Comparative Assessment of Tedizolid Pharmacokinetics and Tissue Penetration between Diabetic Patients with Wound Infections and Healthy Volunteers via In Vivo Microdialysis.

Herein, we present pharmacokinetic and tissue penetration data for oral tedizolid in hospitalized patients with diabetic foot infections (DFI) compared with healthy volunteers. Participants received oral tedizolid phosphate 200 mg every 24 h for 3 doses to achieve steady state. A microdialysis catheter was inserted into the subcutaneous tissue near the margin of the wound for patients or into thigh tissue of volunteers. Following the third dose, 12 blood and 14 dialysate fluid samples were collected over 24 h to characterize tedizolid concentrations in plasma and interstitial extracellular fluid of soft tissue. Mean ± standard deviation (SD) tedizolid pharmacokinetic parameters in plasma for patients compared with volunteers, respectively, were as follows: maximum concentration (Cmax), 1.5 ± 0.5 versus 2.7 ± 1.1 mg/liter (P = 0.005); time to Cmax (Tmax) (median [range]), 5.9 (1.2 to 8.0) versus 2.5 (2.0 to 3.0 h) (P = 0.003); half-life (t1/2), 9.1 ± 3.6 versus 8.9 ± 2.2 h (P = 0.932); and plasma area under the concentration-time curve for the dosing interval (AUC p ), 18.5 ± 9.7 versus 28.7 ± 9.6 mg · h/liter (P = 0.004). The tissue area under the concentration-time curve (AUC t ) for the dosing interval was 3.4 ± 1.5 versus 5.2 ± 1.6 mg · h/liter (P = 0.075). Tissue penetration median (range) was 1.1 (0.3 to 1.6) versus 0.8 (0.7 to 1.0) (P = 0.351). Despite lower plasma Cmax and delayed Tmax values for patients with DFI relative to healthy volunteers, the penetration into and exposure to tissue were similar. Based on available pharmacodynamic thresholds for tedizolid, the plasma and tissue exposures using the oral 200 mg once-daily regimen are suitable for further study in treatment of DFI.

[Value of gastric fluid to start antibiotics in premature babies suspected of having early neonatal bacterial infection]. / Intérêt du liquide gastrique dans la décision d'antibiothérapie du nouveau-né prématuré suspect d'infection bactérienne précoce.

The objective of this study was to evaluate the value of direct examination and culture of gastric fluid in the treatment of early neonatal bacterial infections (INBP) in pre-term infants. MATERIALS AND METHODS: Observational study conducted over 6 months in a Type III center. All hospitalized premature babies who had routine gastric fluid sampling at birth during the period of the study were included. They were classified into two groups: premature infants with probable or suspected infection and treated as such (Group 1) and premature infants with no infection or only having colonization (Group 2). RESULTS AND DISCUSSION: In total, 255 pre-term infants were included in the study. Group 1 consisted of 127 newborns and group 2 consisted of 128 newborns. The direct gastric fluid examination was positive in 51 newborns in Group 1 and in 46 newborns in group 2. The culture was positive in 25 newborns in group 1 and eight newborns in group 2. Direct examination of gastric fluid of the 255 children studied had low sensitivity (40.1%) and low specificity (64%) of INBP, with 52.6% positive predictive value (PPV) and 51.8% negative predictive value (NPV). The gastric fluid culture was specific (93.7%) of the INBP, sensitivity was low (19.6%), with PPV at 75.7% and NPV at 54%. CONCLUSION: These results undermine the relevance of the direct examination of gastric fluid in the delicate diagnosis of INBP. This direct examination has a low PPV and NPV. It is advisable not to start or stop antibiotic therapy solely on this argument; however, it can guide the choice of antibiotic therapy and remains useful for this reason. The culture of gastric fluid has very good specificity (93.7%).

Emergent pattern formation in an interstitial biofilm.

Collective behavior of bacterial colonies plays critical roles in adaptability, survivability, biofilm expansion and infection. We employ an individual-based model of an interstitial biofilm to study emergent pattern formation based on the assumptions that rod-shaped bacteria furrow through a viscous environment and excrete extracellular polymeric substances which bias their rate of motion. Because the bacteria furrow through their environment, the substratum stiffness is a key control parameter behind the formation of distinct morphological patterns. By systematically varying this property (which we quantify with a stiffness coefficient γ), we show that subtle changes in the substratum stiffness can give rise to a stable state characterized by a high degree of local order and long-range pattern formation. The ordered state exhibits characteristics typically associated with bacterial fitness advantages, even though it is induced by changes in environmental conditions rather than changes in biological parameters. Our findings are applicable to a broad range of biofilms and provide insights into the relationship between bacterial movement and their environment, and basic mechanisms behind self-organization of biophysical systems.

Dalbavancina: parámetros farmacocinéticos y farmacodinámicos / Dalbavancin: pharmacokinetic and pharmacodynamic parameters

La dalbavancina es un nuevo antibiótico lipoglucopéptido de tamaño de estructura elevado, lo que condiciona su perfil farmacocinético. No se absorbe tras la administración por vía oral, por lo que se administra por vía intravenosa. Su distribución se produce a través del líquido extracelular, alcanzando concentraciones adecuadas en piel, hueso, líquido de blíster y sinovial. Circula en plasma unido a proteínas en proporción muy elevada. Las concentraciones en tejido cerebral y líquido cefalorraquídeo (LCR) son inadecuadas. Se elimina de forma mixta a través de metabolismo no microsomal con metabolitos inactivos, y renal por filtración glomerular. La eliminación se produce a una velocidad muy reducida, tal y como señala el valor de su aclaramiento y su semivida de eliminación terminal, que supera las 300 h. Esta circunstancia supone la permanencia en el plasma y los tejidos de concentraciones adecuadas durante un prolongado período y justifica la pauta posológica a utilizar: 1 g la primera dosis y 500 mg la segunda, que se administra 7 días después de la primera. La farmacocinética es lineal y presenta escasa variabilidad intra e interindividual. No está implicado en interacciones farmacocinéticas. No son necesarios ajustes de dosis para los pacientes con insuficiencia renal leve o moderada (aclaramiento de creatinina ≥ 30 a 79 ml/min). No se requieren ajustes de dosis para pacientes que reciben regularmente hemodiálisis programada (3 veces por semana) y puede administrarse sin tener en cuenta los tiempos de hemodiálisis. En pacientes con insuficiencia renal crónica, cuyo aclaramiento de creatinina es < 30 ml/min y que no están recibiendo regularmente hemodiálisis programada, la dosis recomendada debe reducirse a 750 mg por semana, seguido 1 semana más tarde de 375 mg. No parece necesario ajustar la dosis en pacientes con insuficiencia hepática ni en ancianos, mientras que en niños no se dispone de información sobre la dosificación más apropiada. El parámetro farmacocinético/farmacodinámico que mejor describe la eficacia de dalbavancina es la relación área bajo la curva/concentración mínima inhibitoria (AU)

Dalbavancin is a new lipoglycopeptide antibiotic whose structure influences its pharmacokinetic profile. It is not absorbed after oral administration and is therefore administered intravenously. It is distributed through intracellular fluid, reaching adequate concentrations in the skin, bone, blister fluid and synovial fluid. Plasma protein binding is very high. Concentrations in brain tissue and cerebrospinal fluid (CSF) are inadequate. Excretion is through non-microsomal metabolism with inactive metabolites and through the kidneys by glomerular filtration. Dalbavancin is eliminated slowly, as shown by its clearance value and its terminal elimination half-life, which exceeds 300 hours. This means that adequate concentrations of the drug remain in plasma and tissues for a prolonged period and explains the dosing regimen: a first dose of 1 g followed 7 days later by a 500 mg dose. The pharmacokinetics are linear and show little intra- and interindividual variability. There are no pharmacokinetic interactions. Dose adjustment is not required for patients with mild or moderate renal insufficiency (creatinine clearance ≥ 30 to 79 ml/min). Dosage adjustment is not required in patients regularly receiving elective haemodialysis (3 times/week) and the drug can be administered without consideration of haemodialysis times. In patients with chronic renal insufficiency, whose creatinine clearance is < 30 ml/min and who are not regularly receiving elective haemodialysis, the recommended dose should be reduced to 750 mg per week, followed 1 week later by 375 mg. Dosage adjustment does not seem necessary in patients with liver failure or in older patients. There is no information on the most appropriate dosage in children. The pharmacokinetic/ pharmacodynamics parameter that best describes the effectiveness of dalbavancin is the ratio between the area under the curve and the minimum inhibitory concentration (AU)

In vitro activity of human-simulated epithelial lining fluid exposures of ceftaroline, ceftriaxone, and vancomycin against methicillin-susceptible and -resistant Staphylococcus aureus.

Staphylococcus aureus, including methicillin-susceptible (MSSA) and -resistant (MRSA) strains, is an important pathogen of bacterial pneumonia. As antibiotic concentrations at the site of infection are responsible for killing, we investigated the activity of human-simulated epithelial lining fluid (ELF) exposures of three antibiotics (ceftaroline, ceftriaxone, and vancomycin) commonly used for treatment of S. aureus pneumonia. An in vitro pharmacodynamic model was used to simulate ELF exposures of vancomycin (1 g every 12 h [q12h]), ceftaroline (600 mg q12h and q8h), and ceftriaxone (2 g q24h and q12h). Four S. aureus isolates (2 MSSA and 2 MRSA) were evaluated over 72 h with a starting inoculum of ∼ 10(6) CFU/ml. Time-kill curves were constructed, and microbiological response (change in log10 CFU/ml from 0 h and the area under the bacterial killing and regrowth curve [AUBC]) was assessed in duplicate. The change in 72-h log10 CFU/ml was largest for ceftaroline q8h (reductions of >3 log10 CFU/ml against all strains). This regimen also achieved the lowest AUBC against all organisms (P < 0.05). Vancomycin produced reliable bacterial reductions of 0.9 to 3.3 log10 CFU/ml, while the activity of ceftaroline q12h was more variable (reductions of 0.2 to 2.3 log10 CFU/ml against 3 of 4 strains). Both regimens of ceftriaxone were poorly active against MSSA tested (0.1 reduction to a 1.8-log10 CFU/ml increase). Against these S. aureus isolates, ELF exposures of ceftaroline 600 mg q8h exhibited improved antibacterial activity compared with ceftaroline 600 mg q12h and vancomycin, and therefore, this q8h regimen deserves further evaluation for the treatment of bacterial pneumonia. These data also suggest that ceftriaxone should be avoided for S. aureus pneumonia.

The CD200-CD200R1 inhibitory signaling pathway: immune regulation and host-pathogen interactions.

The CD200:CD200R1 inhibitory signaling pathway has been implicated in playing a prominent role in limiting inflammation in a wide range of inflammatory diseases. CD200R1 signaling inhibits the expression of proinflammatory molecules including tumor necrosis factor, interferons, and inducible nitric oxide synthase in response to selected stimuli. Unsurprisingly, due to the regulatory role that CD200R1 plays in multiple inflammatory pathways, an increasing number of parasitic, bacterial, and viral pathogens exploit this pathway to suppress host defenses. A complete understanding of the pathways regulated by CD200R1 signaling and the diverse mechanisms that pathogens have evolved to manipulate the CD200:CD200R1 pathway can help identify clinical situations where targeting this interaction can be of therapeutic benefit. In this review, we compare CD200R1 to other pathogen-targeted inhibitory receptors and highlight how this signaling pathway is utilized by a diverse number of pathogens and, therefore, may represent a novel targeting strategy for the treatment of infectious diseases.

Activity and immunodetection of lysozyme in earthworm Dendrobaena veneta (Annelida).

In the present study, lysozyme-like activity against Micrococcus luteus was detected in the coelomic fluid, the extract from coelomocytes, intestine and in the homogenates from cocoons of Dendrobaena veneta. Four hours after immunization with Escherichia coli, the lysozyme activity in the coelomic fluid increased about three times and in the extract of coelomocytes - four times, in comparison to the control. In three cases: of the coelomic fluid, the homogenates from cocoons and the extract from coelomocytes, the antibody against HEWL (hen egg white lysozyme) recognized only one protein with a molecular mass of about 14.4 kDa. In the coelomic fluid, apart from the protein with molecular mass of 14.4 kDa the antibody directed against human lysozyme recognized an additional protein of 22 kDa. Using the bioautography technique after electrophoretic resolution of native proteins in acidic polyacrylamide gels, two lytic zones of M. luteus were observed in the case of the coelomic fluid and three after the analysis of the extract of coelomocytes and the egg homogenates. The results indicated the existence of several forms of lysozyme with a different electric charge in the analyzed D. veneta samples. The highest lysozyme activity in the intestine of D. veneta was observed in the midgut. The antibody directed against human lysozyme indicated a strong positive signal in epidermal and midgut cells of earthworm.

Extracellular ubiquitin: immune modulator and endogenous opponent of damage-associated molecular pattern molecules.

Ubiquitin is a post-translational protein modifier and plays essential roles in all aspects of biology. Although the discovery of ubiquitin introduced this highly conserved protein as a molecule with extracellular actions, the identification of ubiquitin as the ATP-dependent proteolysis factor 1 has focused subsequent research on its important intracellular functions. Little attention has since been paid to its role outside of the cell. During recent years, multiple observations suggest that extracellular ubiquitin can modulate immune responses and that exogenous ubiquitin has therapeutic potential to attenuate exuberant inflammation and organ injury. These observations have not been integrated into a comprehensive assessment of its possible role as an endogenous immune modulator. This review recapitulates the current knowledge about extracellular ubiquitin and discusses an emerging facet of its role in biology during infectious and noninfectious inflammation. The synopsis of these data along with the recent identification of ubiquitin as a CXCR4 agonist suggest that extracellular ubiquitin may have pleiotropic roles in the immune system and functions as an endogenous opponent of DAMPs. Functions of extracellular ubiquitin could constitute an evolutionary conserved control mechanism aimed to balance the immune response and prevent exuberant inflammation. Further characterization of its mechanism of action and cellular signaling pathways is expected to provide novel insights into the regulation of the innate immune response and opportunities for therapeutic interventions.

Extracellular DNA: a major proinflammatory component of Pseudomonas aeruginosa biofilms.

We previously demonstrated that extracellular bacterial DNA activates neutrophils through a CpG- and TLR9-independent mechanism. Biofilms are microbial communities enclosed in a polymeric matrix that play a critical role in the pathogenesis of many infectious diseases. Because extracellular DNA is a key component of biofilms of different bacterial species, the aim of this study was to determine whether it plays a role in the ability of biofilms to induce human neutrophil activation. We found that degradation of matrix extracellular DNA with DNase I markedly reduced the capacity of Pseudomonas aeruginosa biofilms to induce the release of the neutrophil proinflammatory cytokines IL-8 and IL-1beta (>75%); reduced the upregulation of neutrophil activation markers CD18, CD11b, and CD66b (p < 0.001); reduced the number of bacteria phagocytosed per neutrophil contacting the biofilm; and reduced the production of neutrophil extracellular traps. Consistent with these findings, we found that biofilms formed by the lasI rhlI P. aeruginosa mutant strain, exhibiting a very low content of matrix extracellular DNA, displayed a lower capacity to stimulate the release of proinflammatory cytokines by neutrophils, which was not decreased further by DNase I treatment. Together, our findings support that matrix extracellular DNA is a major proinflammatory component of P. aeruginosa biofilms.

Host-derived extracellular nucleic acids enhance innate immune responses, induce coagulation, and prolong survival upon infection in insects.

Extracellular nucleic acids play important roles in human immunity and hemostasis by inducing IFN production, entrapping pathogens in neutrophil extracellular traps, and providing procoagulant cofactor templates for induced contact activation during mammalian blood clotting. In this study, we investigated the functions of extracellular RNA and DNA in innate immunity and hemolymph coagulation in insects using the greater wax moth Galleria mellonella a reliable model host for many insect and human pathogens. We determined that coinjection of purified Galleria-derived nucleic acids with heat-killed bacteria synergistically increases systemic expression of antimicrobial peptides and leads to the depletion of immune-competent hemocytes indicating cellular immune stimulation. These activities were abolished when nucleic acids had been degraded by nucleic acid hydrolyzing enzymes prior to injection. Furthermore, we found that nucleic acids induce insect hemolymph coagulation in a similar way as LPS. Proteomic analyses revealed specific RNA-binding proteins in the hemolymph, including apolipoproteins, as potential mediators of the immune response and hemolymph clotting. Microscopic ex vivo analyses of Galleria hemolymph clotting reactions revealed that oenocytoids (5-10% of total hemocytes) represent a source of endogenously derived extracellular nucleic acids. Finally, using the entomopathogenic bacterium Photorhabdus luminescens as an infective agent and Galleria caterpillars as hosts, we demonstrated that injection of purified nucleic acids along with P. luminescens significantly prolongs survival of infected larvae. Our results lend some credit to our hypothesis that host-derived nucleic acids have independently been co-opted in innate immunity of both mammals and insects, but exert comparable roles in entrapping pathogens and enhancing innate immune responses.

Proteomic analysis of human cervical-vaginal fluids.

The pathophysiology of vaginal conditions is still ill-defined at a molecular level. Because the proteome of the human cervical-vaginal fluid (CVF) has not been reported to date, we undertook the identification of proteins present in the cell-free fraction of these fluids. Proteins were separated bidimensionally (2-D) by isoelectrofocusing (pH 3-11) followed by SDS-polyacrylamide electrophoresis. The proteins of 147 spots were identified by matrix-assisted laser desorption/ ionization-time-of-flight-mass spectrometry (MALDI-TOF/TOF). This approach was supplemented by immunoassays for markers of neutrophils (myeloperoxidase, MPO; neutrophil gelatinase-associated lipocalin, NGAL/HNL) and eosinophils (eosinophil cationic protein: ECP) and by immunoblotting (lactoferrin, calgranulins A and B and annexins A1 and A3. Nearly half of the proteins (69/147) and protein fragments detected were found to be plasma components, on the basis of which the human CVF can be broadly considered a plasma transudate. Although the pattern of protein spots was very similar for all fluids analyzed, a relative overabundance of major plasma proteins such as albumin, transferrin, immunoglobulins, apolipoproteins, alpha-1-acid glycoprotein 1, and calgranulins was associated with the presence of a high number of polymorphonuclear leukocytes in the lavages from which those cell-free fluids had been obtained. Instead, fluids from women experiencing vulvovaginal candidiasis did not show differences in the protein maps compared with asymptomatic individuals. Neutrophil and eosinophil granule secretion proteins were also detected in variable amounts in the lavage fluids by both immunoassay and immunoblotting, indicating polymorphonuclear cell activation.

Detection of Mycobacterium tuberculosis DNA in respiratory and nonrespiratory specimens by the Amplicor MTB PCR.

To evaluate the diagnostic performance of a commercially available Mycobacterium tuberculosis PCR assay (Amplicor MTB-ROCHE), 2296 respiratory and nonrespiratory specimens from 2296 patients with suspected tuberculosis (TB) were collected prospectively in an 8-year period. Clinical data for each patient were abstracted, and all samples were examined blindly by direct microscopy, culture, and PCR. M. tuberculosis DNA was detected in 93 of 113 culture-positive samples and in 29 of 38 samples from patients with probable TB. The lowest sensitivity was observed in pleural fluid and abscess aspirates. The sensitivity, specificity, and positive predictive value of the assay were 97.2%, 100%, and 100% for smear-positive specimens and 75.3%, 97.0%, and 47.5% for smear-negative specimens, respectively. The PCR cost per additional correct clinical decision was Euro 2826 but would have declined to Euro 308 if the test was applied only to smear-positive specimens. The overall performance of Amplicor MTB test was excellent for smear-positive, but suboptimal for smear-negative specimens.

Intercellular spreading of Porphyromonas gingivalis infection in primary gingival epithelial cells.

Porphyromonas gingivalis, an important periodontal pathogen, is an effective colonizer of oral tissues. The organism successfully invades, multiplies in, and survives for extended periods in primary gingival epithelial cells (GECs). It is unknown whether P. gingivalis resides in the cytoplasm of infected cells throughout the infection or can spread to adjacent cells over time. We developed a technique based on flow cytofluorometry and fluorescence microscopy to study propagation of the organism at different stages of infection of GECs. Results showed that P. gingivalis spreads cell to cell and that the amount of spreading increases gradually over time. There was a very low level of propagation of bacteria to uninfected cells early in the infection (3 h postinfection), but there were 20-fold and 45-fold increases in the propagation rate after 24 h and 48 h, respectively, of infection. Immunofluorescence microscopy of infected cells suggested that intercellular translocation of P. gingivalis may be mediated through actin-based membrane protrusions, bypassing the need for release of bacteria into extracellular medium. Consistent with these observations, cytochalasin D treatment of infected cells resulted in significant inhibition of bacterial spreading. This study shows for the first time that P. gingivalis disseminates from cell to cell without passing through the extracellular space. This mechanism of spreading may allow P. gingivalis to colonize oral tissues without exposure to the humoral immune response.

Medical management of parapneumonic pleural disease.

Considerable heterogeneity exists in the management of parapneumonic pleural disease. A randomized controlled trial (RCT) demonstrated the effectiveness of small-catheter drainage with fibrinolysis, but surgical devotees suggest this may only be applicable to "early" cases. We examined evidence-based medical management in "all-comers." We performed a retrospective database analysis of the management of all children with complex pleural effusion admitted to the John Radcliffe Hospital over the 7-year period 1996-2003. One hundred and ten children were admitted. Ten were excluded as they were part of a multicenter RCT and had received intrapleural saline instead of urokinase. Of the remaining 100, 51 were female and 49 male. Median age on admission was 5.8 years (range, 0.3-16.5). Symptoms preadmission averaged 11 days, with December the most common month for presentation. Ninety-six underwent chest ultrasound, confirming an effusion in all, described as loculated/septated (68) or echogenic (11). In 17 cases, no specific comment was made regarding the nature of the fluid seen on ultrasound. Ninety-five had subsequent chest tube drainage and then received intrapleural fibrinolysis with urokinase. An etiological organism was identified in 21 cases (21%) (Streptococcus pneumoniae in 10, group A Streptococcus in 5, Staphylococcus aureus in 4, Haemophilus influenzae in 1, and coliform in 1). In a further 9 cases (9%), Gram-positive organisms were seen on pleural fluid microscopy, but did not grow on culture. Two (2%) required surgery due to the persistence of symptoms and an inadequate response to medical management. Median duration of admission was 7 days (range, 2-21 days); median duration of stay from intervention was 5 days (range, 2-19 days). At median follow-up of 8 weeks (range, 3-20 weeks), all children were symptom-free, with minimal pleural thickening on chest X-ray. In conclusion, antibiotic therapy with chest drain insertion and intrapleural urokinase is effective in treating complex parapneumonic effusion and is associated with a good long-term outcome.

Effects of pH on Helicobacter pylori binding to human gastric mucins: identification of binding to non-MUC5AC mucins.

Helicobacter pylori causes gastritis, peptic ulcer disease and gastric cancer. The microbe is found in the gastric mucus layer where a pH gradient ranging from acidic in the lumen to neutral at the cell surface is maintained. The aim of the present study was to investigate the effects of pH on H. pylori binding to gastric mucins from healthy individuals. At pH 3, all strains bound to the most charged MUC5AC glycoform and to a putative mucin of higher charge and larger size than subunits of MUC5AC and MUC6, irrespective of host blood-group. In contrast, at pH 7.4 only Le(b)-binding BabA-positive strains bound to Le(b)-positive MUC5AC and to smaller mucin-like molecules, including MUC1. H. pylori binding to the latter component(s) seems to occur via the H-type-1 structure. All strains bound to a proteoglycan containing chondroitin sulphate/dermatan sulphate side chains at acidic pH, whereas binding to secreted MUC5AC and putative membrane-bound strains occurred both at neutral and acidic pH. The binding properties at acidic pH are thus common to all H. pylori strains, whereas mucin binding at neutral pH occurs via the bacterial BabA adhesin and the Le(b) antigen/related structures on the glycoprotein. Our work shows that microbe binding to membrane-bound mucins must be considered in H. pylori colonization, and the potential of these glycoproteins to participate in signalling events implies that microbe binding to such structures may initiate signal transduction over the epithelial layer. Competition between microbe binding to membrane-bound and secreted mucins is therefore an important aspect of host-microbe interaction.

Levels of nicotinamide adenine dinucleotide in extracellular body fluids of pigs may be growth-limiting for Actinobacillus pleuropneumoniae and Haemophilus parasuis.

During infection, nutrient deprivation can alter bacterial phenotype. This, in turn, may have implications for pathogenesis and prophylaxis. Actinobacillus pleuropneumoniae (biotype 1) and Haemophilus parasuis, respiratory tract pathogens of swine, are both V-factor-dependent. The concentrations of V factor in the extracellular fluids of pigs are unknown and may limit the growth of these bacteria in vivo. The aim of this study was to determine the concentrations of nicotinamide adenine dinucleotide (NAD) in select porcine body fluids and to compare the availability of NAD in vivo with the affinities of the organisms for this compound. Levels in plasma, tissue fluids (peritoneal, pleural, synovial, and cerebrospinal), and laryngeal, tracheal, and lung washings were determined with an enzymatic cycling assay. We concluded that, although the NAD supply in the respiratory tract is probably not growth-limiting, it may become limiting if the organisms are disseminated.