Generation of an intelligent medical system, using a real database, to diagnose bacterial infection in hospitalized patients.

The initial diagnosis of bacterial infections in the absence of laboratory microbiological data requires physicians to use clinical algorithms based on symptoms, patient history and infection site. Optimization of such algorithms would be achieved by including as many variables associated with bacterial infection as possible. Demographic data are easily available and frequently used to sub-group human populations. A prospective investigation was, therefore, undertaken to examine the influence of demographic variables on bacterial infection rates, using data obtained from 173 patients presenting to Albert Einstein Medical Center. Data was randomly selected from 149 of these patients and used to generate fuzzy rules to model an intelligent medical system. To test the accuracy of this system at determining bacterial infection, based solely on demographic data, the program was given the remaining 24 patients' information. All 18 patients with either streptococcal, staphylococcal or Escherichia coli infections were correctly diagnosed. Non-E.coli GNR were misdiagnosed as E. coli infections in two patients resulting in an overall prediction rate for the 24 patients of 91.66%. This study suggests that the direct correlation of demographic variables with a predisposition to bacterial infection allow the design of an intelligent medical system, which shows great future potential as a diagnostic tool for all physicians.

Using fuzzy sets to analyze putative correlates between age, blood type, gender and/or race with bacterial infection.

Previous studies have suggested that the demographic variables of age and blood type may serve as "risk factors" for infection by specific bacterial species. Since both demographic variables and bacterial species are defined using generally accepted parameters, they constitute highly suitable variables for the generation of a fuzzy logic program. A prospective study was therefore undertaken to examine the influence of age, blood type, gender and race on bacterial infection rates using a real database generated from 187 bacteremic patients admitted to Albert Einstein Medical Center. A fuzzy logic program was created using 155 randomly selected patients' data with four input (demographic variables) and four output classes (infections with "staphylococci", "streptococci", "Escherichia coli" or "non-E. coli gram negative rods (non-E.coli GNR)"). To see whether bacterial infection could be predicted based on demographic data alone, the program was tested using the remaining 32 patients' data. The program was able to correctly determine the bacterial output group of 27 of 32 randomly selected patients, giving an overall correlation of 84.38%. This study suggests that the direct correlation of demographic variables with a predisposition to bacterial infection allow the design of an intelligent medical system, which shows great future potential as a powerful diagnostic tool for all physicians.

The effect of bacterial toxins on levels of intracellular adenosine nucleotides and human ciliary beat frequency.

Toxins that slow ciliary beat are virulence determinants of bacteria that infect or invade ciliated epithelial surfaces. We have previously shown that the effect of the Pseudomonas aeruginosa toxin pyocyanin on ciliary beat is associated with a fall in intracellular cAMP and ATP. We have now investigated whether reduction in intracellular adenosine nucleotides might be a common mechanism of action of other bacterial toxins which slow ciliary beat. Two other P. aeruginosa toxins, 1-hydroxyphenazine (1-HP) and rhamnolipid, and two Haemophilus influenzae fractions produced by gel filtration of broth cultures were tested. The effect on human nasal epithelium ciliary beat frequency (CBF), and intracellular cAMP and ATP were measured, and the effect of two pharmacological agents, dibutyryl cAMP and salmeterol, on these changes was assessed. 1-HP, rhamnolipid and the two H. influenzae fractions slowed CBF before there was significant release of lactate dehydrogenase from the cells. The toxins also caused a fall in intracellular cAMP and ATP. Dibutyryl cAMP and salmeterol at the concentrations used do not increase baseline CBF, but diminished the fall in CBF and intracellular adenosine nucleotides. The cAMP and ATP levels in these studies were combined with those previously obtained with pyocyanin. there was a good correlation between cAMP and ATP levels and CBF. Bacterial toxins which slow CBF may act by causing a fall in intracellular adenosine nucleotides, and agents which stimulate cAMP may prevent toxin-induced slowing of ciliary beat.

Pyruvate oxidase, as a determinant of virulence in Streptococcus pneumoniae.

Pneumococcus has been shown to bind to epithelial cells of the nasopharynx and lung, and to endothelial cells of the peripheral vasculature. To characterize bacterial elements required for attachment to these cell types, a library of genetically altered pneumococci with defects in exported proteins was screened for the loss of attachment to glycoconjugates representative of the nasopharyngeal cell receptor, type II lung cells (LC) and human endothelial cells (EC). A mutant was identified which showed a greater than 70% loss in the ability to attach to all cell types. This mutant also showed decreased adherence to the glycoconjugates containing the terminal sugar residues GalNAcbeta1-3Gal, GalNAcbeta1-4Gal and the carbohydrate GlcNAc, which are proposed components of the pneumococcal receptors specific to the surfaces of LC and EC. Analysis of the locus altered in this mutant revealed a gene, spxB, that encodes a member of the family of bacterial pyruvate oxidases which decarboxylates pyruvate to acetyl phosphate plus H2O2 and CO2. This mutant produced decreased concentrations of H2O2 and failed to grow aerobically in a chemically defined medium, unless supplemented with acetate which presumably restores acetyl phosphate levels by the action of acetate kinase, further suggesting that spxB encodes a pyruvate oxidase. The addition of acetate to the growth medium restored the adherence properties of the mutant indicating a link between the enzyme and the expression of bacterial adhesins. A defect in spxB corresponded to impaired virulence of the mutant in vivo. Compared to the parent strain, an spxB mutant showed reduced virulence in animal models for nasopharyngeal colonization, pneumonia, and sepsis. We propose that a mutation in spxB leads to down-regulation of the multiple adhesive properties of pneumococcus which, in turn, may correlate to diminished virulence in vivo.

PAf receptor anchors Streptococcus pneumoniae to activated human endothelial cells.

Streptococcus pneumoniae can produce asymptomatic colonization or aggressive sepsis. We sought to differentiate the molecular mechanisms of these disparate courses. Cytokine or thrombin activation of human vascular endothelial cells and type II pneumocytes enhanced pneumococcal adherence relative to resting cells. Adherence and subsequent invasion was dramatically reduced by PAF receptor antagonists. Cells transfected with the PAF receptor gained the ability to support pneumococcal adherence. PAF or PAF receptor antagonists inhibited attachment and invasion. Adherence involved phosphorylcholine on the pneumococcal teichoic acid. Virulent pneumococci target the PAF receptor on activated human cells, a necessary step to facilitate subsequent invasion.

Streptococcus pneumoniae anchor to activated human cells by the receptor for platelet-activating factor.

The Gram-positive bacterium Streptococcus pneumoniae is a major cause of pneumonia, sepsis and meningitis. Although the invasive disease is severe, some 40% of individuals harbour the pneumococcus in the nasopharynx asymptomatically. Here we investigate the molecular elements of the encounter between host and pathogen that distinguish these different outcomes. We show that inflammatory activation of human cells shifts the targeting of the pneumococcus to a new receptor, that for the G-protein-coupled platelet-activating factor (PAF). Only virulent pneumococci engage the PAF receptor. Attachment of the bacterial phosphorylcholine to the PAF receptor enhanced adherence, which was coupled to invasion of endothelial, epithelial and PAF-receptor-transfected cells. This progression could be arrested in vitro and in vivo by PAF-receptor-specific antagonists, suggesting a possible approach to therapy.

The role of N-glycosylation for functional expression of the human platelet-activating factor receptor. Glycosylation is required for efficient membrane trafficking.

Streptococcus pneumoniae has been shown to utilize the platelet activating factor receptor for binding and invasion of host cells (Cundell, D. R., Gerard, N. P., Gerard, C., Idanpaan-Heikkila, I., and Tuomanen, E. I. (1995) Nature, in press). Because bacterial binding is in part carbohydrate dependent, and the human platelet-activating factor (PAF) receptor bears a single N-linked glycosylation sequence in the second extracellular loop, we undertook studies to determine the role of this epitope in PAF receptor function. Binding of pneumococci to COS cells transfected with the human PAF receptor is greatly reduced for a receptor mutant that bears no N-linked glycosylation site. Immunohistochemical and binding analyses show decreased expression of the non-glycosylated molecule on the cell membrane relative to the wild type receptor; however, metabolic labeling and immunopurification indicate it is synthesized intracellularly at a level similar to the native molecule. A mutant receptor encoding a functional glycosylation site at the NH2 terminus is better expressed at the cell surface compared with the non-glycosylated form, indicating that trafficking to the cell surface is facilitated by glycosylation, but its location is relatively unimportant. The binding affinity for PAF is not significantly effected by the presence or location of the carbohydrate, and variations in cell surface expression have little influence on signal transduction, as the non-glycosylated PAF receptor is equally effective for activation of phospholipase C as the native molecule. These data are supportive of pneumococcal binding on protein moiety(ies) of the PAF receptor and indicate that N-glycosylation facilitates expression of the protein on the cell membrane.

Peptide permeases from Streptococcus pneumoniae affect adherence to eucaryotic cells.

To gain access to tissues within the human host, Streptococcus pneumoniae initially colonizes the nasopharynx and then interacts with glycoconjugates on the surfaces of target cells at various sites of infection. Although pneumococcal adhesins are currently unknown, exported proteins on the bacterial surface are potential candidates. To identify bacterial elements involved in this process, mutants of S. pneumoniae with defects in exported proteins were screened for the inability to adhere to cells representative of three in vivo niches: (i) agglutination of bovine erythrocytes, which reflects adherence to cells which reside in the nasopharynx; (ii) human type II pneumocytes (lung cells [LC]), representing the alveolar site of infection; and (iii) human vascular endothelial cells (EC), representing the endovascular site. The capacity of the mutants to adhere during the course of pneumococcal disease was also assessed by using cytokine-activated LC and EC. All of the 30 mutants analyzed produced hemagglutination values comparable with those of the parent strain. Four independent mutants demonstrated a greater than 50% decrease in adherence to both LC and EC. Sequence analysis of the altered alleles from these strains showed that mutations had occurred in two previously identified loci, plpA and ami, which belong to the family of genes encoding protein-dependent peptide permeases. Mutations in the ami locus resulted in an inability to recognize the GalNAc beta 1-4Gal glycoconjugate receptor present on resting LC and EC, whereas mutations in plpA resulted in a failure to recognize a GalNAc beta 1-3Gal glycoconjugate receptor also present on resting cells. Mutations in neither allele affected recognition of GlcNAc receptors present on cytokine-activated LC and EC. These results suggest that peptide permeases modulate pneumococcal adherence to epithelial and endothelial cells either by acting directly as adhesins or by modulating the expression of adhesins on the pneumococcal surface during the initial stages of colonization of the lung or the vascular endothelium.

Relationship between colonial morphology and adherence of Streptococcus pneumoniae.

Phase variants in colonial opacity of pneumococci differ in the ability to colonize the nasopharynx of infant rats. To explain this observation at a cellular level, we compared the ability of opacity variants to adhere to buccal epithelial cells, type II pneumocytes, or vascular endothelial cells and to the glycoconjugates that represent the cognate receptors at each of these sites. The transparent phenotype was associated with enhanced adherence to buccal cells (approximately 100%) and their receptor relative to that of the opaque variants. Only modest differences in adherence (< 45%) were demonstrated to resting lung and vascular cells. In contrast, adherence of transparent variants increased by 90% to lung cells stimulated with interleukin-1 and by 130% to endothelial cells stimulated with tumor necrosis factor. In contrast, cytokine stimulation did not influence the adherence of opaque pneumococci. This difference correlated with the unique ability of transparent variants to adhere to immobilized GlcNAc and to cells bearing transfected platelet-activating factor receptors. These results suggest that the mechanism of enhanced colonization of the nasopharynx in vivo by transparent as compared with opaque phase variants involves a greater ability to adhere to both GlcNAc beta 1-3Gal on buccal epithelial cells and GlcNAc and PAF receptors on cytokine-activated, as opposed to resting, lung and endovascular cells.

Receptor specificity of adherence of Streptococcus pneumoniae to human type-II pneumocytes and vascular endothelial cells in vitro.

The adherence of S. pneumoniae to human type-II pneumocytes and endothelial cells (EC) is critical to the pathogenesis of pneumococcal pneumonia and bacteremia. We established that the preferred target cell to which pneumococci adhere in the lung is the type-II lung cell (LC) and have developed an in vitro adherence assay to determine the molecular details of this interaction. Pneumococcal receptors on cultured human LC and EC appeared to be glycoproteins since treatment of the monolayers with tunicamycin significantly impaired bacterial adherence. Inhibition of adherence to LC and EC occurred following incubation with several carbohydrates including GalNAc, mannose and GalNAc beta-4Gal- and GalNAc beta 1-3Gal-containing glycoconjugates. Pneumococci could bind directly to these immobilized sugars and their addition to adherent pneumococci could elute the bacteria from LC and EC. Combinations of glycoconjugates indicated that two independent classes of pneumococcal receptor existed on both cell types. These were defined by the minimal receptor units GalNAc beta 1-4Gal and GalNAc beta 1-3Gal which participate in pneumococcal cell wall and protein-dependent mechanisms of adherence, respectively.

Effect of salmeterol on human nasal epithelial cell ciliary beating: inhibition of the ciliotoxin, pyocyanin.

1. Patients with airway infection by Pseudomonas aeruginosa have impaired mucociliary clearance. Pyocyanin is a phenazine pigment produced by P. aeruginosa which is present in the sputum of colonized patients, slows human ciliary beat frequency (CBF) in vitro and slows mucociliary transport in vivo in the guinea-pig. 2. We have investigated the effect of salmeterol, a long-acting beta 2-adrenoceptor agonist, on pyocyanin-induced slowing of human CBF in vitro. Salmeterol (2 x 10(-7) M) was found to reduce pyocycanin (20 micrograms ml-1)-induced slowing of CBF by 53% and the fall in intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) by 26% and ATP by 29%. 3. Another beta 2-adrenoceptor agonist, isoprenaline (2 x 10(-7) M), also inhibited pyocyanin-induced slowing of CBF by 39%. 4. The effects of salmeterol (30 min preincubation) persisted after washing the cells. 5. Propranolol (10(-7) M) and the beta 2-specific antagonist, ICI 118551 (10(-6) M) blocked the protective effects of salmeterol completely, but atenolol (10(-6) M) was less effective. These results suggested that the effects of salmeterol on pyocyanin-induced effects were mediated primarily via the stimulation of beta 2-adrenoceptors. 6. Pyocyanin-induced ciliary slowing is associated with a substantial fall in intracellular cyclic AMP and ATP. Salmeterol reversed the effects of pyocyanin on cyclic AMP and ATP. 7. Mucociliary clearance is an important defence mechanism of the airways against bacterial infection. Salmeterol may benefit patients colonized by P. aeruginosa, not only by its bronchodilator action, but also by protecting epithelial cells from pyocyanin-induced slowing of CBF.

The effect of erythromycin on Pseudomonas aeruginosa and neutrophil mediated epithelial damage.

Erythromycin therapy for long periods may benefit patients with chronic bronchial sepsis colonized by Pseudomonas aeruginosa despite the lack of antibacterial activity. We have investigated the effect of filtrates of 24 h P. aeruginosa cultures (CF) with or without erythromycin 0.5, 5, 20 mg/L on human nasal epithelium in the absence or presence of polymorphonuclear leucocytes (PMN). Ciliary beat frequency (CBF) and epithelium integrity were examined for 4 h. Erythromycin (20 mg/L) alone did not affect epithelium. CF without erythromycin slowed CBF by 63.5% of control at 4 h, and caused disruption of surface integrity in 80% of the epithelium. Addition of erythromycin to CF did not inhibit these effects. Erythromycin did not affect growth of P. aeruginosa. Filtrates of P. aeruginosa cultured with erythromycin (5 and 20 mg/L) caused less CBF slowing (37.2% and 19.2% of control, respectively) and epithelial disruption (4.2% and 6.7%, respectively). Unstimulated PMN (10(7)/mL) slowed CBF by 13% of control at 4 h but did not cause epithelial disruption. PMN and CF together slowed CBF (95.4% of control) and damaged epithelium (93.3% of epithelium disrupted) synergically. Pre-incubation of PMN with erythromycin did not inhibit these effects. PMN and filtrates of P. aeruginosa cultured with erythromycin (5 and 20 mg/L) caused less CBF slowing (58.0% and 33.6% of control, respectively) and epithelial disruption (40.0% and 13.3%, respectively). Erythromycin may benefit patients by reducing P. aeruginosa production of factors which damage epithelium and stimulate neutrophil mediated cytotoxicity.

Effect of tracheal cytotoxin from Bordetella pertussis on human neutrophil function in vitro.

The infiltration of neutrophils which phagocytose and kill microorganisms is an important defense mechanism against infections of the airways. Bordetella pertussis is a human respiratory pathogen which colonizes ciliated epithelium, causing whooping cough. We have investigated the effects of the peptidoglycan fragment tracheal cytotoxin (TCT) of B. pertussis on human neutrophil function in vitro. TCT (10(-6) to 10(-8) M) was toxic for human neutrophils, as measured by lactate dehydrogenase release and levels of intracellular ATP. TCT (10(-9) to 10(-15) M) did not stimulate neutrophil migration or chemiluminescence and did not affect neutrophil phagocytosis. Incubation of neutrophils for 20 min with TCT (10(-9) to 10(-11) M) significantly inhibited (P < 0.05) their subsequent migration toward the chemotactic factor N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP; 10(-9) M). Incubation of neutrophils for 20 min with TCT (10(-9) to 10(-15) M) significantly inhibited (P < 0.05) chemiluminescence stimulated by FMLP (10(-5) M). TCT (10(-6) to 10(-12) M) did not stimulate interleukin-1 alpha production by neutrophils or serum complement activation by the alternate pathway. We conclude that TCT at concentrations of < 10(-8) M affects important neutrophil functions and at higher concentrations is toxic. TCT may therefore contribute to the survival of B. pertussis within the airways in vivo.

Human bronchial epithelial cell dysfunction following in vitro exposure to nitrogen dioxide.

Nitrogen dioxide (NO2), is a major air pollutant, that causes bronchoconstriction and bronchial hyperreactivity, and may also lead to damage and inflammation of the airway epithelium. We have cultured human bronchial epithelial cells and investigated the effect of exposure to NO2, for 20 min on epithelial cell membrane integrity and function in vitro. Epithelial cell membrane damage and permeability were assessed by release of 51Cr from prelabelled cells, and movement of 14C-labelled bovine serum albumin (BSA) across the bronchial epithelial cell monolayers. Ciliary beat frequency (CBF) of the cells was monitored by the analogue contrast enhancement technique, and arachidonic acid (AA) metabolism was investigated by analysis of radiolabelled AA metabolites generated from cultures prelabelled by incubation with [3H]-arachidonic acid. Exposure to 400 and 800 parts per billion (ppb) NO2 significantly increased the release of 51Cr from 0.9 +/- 0.4%, in control cultures exposed to 5% CO2 in air, to 9.7 +/- 3.2% and 13.9 +/- 3.5%, respectively. Similarly, NO2 also significantly increased the movement of 14C-BSA across the epithelial monolayers from 1.3 +/- 0.2%, in control cultures, to 2.7 +/- 0.2%, 3.8 +/- 0.4% and 5.1 +/- 0.5%, respectively, in cultures exposed to 100, 400 and 800 ppb NO2. Although NO2 attenuated the CBF of the cells at all concentrations investigated, this was significant only at the concentration of 2,000 ppb NO2.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of action of Pseudomonas aeruginosa pyocyanin on human ciliary beat in vitro.

Pyocyanin is a blue redox active pigment produced by Pseudomonas aeruginosa. It is present at concentrations of up to 10(-4) M in sputa from patients with cystic fibrosis and bronchiectasis who are heavily colonized with this organism. Pyocyanin, at physiologically relevant concentrations, slows human nasal ciliary beat frequency (CBF) in vitro and leads to disruption of the epithelium. Pyocyanin-induced slowing of CBF after 2 h was associated with a significant fall in intracellular cyclic AMP (cAMP) (90%) and ATP (66%) and was reversible after the pyocyanin was removed by washing. These effects were not mediated through interaction with neutrophils. The pyocyanin-induced fall in CBF was not affected by EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], pyrazinamide, 8-phenyltheophylline, indomethacin, or antioxidants, including catalase (500 U/ml), superoxide dismutase, and N-acetylcysteine. Ciliary slowing was, however, prevented (> 70%) by isobutylmethylxanthine and forskolin, both of which increase intracellular cAMP, and also by the cAMP analog, dibutyryl cAMP. There was also a concomitant protection against the fall in both cAMP and ATP. These agents also delayed the onset of epithelial disruption associated with pyocyanin treatment. In contrast, treatment with the iron chelator desferrioxamine prevented epithelial disruption, although it had no effect on pyocyanin-induced slowing of CBF. It appears that ciliary slowing can be dissociated from epithelial disruption and that the effects of pyocyanin on CBF are associated with a fall in both intracellular cAMP and ATP.

Inhibition of human neutrophil migration in vitro by low-molecular-mass products of nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae commonly causes infections in the lower and upper respiratory tract, although the mechanisms of its colonization and persistence in the airways are unclear. Culture filtrates from six clinical isolates of this bacterium were assessed for their abilities to influence neutrophil function in vitro. Each culture filtrate was assessed on six separate occasions with neutrophils obtained from six different donors. During the log and early stationary phases of growth (0 to 18 h), culture filtrates contained primarily neutrophil chemokinetic activity but no activity affecting neutrophil migration toward the chemotactic factors N-formyl-L-methionyl-L-leucyl-L-phenylalanine and leukotriene B4. In contrast, filtrates obtained after 24 h of culture contained factors which inhibited neutrophil migration toward both of these chemotactic factors. This chemotaxis-inhibitory activity persisted between 24 and 72 h of bacterial culture, and it was not associated with the presence of either chemotactic or chemokinetic activity as assessed by checkerboard analysis. Gel filtration of pooled 72-h filtrates yielded three major peaks of chemotaxis-inhibitory activity. Endotoxin was present together with two other low-molecular-mass hydrophobic factors of approximately 8 and 2 kDa. These low-molecular-mass factors are chloroform insoluble and heat stable, and they are inactivated by protease, periodate, and diborane reduction. Activity was completely retained on a wheat germ agglutinin column, and it could be eluted with N-acetyl-D-glucosamine. These data suggest that inhibitory activity is associated with N-acetyl-D-glucosamine-containing glycopeptides, possibly derived from the bacterial cell wall. The production of these compounds may contribute to the persistence of this bacterium in vivo by inhibiting neutrophil chemotaxis in the microenvironment of the respiratory mucosa.

Histidine decarboxylases from bacteria that colonise the human respiratory tract.

We investigated whether production of histamine by bacteria isolated from sputum of patients with infective lung diseases could be attributed to the presence of histidine decarboxylase (HD). Twenty gram-positive and 20 gram-negative organisms were studied for their ability to decarboxylate 14C-histidine in vitro over the pH range 4.5-7.5. Of the bacteria investigated, lysates from the gram-negative species Haemophilus influenzae, H. parainfluenzae, Moraxella (Branhamella) catarrhalis and Pseudomonas aeruginosa liberated 14CO2 and histamine from 14C-histidine in the presence of the cofactor pyridoxal phosphate. In contrast, results obtained in the absence of cofactor were similar to those of negative (lysate-free) controls suggesting that the HD enzymes of these species resembled those previously described in other gram-negative bacteria. No HD activity was detected over this pH range in lysates from gram-positive species. This finding correlated with earlier observations that these gram-positive organisms did not produce histamine in vitro.

The daily variability of bronchial responsiveness to methacholine.

Ten mild atopic asthmatics on inhaled beta 2-agonists alone were studied in order to determine the repeatability of methacholine inhalation provocation tests at 24 h intervals over a period of 5 days. Such patients are most frequently studied in therapeutic trials of anti-asthmatic medications. There were no significant differences in results obtained on any of the days and no evidence for the development of tolerance to methacholine in this group of patients at one day intervals. The 95% confidence interval for repeatability of the results was +/- 1.05 doubling doses of methacholine, and 95% range +/- 2.36 doubling doses, comparable to the results obtained by other investigators on similar patients. Some investigators have produced more highly repeatable results but these have generally been obtained using highly selected groups of patients.