P. aeruginosa (CF-PA). We found that when isolated alone, CF-PA and CF-SA expressed varying levels of PYO and norA transcripts, but all four CF-PA/CF-SA pairs isolated concurrently from CF patients produced a low level of PYO and low norA transcript levels, respectively, suggesting a partial adaptation of the two bacteria in circumstances of persistent co-colonization.

Phenazine-1 carboxylic acid of Pseudomonas aeruginosa induces the expression of Staphylococcus aureus Tet38 MDR efflux pump and mediates resistance to phenazines and antibiotics.

In this study, we showed that phenazine-1 carboxylic acid (PCA) of Pseudomonas aeruginosa induced the expression of Tet38 efflux pump triggering Staphylococcus aureus resistance to tetracycline and phenazines. Exposure of S. aureus RN6390 to supernatants of P. aeruginosa PA14 and its pyocyanin (PYO)-deficient mutants showed that P. aeruginosa non-PYO phenazines could induce the expression of Tet38 efflux pump. Direct exposure of RN6390 to PCA compound at 0.25× MIC led to a five-fold increase in tet38 transcripts. Expression of Tet38 protein was identified through confocal microscopy using RN6390(pRN-tet38p-yfp) that expressed YFP under control of the tet38 promoter by PCA at 0.25× MIC. The MICs of PCA of a Tet38-overexpressor and a Δtet38 mutant showed a three-fold increase and a two-fold decrease, respectively, compared with that of wild-type. Pre-exposure of RN6390 to PCA (0.25× MIC) for 1 hour prior to addition of tetracycline (1× or 10× MIC) improved bacteria viability of 1.5-fold and 2.6-fold, respectively, but addition of NaCl 7% together with tetracycline at 10× MIC reduced the number of viable PCA-exposed RN6390 of a 2.0-log10 CFU/mL. The transcript levels of tetR21, a repressor of tet38, decreased and increased two-fold in the presence of PCA and NaCl, respectively, suggesting that the effects of PCA and NaCl on tet38 production occurred through TetR21 expression. These data suggest that PCA-induced Tet38 protects S. aureus against tetracycline during coinfection with P. aeruginosa; however, induced tet38-mediated S. aureus resistance to tetracycline is reversed by NaCl 7%, a nebulized treatment used to enhance sputum mobilization in CF patients.

Staphylococcus aureus AbcA transporter enhances persister formation under ß-lactam exposure.

We evaluated the role of Staphylococcus aureus AbcA transporter in bacterial persistence and survival following exposure to the bactericidal agents nafcillin and oxacillin at both the population and single-cell levels. We show that AbcA overexpression resulted in resistance to nafcillin but not oxacillin. Using distinct fluorescent reporters of cell viability and AbcA expression, we found that over 6-14 hours of persistence formation, the proportion of AbcA reporter-expressing cells assessed by confocal microscopy increased sixfold as cell viability reporters decreased. Similarly, single-cell analysis in a high-throughput microfluidic system found a strong correspondence between antibiotic exposure and AbcA reporter expression. Persister cells grown in the absence of antibiotics showed neither an increase in nafcillin MIC nor in abcA transcript levels, indicating that survival was not associated with stable mutational resistance or abcA overexpression. Furthermore, persister cell levels on exposure to 1×MIC and 25×MIC of nafcillin decreased in an abcA knockout mutant. Survivors of nafcillin and oxacillin treatment overexpressed transporter AbcA, contributing to an enrichment of the number of persisters during treatment with pump-substrate nafcillin but not with pump-non-substrate oxacillin, indicating that efflux pump expression can contribute selectively to the survival of a persister population.

Role of Staphylococcus aureus Tet38 in Transport of Tetracycline and Its Regulation in a Salt Stress Environment.

Staphylococcus aureus Tet38 efflux pump has multiple functions, including conferring resistance to tetracycline and other compounds and enabling internalization and survival within epithelial cells. In this study, we evaluated the effects of sodium and potassium on tet38 expression. These monovalent cations are known to play a role in transport by the related S. aureus TetK and B. subtilis TetL transporters. tet38 transcription decreased with increasing sodium concentrations by means of direct repression by the salt stress-dependent KdpD/E regulator. tet38 transcription increased 20-fold and tetracycline minimum inhibitory concentration (MIC) increased 4-fold in a ΔkdpD mutant. KdpE bound specifically to the tet38 promoter. Under extreme salt stress, the survival of S. aureus with intact tet38 was reduced compared to that of a Δtet38 mutant. To study the effect of sodium on Tet38 function, we generated constructs overexpressing tet38 and tetK and introduced them into Escherichia coli TO114, which is deficient in major sodium transporters. Tet38 tetracycline efflux was directly demonstrated in a fluorescence assay, and tetracycline efflux of both Tet38 and TetK was abolished by the protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP). In contrast, NaCl inhibited efflux by Tet38 but not TetK, whereas KCl inhibited efflux by TetK but not Tet38. Cell-associated Na increased with heterologous overexpression of Tet38. These data indicate that S. aureus Tet38 is a tetracycline efflux pump regulated by the KdpD/E regulator. Under salt stress, S. aureus adjusted its survival in part by reducing the expression of tet38 through KdpD/E. The mechanisms by which Tet38 is detrimental to salt tolerance in S. aureus and inhibited by sodium remain to be determined. IMPORTANCE This study shows that S. aureus Tet38 is a tetracycline efflux pump regulated by KdpD/E regulator. These findings are the first direct demonstration of Tet38-mediated tetracycline efflux, which had previously been inferred from its ability to confer tetracycline resistance. Under salt stress, S. aureus adjusts its survival in part by reducing the expression of tet38 through KdpD/E. We demonstrated the differences in the respective functions of S. aureus Tet38 and other tetracycline efflux transporters (S. aureus TetK, B. subtilis TetL) regarding their transport of tetracycline and Na+/K+. Notably, sodium selectively reduced tetracycline efflux by Tet38, and potassium selectively reduced tetracycline efflux by TetK. The multiple functions of Tet38 emphasize its importance in bacterial adaptation to and survival in diverse environments.

Staphylococcus aureus Efflux Pumps and Tolerance to Ciprofloxacin and Chlorhexidine following Induction by Mupirocin.

Mupirocin induced expression of genes encoding efflux pumps NorA and MepA as well as a yellow fluorescent protein (YFP) fluorescence reporter of NorA. Mupirocin exposure also produced reduced susceptibility to pump substrates ciprofloxacin and chlorhexidine, a change that was dependent on intact norA and mepA, respectively.

Staphylococcus aureus Tet38 Efflux Pump Structural Modeling and Roles of Essential Residues in Drug Efflux and Host Cell Internalization.

The Staphylococcus aureus Tet38 membrane protein has distinct functions, including drug efflux and host cell attachment and internalization mediated by interaction with host cell CD36. Using structural modeling and site-directed mutagenesis, we identified key amino acids involved in different functions. Tet38, a member of the major facilitator superfamily, is predicted to have 14 transmembrane segments (TMS), 6 cytoplasmic loops, and 7 external loops. Cysteine substitutions of arginine 106 situated at the junction of TMS 4 and external loop L2, and glycine 151 of motif C on TMS 5, resulted in complete or near-complete (8- to 16-fold) reductions in Tet38-mediated resistance to tetracycline, with minimal to no effect on A549 host cell internalization. In contrast, a three-amino-acid deletion, F411P412G413, in external loop L7 situated between TMS 13 and 14 led to a decrease of 4-fold in S. aureus internalization by A549 cells and a partial effect on tetracycline resistance (4-fold reduction). A three-amino-acid deletion, D38D39L40, in external loop L1 situated between TMS-1 and TMS-2, had a similar partial effect on tetracycline resistance but did not affect cell internalization. Using an Ni column retention assay, we showed further that the L7, but not the L1, deletion impaired binding to CD36. Thus, the L7 domain of Tet38 is key for interaction with CD36 and host cell internalization, and amino acids R106 and G151 (TMSs 4 and 5) are particularly important for tetracycline resistance without affecting internalization.

Tet38 of Staphylococcus aureus Binds to Host Cell Receptor Complex CD36-Toll-Like Receptor 2 and Protects from Teichoic Acid Synthesis Inhibitors Tunicamycin and Congo Red.

Using an affinity column retention assay, we showed that the purified Tet38 membrane transporter of Staphylococcus aureus bound specifically to host cell CD36 and to the complex CD36-Toll-like receptor 2 (TLR-2), but not to TLR-2 alone or TLR-2 and S. aureus lipoteichoic acid (LTA). We tested the effect of LTA on the internalization of S. aureustet38 mutant QT7 versus RN6390 by A549 epithelial cells. Addition of anti-LTA antibody to the bacteria prior to adding to A549 cells reduced internalization of QT7 2-fold compared to that with nonspecific antibody treatment. QT7 internalized 4- to 6-fold less than RN6390 with or without anti-LTA antibody. These data suggested that Tet38 and LTA were independently involved in the invasion process. The wall teichoic acid (WTA) inhibitor tunicamycin had an 8-fold decrease in activity with overexpression of tet38 and a 2-fold increase in activity in QT7 (tet38). Reserpine (an inhibitor of efflux pumps) reduced the effect of tet38 overexpression on tunicamycin resistance 4-fold. In addition, tet38 affected growth in the presence of LTA inhibitor Congo red, with overexpression increasing growth and deletion of tet38 reducing growth. In conclusion, Tet38 contributes to S. aureus invasion of A549 via direct binding to CD36 of the complex CD36-TLR-2, and LTA independently bound to TLR-2. The reduction of tunicamycin resistance in the presence of reserpine and the survival ability of the tet38 overexpressor in the presence of Congo red suggest that Tet38 can also protect the synthesis of LTA and WTA in S. aureus against their inhibitors, possibly functioning as an efflux pump.

Tet38 Efflux Pump Contributes to Fosfomycin Resistance in Staphylococcus aureus.

Fosfomycin inhibits MurA following uptake by the GlpT transporter of glycerol-3-phosphate in Escherichia coli In Staphylococcus aureus, plasmid overexpression of the Tet38 efflux pump and a glpT mutant resulted in increased MICs and decreased accumulation of fosfomycin, with MICs affected by glycerol-3-phosphate. In contrast, a tet38 mutant had a lower MIC and increased accumulation of fosfomycin, suggesting that Tet38 acts as an efflux transporter of fosfomycin.

Tet38 Efflux Pump Affects Staphylococcus aureus Internalization by Epithelial Cells through Interaction with CD36 and Contributes to Bacterial Escape from Acidic and Nonacidic Phagolysosomes.

We previously reported that the Tet38 efflux pump is involved in internalization of Staphylococcus aureus by A549 lung epithelial cells. A lack of tet38 reduced bacterial uptake by A549 cells to 36% of that of the parental strain RN6390. Using invasion assays coupled with confocal microscopy imaging, we studied the host cell receptor(s) responsible for bacterial uptake via interaction with Tet38. We also assessed the ability of S. aureus to survive following alkalinization of the phagolysosomes by chloroquine. Antibody to the scavenger receptor CD36 reduced the internalization of S. aureus RN6390 by A549 cells, but the dependence on CD36 was reduced in QT7 tet38, suggesting that an interaction between Tet38 and CD36 contributed to S. aureus internalization. Following fusion of the S. aureus-associated endosomes with lysosomes, alkalinization of the acidic environment with chloroquine led to a rapid increase in the number of S. aureus RN6390 bacteria in the cytosol, followed by a decrease shortly thereafter. This effect of chloroquine was not seen in the absence of intact Tet38 in mutant QT7. These data taken together suggest that Tet38 plays a role both in bacterial internalization via interaction with CD36 and in bacterial escape from the phagolysosomes.

Transcriptional Regulator TetR21 Controls the Expression of the Staphylococcus aureus LmrS Efflux Pump.

TetR21 controls the expression of Tet38 and LmrS efflux pumps. A tetR21 mutant, QT21, exhibited a 4-fold increase in the transcription level of lmrSStaphylococcus aureuslmrS overexpressor showed increases of 4-fold and 2-fold, respectively, in the MICs of chloramphenicol and erythromycin, while the MICs of lmrS mutant QT18 and lmrS-tetR21 mutant QT1821 remained similar to those of parental strain RN6390. TetR21 does not bind to the promoter of lmrS, suggesting indirect regulation of lmrS.

Role of the Tet38 Efflux Pump in Staphylococcus aureus Internalization and Survival in Epithelial Cells.

We previously identified the protein Tet38 as a chromosomally encoded efflux pump of Staphylococcus aureus that confers resistance to tetracycline and certain unsaturated fatty acids. Tet38 also contributes to mouse skin colonization. In this study, we discovered a novel regulator of tet38, named tetracycline regulator 21 (TetR21), that bound specifically to the tet38 promoter and repressed pump expression. A ΔtetR21 mutant showed a 5-fold increase in tet38 transcripts and an 8-fold increase in resistance to tetracycline and fatty acids. The global regulator MgrA bound to the tetR21 promoter and indirectly repressed the expression of tet38. To further assess the full role of Tet38 in S. aureus adaptability, we tested its effect on host cell invasion using A549 (lung) and HMEC-1 (heart) cell lines. We used S. aureus RN6390, its Δtet38, ΔtetR21, and ΔmgrA mutants, and a Δtet38 ΔtetR21 double mutant. After 2 h of contact, the Δtet38 mutant was internalized in 6-fold-lower numbers than RN6390 in A549 and HMEC-1 cells, and the ΔtetR21 mutant was internalized in 2-fold-higher numbers than RN6390. A slight increase of 1.5-fold in internalization was found for the ΔmgrA mutant. The growth patterns of RN6390 and the ΔmgrA and ΔtetR21 mutants within A549 cells were similar, while no growth was observed for the Δtet38 mutant. These data indicate that the Tet38 efflux pump is regulated by TetR21 and contributes to the ability of S. aureus to internalize and replicate within epithelial cells.

Transcriptional profiling analysis of the global regulator NorG, a GntR-like protein of Staphylococcus aureus.

The GntR-like protein NorG has been shown to affect Staphylococcus aureus genes involved in resistance to quinolones and ß-lactams, such as those encoding the NorB and AbcA transporters. To identify the target genes regulated by NorG, we carried out transcriptional-profiling assays using S. aureus RN6390 and its isogenic norG::cat mutant. Our data showed that NorG positively affected the transcription of global regulators mgrA, arlS, and sarZ. The three putative drug efflux pump genes most positively affected by NorG were the NorB efflux pump (5.1-fold), the MmpL-like protein SACOL2566 (5.2-fold), and the BcrA-like drug transporter SACOL2525 (5.7-fold) genes. The S. aureus predicted MmpL protein showed 53% homology with the MmpL lipid transporter of Mycobacterium tuberculosis, and the putative SACOL2525 protein showed 87% homology with the bacitracin drug transporter BcrA of Staphylococcus hominis. Two pump genes most negatively affected by NorG were the NorC (4-fold) and AbcA (6-fold) genes. Other categories of genes, such as those participating in amino acid, inorganic ion, or nucleotide transporters and metabolism, were also affected by NorG. Real-time reverse transcription (RT)-PCR assays for mgrA, arlS, sarZ, norB, norC, abcA, mmpL, and bcrA-like were carried out to verify microarray data and showed the same level of up- or downregulation by NorG. The norG mutant showed a 2-fold increase in resistance to norfloxacin and rhodamine, both substrates of the NorC transporter, which is consistent with the resistance phenotype conferred by overexpression of norC on a plasmid. These data indicate that NorG has broad regulatory function in S. aureus.

Plant vaccines: an immunological perspective.

The advent of technologies to express heterologous proteins in planta has led to the proposition that plants may be engineered to be safe, inexpensive vehicles for the production of vaccines and possibly even vectors for their delivery. The immunogenicity of a variety of antigens of relevance to vaccination expressed in different plants has been assessed. The purpose of this article is to examine the utility of plant-expression systems in vaccine development from an immunological perspective.

IL-4(-/-) mice with lethal Mesocestoides corti infections--reduced Th2 cytokines and alternatively activated macrophages.

Protection against Mesocestoides corti, a cestode that invades vital organs, is dependent on the production of IL-4, as IL-4(-/-) mice were found to have higher parasite burdens when compared with wild-type mice. The goal of this study was to investigate the role of IL-4 in immunity to M. corti, focusing on the immunological profile and on potential mediators of pathology. IL-4(-/-) mice infected with M. corti showed 100% mortality by 32 days, whereas wild-type mice survived for approximately 1 year. Parasite burdens were significantly increased in the liver, peritoneal, and thoracic cavities of IL-4(-/-) mice, associated with impaired recruitment of inflammatory cells and a reduction in monocytes and macrophages. IL-5 production by splenocytes and expression in liver tissue was decreased in infected IL-4(-/-) mice compared with wild-type mice. In contrast, IL-4(-/-) mice produced increased amounts of IFNgamma and TNFalpha. Alternatively activated macrophages were a major feature of liver granulomas in wild-type mice evidenced by Arginase I expression, while livers from infected IL-4(-/-) mice showed impaired alternative macrophage activation without increased classical macrophage activation. Thus, lethality during M. corti infection of IL-4(-/-) mice is associated with decreased Th2 cytokines, increased Th1 cytokines and impairment of alternatively activated macrophages.

Characterization of ileal vitamin B12 Binding using homogeneous human and hog intrinsic factors.

Elucidation of the mechanism of intrinsic factor (IF)-mediated vitamin B(12) (B(12)) binding to ileal binding sites has been hampered by the use of crude or only partially purified preparations of IF in previous studies. We have used homogeneous human IF and hog IF isolated by affinity chromatography to study [(57)Co]B(12) binding to ileal mucosal homogenates. The following observations were made: (a) Human IF-B(12) and hog IF-B(12) were bound to human, monkey, hog, dog, rabbit, mouse, hamster, and guinea pig ileal, but not jejunal, homogenates in amounts significantly greater than free B(12) or B(12) bound to five other homogeneous B(12)-binding proteins; (b) only IF-mediated B(12) binding was localized to ileal homogenates and was inhibited by EDTA; (c) values for the association constant (K(a)) for the various ileal homogenates mentioned above and human IF-B(12) and hog IF-B(12) ranged from 0.3 x 10(9) M(-1) to 13.0 x 10(9) M(-1). Apparent differences in the K(a) for human IF-B(12) and hog IF-B(12) existed in most species; (d) the number of ileal IF-B(12) binding sites per gram (wet weight) of ileal mucosa ranged from 0.3 x 10(12) to 4.9 x 10(12). The same value was always obtained with human IF-B(12) and hog IF-B(12) for any given homogenate preparation; (c) 100-fold excesses of free B(12) or human IF and hog IF devoid of B(12) did not significantly inhibit human IF-B(12) and hog IF-B(12) binding to human and hog ileal homogenates. THESE EXPERIMENTS PERFORMED WITH HOMOGENEOUS IF INDICATE THAT: (a) gastric factors other than IF are not required for B(12) binding to ileal IF-B(12)-binding sites: (b) the mechanism of ileal IF-B(12) binding is different from that of free B(12) or of B(12) bound to non-IF-B(12)-binding proteins; (c) human IF and hog IF have different structures; (d) human IF-B(12) and hog IF-B(12) bind to the same ileal binding sites; and (c) human and hog ileal IF-B(12) binding sites bind free B(12) and human and hog IF devoid of B(12) poorly, if at all.

Circadian distribution of hematology variables in subjects with multiple sclerosis.

Hematology variables were measured in blood samples obtained every 3h (8/24h) from 10 multiple sclerosis (MS) patients and 34 healthy subjects and analyzed for circadian characteristics using the population multiple-components method. Red blood cell (RBC) and hemoglobin levels as well as hematocrits exhibited circadian rhythms with minimal amplitudes in healthy individuals and insignificant variability in the smaller group of MS patients. In contrast the total white blood cell (WBC) and platelet counts for MS patients and healthy individuals both showed significant circadian characteristics while the mean 24h WBC and platelet levels did not significantly differ between the two groups. When the different WBC subsets were examined independently, statistically significant circadian rhythms were seen for lymphocytes and eosinophils for both MS patients and healthy individuals and for neutrophils only in the latter. Moreover, the 24h mean levels of lymphocytes, basophils, and eosinophils were significantly higher for the healthy controls while those of monocytes were higher for the MS patients. However, of all the variables tested with significant circadian rhythms in both groups of individuals, only those of lymphocyte numbers exhibited different patterns with somewhat higher amplitude in healthy individuals and a peak level occurring over an hour after that of MS patients. These changes may be the reflection of a disturbance in the regulation of patterns of lymphocyte activity and migration in MS patients. In addition, the elevation in circulating monocytes in MS patients is consistent with the inflammatory nature of the disease.

Clinical applications of quinolones.

The quinolone antimicrobials are the class of inhibitors of bacterial topoisomerases that has been developed most fully for clinical use in human medicine. Initial members of the class had their greatest potency against Gram-negative bacteria, but newly developed members have exhibited increased potency against Gram-positive bacteria and soon agents will be available with additional activity against anaerobic bacteria, providing a broad spectrum of potency. After nalidixic acid, the earliest member of the class which was used for treatment of urinary tract infections, the later fluoroquinolone congeners have had sufficient potency, absorption, and distribution into tissue for additional uses in treatment of sexually transmitted diseases, infections of the gastrointestinal tract, respiratory tract, skin, and bones and joints. Tolerability of these agents in usual doses has been good. Acquired bacterial resistance resulting from clinical uses has occurred in particular among staphylococci and Pseudomonas aeruginosa. Intense drug use and ability of resistant pathogens to spread have also contributed to development of resistance in initially more susceptible pathogens such as Escherichia coli and Neisseria gonorrhoeae in certain settings. Preservation of the considerable clinical utility of the quinolone class for the long term will be affected by the extent to which their use is judicious.

Haptoglobin and albumin synthesis in isolated rat hepatocytes. Response to potential mediators of the acute-phase reaction.

Incorporation of [3H]leucine into haptoglobin and albumin by isolated rat hepatocyte suspensions was studied to assess the role of potential mediators of the acute-phase reaction in promoting haptoglobin synthesis. In vitro, in the presence of insulin, the addition of a hormone mixture containing hydrocortisone, glucagon, somatotropin, and triiodothyronine resulted in a 70% increase in leucine incorporation into haptoglobin relative to albumin at 48 h incubation. A variety of agents, selected because they are considered to play some part in the acute-phase reaction, were added to the medium, and similar measurements of leucine incorporation were made. The specific binding to hepatocytes by asialo- and asialo, agalacto-derivatives of haptoglobin or orosomucoid did not affect synthesis of haptoglobin or albumin. Epinephrine, prostaglandins E1 and E2, bacterial lipopolysaccharide, and sera containing active complement components also failed to stimulate relative haptoglobin synthesis. A partially purified preparation of human leukocytic pyrogen, however, caused a 70% increase in leucine incorporation into haptoglobin relative to albumin in the present of the hormone mixture, suggesting that this substance may affect acute-phase protein synthesis.

Solid-phase radioimmunoassay for IgG antibodies to Staphylococcus epidermidis. Use in serious coagulase-negative staphylococcal infections.

A radioimmunoassay (RIA) for human IgG antibodies to Staphylococcus epidermidis was compared with an agar-gel-diffusion assay in patients with a variety of infections. The RIA was sensitive and reproducible and discriminated between endocarditis and uncomplicated bacteremias due to coagulase-negative staphylococci. Anti-S epidermidis antibodies by RIA were elevated in 16 (89%) of 18 patients with coagulase-negative staphylococcal endocarditis but in none of 28 patients with uncomplicated bacteremia (n = 18) or with blood culture contaminated with these organisms (n = 10). Cross-reacting IgG antibodies to S epidermidis antigens were also detected by RIA in 13 (76%) of 17 patients with Staphylococcus aureus endocarditis but in none of 17 patients with nonvalvular S aureus bacteremias and in none of 25 patients with endocarditis or bacteremia caused by other pathogens. Agar-gel-diffusion assay was less sensitive than RIA for detecting coagulase-negative staphylococcal endocarditis, being positive in nine (50%) of 18 such patients. This RIA may be useful in distinguishing patients with endocarditis from those with nonvalvular staphylococcemias or blood culture contamination.

Circulating immune complexes in prosthetic valve endocarditis.

Using the Raji cell radioimmunoassay, we have determined circulating immune complex (CIC) levels in 36 patients with prosthetic valves during 38 episodes of fever. Fever resulted from prosthetic valve endocarditis (PVE) in 27 instances and from other causes in 11 instances. Peak initial CIC levels higher than 100 micrograms/mL occurred more frequently in the group with PVE, while peak initial CIC values less than 30 micrograms/mL were more frequent in the control group. Circulating immune complex levels fell substantially with completion of antibiotic therapy in 28 (78%) of the patients with PVE. Late CIC elevations were associated with drug-related rashes and replacement of persistently infected prostheses. Our data suggest that the predictive value of measurement of CIC levels in patients with fever and prosthetic valves is in excluding PVE in patients with CIC levels persisting below 30 micrograms/mL.