Neutralization of human serum beta-lysin by sodium polyanetholsulfonate and sodium amylosulfate.

Normal fresh and heat-inactivated (56 degrees C, 30 min) human sera (80 vol%, i.e., 80% [vol/vol] of a 2-ml assay volume) killed Bacillus subtilis ATCC 6633 cell inocula of 1.5 x 10(4) colony-forming units per ml within 1 to 2 h after exposure. The B. subtilis assay strain proved slightly and reversibly susceptible to 5 mug of egg white lysozyme per ml. Seitz filtration of fresh human serum completely removed beta-lysin activity; significant amounts of serum lysozyme were removed as well, as determined with the bioassay strain Micrococcus lysodeikticus ATCC 4698. However, bactericidal activity of human serum via classical or alternative complement pathway activation remained intact. Addition of 0.01 M dithiothreitol to fresh human serum abolished beta-lysin activity, but not that of serum lysozyme. Chelation of fresh and heat-inactivated human serum with 0.01 M MgCl(2) + 0.01 M ethylene glycol tetraacetic acid, but not with 0.01 M ethylenediaminetetraacetic acid, markedly retarded beta-lysin activity; however, lysozyme activity remained unaffected. Chelation of serum with 0.01 M MgCl(2) + 0.01 M ethylene glycol tetraacetic acid + 0.01 M CaCl(2) completely abrogated beta-lysin activity, but not that of lysozyme. Absorption of human serum with 10 mg of bentonite per ml (10 min, 37 degrees C) completely removed beta-lysin and lysozyme activity, but failed to affect serum bactericidal activity against Escherichia coli control strain C. Reconstitution of 50 vol% of bentonite-absorbed serum with 40 vol% of heat-inactivated human serum restored both beta-lysin and lysozyme activity. Addition of either 63 to 500 mug of sodium polyanetholsulfonate per ml or 63 to 500 mug of sodium amylosulfate per ml to 80 vol% of fresh human serum completely neutralized beta-lysin activity for the entire observation period of 22 h.     

Effect of sodium polyanethol sulfonate in blood cultures.

Fifteen-hundred hospital blood cultures were made in duplicate, with and without 0.05% sodium polyanethol sulfonate in the broth medium. A significantly higher rate and speed of recovery of both gram-positive cocci and gram-negative bacilli was accomplished in sodium polyanethol sulfonate broth. The effect was independent of the content of 0.1% agar in the growth medium. In the cases of Neisseria meningitidis septicemia examined, however, a detrimental result on recoveries was observed. The addition of sodium polyanethol sulfonate also resulted in an increased frequency of recoveries of contaminating organisms.     

Studies on neutralization of human serum bactericidal activity by sodium amylosulfate.

The synthetic anticoagulant sodium amylosulfate (SAS) at concentrations of 125 to 2,000 microgram/ml failed to completely neutralize the bactericidal activity of 80 and 50% (by volume) fresh human serum. Furthermore, SAS failed to inhibit the alternative pathway of complement activation in 80% (by volume) fresh human serum that had been chelated with 0.01 M magnesium ions plus 0.01 M ethyleneglycol-bis(beta-aminoethylether)-N,N-tetraacetic acid. However, SAS at 250 to 1,000 microgram/ml effectively neutralized the bactericidal activity of 20% (by volume) fresh human serum. Therefore, SAS (at 250 to 1,000 microgram/ml) should be used only in blood samples that have been diluted at least fivefold (less than or equal to 20% [by volume]) in suitable broth media.     

Inhibitory effect in vitro of sodium polyanethol sulfonate on the growth of Neisseria meningitidis.

The influence of 0.05% sodium polyanethol sulfonate on the growth of 24 strains of Neisseria meningitidis in broth medium was examined; several of the strains were markedly inhibited. A paper disk method was evolved for screening the sensitivity of various bacteria to sodium polyanethol sulfonate on solid medium; the highest sensitivity was observed in N. meningitidis and Neisseria gonorrhoeae. Zones of growth inhibition were also observed in a proportion of strains in certain species of gram-positive cocci, whereas all gram-negative bacilli were uniformly resistant. The implications of these observations for the routine use of sodium polyanethol sulfonate in blood culture media are discussed.     

Evaluation of the sodium polyanethol sulfonate disk test for the identification of Peptostreptococcus anaerobius.

The previously reported sodium polyanethol sulfonate disk test for the identification of Peptostreptococcus anaerobius (Graves et al., 1974) was evaluated, with modifications. Three bands of brucella agar, three inoculum sizes, and two inoculum sources were compared. Nine stock cultures of P. anaerobius (eight normal flora isolates and ATCC 27337) and 16 fresh clinical isolates were used. All cultures of P. anaerobius showed inhibition zones of 12 to 30 mm in diameter, regardless of test conditions. Out of 103 clinical isolates of other species of anaerobic gram-positive cocci tested, only two had an inhibition zone size in this range (one P. micros of 11 studied had a zone of 12 mm and one P. prevotii of 14 studied had a zone of 16). The test had an overall accuracy of 98% in the identification of P. anaerobius from clinical specimens. Since P. anaerobius accounts for one-fifth to one-third of all anaerobic gram-positive cocci encountered in clinical specimens, this simple and rapid technique can be very useful for presumptive identification.     

Effect of blood dilution on recovery of organisms from clinical blood cultures in medium containing sodium polyanethol sulfonate.

This clinical study was designed to evaluate the standard laboratory protocol that requires blood specimens be diluted with greater than or equal to 10 volumes of media. Blood was collected from hospitalized patients, and 1 ml was inoculated into each of three vials containing 2.3, 7.3, and 24 ml of BACTEC 6B aerobic medium resulting in dilutions of 1:4, 1:10, and 1:30, respectively. The three test vials were treated identically, and the study was carried out at four hospitals. Of the 2,550 sets of vials inoculated, 174 were positive with clinically significant isolates from 105 patients. There was no difference in the number of positive cultures recovered by 24 h (67%) or 48 h (90%) from any dilution. These percentages agreed with other reports from BACTEC users. The number of positive vials (139, 144, 147, respectively) at each dilution was not significantly different, indicating that all three dilutions showed equal recovery of pathogenic microorganisms. Despite this overall equality, two patients, one on antibiotic therapy, were found to have correlated cultures which failed to grow at the 1:4 dilution. This finding implies that a 1:4 dilution of blood cannot be recommended unequivocably despite the higher overall recovery rate of positive cultures.     

Comparison of recovery rates of various organisms from clinical hypertonic blood cultures by using various concentrations of sodium polyanethol sulfonate.

By using parallel culture techniques, the recovery rates of a wide spectrum of organisms encountered in hypertonic clinical blood cultures was determined from four different blood culture bottles. Each bottle was identical except for the amount of sodium polyanethol sulfonate (SPS) present. Flasks A, B, C, and D contained SPS in final concentrations of 0.025, 0.05, 0.075, and 0%, respectively. Of 144 patients found to have clinically relevant organisms in their blood cultures, 127 had positive A flasks, 144 had positive B flasks, 140 had positive C flasks, and 110 had positive D flasks. There was no significant difference in the time required to obtain organism recovery from the A, B, or C flasks; however, the time required to obtain organism recovery from the D flask was considerably longer, ranging up to 5 days in many cases. Of the various organisms recovered, 3 of 7 strains of anaerobic streptococci and 1 of 28 strains of Streptococcus pneumoniae appeared to be inhibited by SPS when the concentration was 0.075%. In no case was an organism recovered from either the A or D flask but not from the B flask, indicating that a concentration of 0.05% SPS in hypertonic media does not inhibit the growth of a wide spectrum of organisms in clinical blood cultures.     

Gelatin neutralization of the inhibitory effect of sodium polyanethol sulfonate on Neisseria meningitidis in blood culture media.

The inhibitory effect of sodium polyanethol sulfonate (0.05%) upon growth of Neisseria meningitidis was found to be neutralized by adding gelatin (l.1%) to the growth medium. The neutralizing effect was demonstrated in solid medium, as well as in nutrient broth for blood cultures. The findings parallel those of Wilkins and West (6) regarding gelatin neutralization of the inhibitory effect of sodium polyanethol sulfonate on Peptostreptococcus anaerobius.     

Comparison of sodium amylosulfate and sodium polyanetholsulfonate in blood culture media.

A comparison between sodium polyanetholsulfonate and sodium amylosulfate in unvented vacuum blood culture bottles containing tryptic soy broth was made with 5,800 sets of blood cultures. No statistically significant differences in isolation rates of bacteria were noted.     

Neutralization of bacteria- and endotoxin-induced hypotension by lipoprotein-free human serum.

Normal human serum and a fraction rich in lipoprotein, Cohn fraction IV1, have been shown in previous studies to detoxify native endotoxin by decreasing lethality for mice, fever in rabbits, and by the alteration of the characteristic endotoxin-anti-endotoxin precipitin pattern in gels. These studies are extended herein and document the ability of normal human serum and fraction IV1 to neutralize the induction of hypotension in rabbits by viable gram-negative bacilli. Further fractionation of serum, using an ultracentrifugal flotation method for producing lipoprotein-free human serum and purified high-density lipoproteins, revealed the lipoprotein-free fraction to be capable of inhibiting endotoxin hypotensive activity and to alter diffusion of endotoxin in gels. On the other hand, the purified high-density lipoproteins failed to negate either activity.     

Neutralization of bacterial lipopolysaccharides by human plasma.

To quantify the neutralization of bacterial lipopolysaccharide (LPS) by human plasma, dilutions of Escherichia coli O113 LPS were incubated with plasma, followed by the addition of Limulus amebocyte lysate (LAL). The reaction between the LPS and LAL was monitored spectrophotometrically, and the concentration of LPS resulting in 50% lysate response (LR50) was determined. Analysis of 145 outdated plasma samples yielded a range of LR50 between 6 and 1,500 ng/ml. Pools of plasma with high and low LR50 were prepared. The pool with high LR50 neutralized 166-fold more E. coli 0113 LPS, 190-fold more E. coli 0111B4 LPS, 42-fold more Klebsiella pneumoniae LPS, and 29-fold more Salmonella typhimurium LPS than did the pool with low LR50. Each pool had similar immunoglobulin G (IgG) and IgM antibody levels to homologous LPS, measured by an enzyme-linked immunosorbent assay. Analysis of 212 fresh-frozen plasma units revealed a range of LR50 between 48 and 6,000 ng/ml. Incubation of LPS in a pool of fresh-frozen plasma with high LR50 elicited significantly less fever in the rabbit pyrogen test than did LPS incubated in plasma with low LR50 (fever index, 2.68 +/- 0.61 degrees C X h and 3.52 +/- 0.66 degrees C X h, respectively; P = 0.003). We conclude that there is a 100-fold range in the endotoxin-neutralizing capacity of human plasma and that this variation is not due to LPS-specific IgG or IgM antibodies. Further investigations are needed to determine whether differing susceptibility of patients to the effects of LPS is due to differences in the endotoxin-neutralizing capacity of their plasma and whether plasma screened for high endotoxin-neutralizing capacity may be therapeutically useful in endotoxemia.     

Comparative evaluation of supplemented peptone broth with sodium polyanetholesulfonate and trypticase soy broth with sodium amylosulfate for detection of septicemia.

We compared the yield and speed of detection of clinically important microorganisms from 10,156 paired 5-ml samples of blood cultured in supplemented peptone broth (SPB) with 0.03% sodium polyanetholesulfonate (SPS) or Trypticase soy broth (TSB) with 0.5% sodium amylosulfate (SAS). The atmosphere of incubation (open venting units) and ratio of blood to broth (1:10) were the same for both samples. Only cultures with adequate blood samples (greater than or equal to 80% of stated volume) were compared statistically. Overall, SPB/SPS outperformed TSB/SAS. Bacteroidaceae and Eubacterium were found more often (P less than 0.05) and viridans streptococci were found sooner (P less than 10(-4)) in SPB/SPS than in TSB/SAS. Most importantly, staphylococci were found both more often (P less than 0.03) and sooner (P less than 10(-7)) in SPB/SPS than in TSB/SAS. In a separate experiment, SAS slowed the growth of a clinical strain of Staphylococcus aureus in TSB. Unless important advantages can be confirmed for SAS in controlled clinical trials, SAS cannot be recommended for routine use as an anticoagulant in blood culture media.     

Prevention of carbon tetrachloride-induced liver necrosis by the chelator alizarin sodium sulfonate.

The administration of the calcium chelator alizarin sodium sulfonate (ASR) (100 mg/kg ip in saline) 30 min before or 6 or 10 hr after CCl4 (1 ml/kg ip as a 20% v/v solution in olive oil) partially prevents the necrogenic effect of the hepatotoxin at 24 hr, but prevention of CCl4 fat accumulation was not observed. Protective action cannot be attributed to potential decreasing effects of ASR on CCl4 levels reaching the liver, on the covalent binding of CCl4-reactive metabolites to cellular components, or on CCl4-induced lipid peroxidation because ASR does not modify these parameters significantly. ASR administration increases GSH levels in livers of both control and CCl4-poisoned animals and decreases the calcium content of intoxicated animals at 24 hr of poisoning. ASR significantly lowers the body temperature of CCl4-treated animals at different times of the intoxication process. Present and previous results from our laboratory on the preventive effects of another very specific calcium chelator, calcion, and several anticalmodulins suggest that the beneficial effects of ASR might be associated with its calcium chelating ability. Other protective effects of ASR, such as lowering body temperature or increasing GSH content in liver, cannot be excluded.     

Controlled clinical comparison of BacT/ALERT standard aerobic and standard anaerobic blood culture bottles inoculated directly or after transport in sodium polyanethol sulfonate tubes

To assess relative performances in the BacT/ALERT blood culture system, we compared results from the direct inoculation of standard media and inoculation after the transport of blood samples in Vacutainer tubes with sodium polyanethol sulfonate. No significant differences in yields or times to detection were found for 387 clinically important isolates from 4,306 blood culture sets.     

Acute but not chronic gastric sodium administration regulates vasoactive intestinal peptide metabolism by the liver.

We have shown previously that gastric sodium loading releases vasoactive intestinal peptide from the intestine and in rabbits on a low sodium diet it appears to decrease vasoactive intestinal peptide metabolism by the liver. To determine the contributions of the low sodium diet and the acute sodium load to changes in vasoactive intestinal peptide metabolism, metabolic clearance studies of vasoactive intestinal peptide infused intraportally were performed. These studies were performed in male New Zealand white rabbits equilibrated on normal and low sodium diets before and after an acute gastric sodium load of 1.5 mmol kg-1. No difference was detectable in metabolic clearance rates between normal and low salt diets, however, decreases in metabolic clearance rates were observed in response to the sodium load (normal diet P less than 0.005, low salt P less than 0.0005). Secretion rates also decreased following the gastric sodium load (normal P less than 0.005, low salt P less than 0.05). We conclude that hepatic VIP metabolism is decreased by acute gastric sodium loading but it is not affected by chronic sodium intake.     

Improving detection of "Viridans streptococcus" bacteraemia by adding sodium polyanethol sulphonate to blood cultures

To detect streptococcal bacteraemia in patients undergoing dental extraction blood cultures containing glucose broth with 0.05% sodium polyanethol sulphonate (Liquoid) were compared with identical cultures without Liquoid.     

In vitro evidence that human airway lysozyme is cleaved and inactivated by Pseudomonas aeruginosa elastase and not by human leukocyte elastase.

The in vitro effects of Pseudomonas aeruginosa elastase (P. aeruginosa E) and of human leukocyte elastase on human airway lysozyme (HAL) were investigated. P. aeruginosa E inactivated and cleaved HAL, whereas human leukocyte elastase had no effect. Total inactivation of HAL by P. aeruginosa E was observed after 120 min of incubation at 37 degrees C, for an elastase-to-lysozyme molar ratio of 1:5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of reaction mixtures containing HAL and P. aeruginosa E in an elastase-to-lysozyme molar ratio of 1:10 showed a progressive disappearance of the HAL band upon increasing the incubation time with P. aeruginosa E. Gel filtration chromatography indicated that HAL was cleaved into at least three peptide fragments. The cleavage of HAL by P. aeruginosa E was accompanied by parallel losses of its bacteriolytic activity and its immunoreactive property.     

The neoglycoprotein mannose-bovine serum albumin, but not progesterone, activates T-type calcium channels in human spermatozoa.

The neoglycoproteins alpha-D-mannose-bovine serum albumin (mannose-BSA) and N-acetyl-alpha-D-glucosamine-BSA (glucNAc-BSA) were shown to rapidly increase intracellular free calcium ([Ca2+]i) in human spermatozoa. The increase in [Ca2+]i induced by these neoglycoproteins accounts for the known ability of these compounds to induce the acrosome reaction in human spermatozoa. Our data support the hypothesis that mannose-BSA, but not progesterone, activates T-type Ca2+ channels in human spermatozoa for the following reasons: (i) the capacity of mannose-BSA to increase [Ca2+]i was inhibited by the specific T-type Ca2+ channel blocker mibefradil (IC50 = 10(-6) mol/l) while progesterone was not inhibited by 10(-5) M mibefradil; (ii) the effect of mannose-BSA to elevate [Ca2+]i was inhibited more potently by Ni2+ (IC50 = 0.1 mmol/l) than Cd2+ (IC50 = 0.5 mmol/l), whereas the effect of progesterone to elevate [Ca2+]i was inhibited equally by Ni2+ and Cd2+ (IC50 = 0.25 mmol/l); (iii) the effects of mannose-BSA and progesterone to increase [Ca2+]i were greater than additive. These data support the idea that mannose-BSA and progesterone were activating distinct Ca2+ channels, one of which was a T-type Ca2+ channel activated by mannose-BSA whereas the Ca2+ channel that was activated by progesterone has yet to be defined at the molecular level.