Polymyxin B sulfate-induced pH-dependent increase in calcium influx in cultured fibroblasts

Polymyxin B sulfate treatment induced an increase in calcium influx in mouse fibroblasts (3T6) and normal human skin fibroblasts. This increase in calcium influx occurred in a dose- and time-dependent fashion and was dependent on pH but independent of the electrochemical gradient of calcium across the plasma membrane. This effect was prevented when cycloheximide (20 micrograms/ml) was added with polymyxin B sulfate. Addition of actinomycin D (2 micrograms/ml) also remarkably reduced this effect. In view of these findings, it is possible that polymyxin B sulfate therapy-induced side effects, such as neuromuscular blockade and kidney dysfunction, are conditional and may be due to an increase in calcium influx.     

Lethal effect of polymyxin B sulfate in experimental Proteus rettgeri infection in mice

The mouse model of intraperitoneal sepsis with Proteus rettgeri was used to evaluate the anti-endotoxic effect of polymyxin B sulfate. An unexpected reversal of the usual protective effect of polymyxin in experimental enterobacterial sepsis was observed in which the lethality of the infection was enhanced.     

Protective effect of polymyxin B sulfate in experimental enterobacterial infection in mice.

The mouse model of intraperitoneal enterobacterial sepsis was used to evaluate the anti-endotoxic effect of polymyxin B sulfate. Single or multiple therapeutic doses of polymyxin, administered either before or after lethal challenge with Serratia marcescens, produced statistically and clinically significant protective effects.     

Results of an experimental study of the pharmacokinetics of polymyxin B sulfate]

Pharmacokinetics of polymyxin B sulfate of Soviet production was studied in various species of animals with the use of different administration routes and dosage. After a single intramuscular administration of the drug to dogs in doses of 1.1 and 2.2 mg/kg the antibiotic was detected within 5 hours at the maximum level during the 1st hour. A two-fold increase of the dose was accompanied by 1.5 times increase in the antibiotic level. Repeated administrations of polymyxin B sulfate in a dose of 4.5 mg/kg did not result in an increase in the blood level as compared to a single use of the drug. When polymyxin B sulfate was administered intravenously, the concentration peak was observed in 15 minutes independent of the dosage. Later the antibiotic level decreased. The maximum level of the drug in the mice was observed 1 hour after its intramuscular administration in a dose of 8 mg/kg, the highest levels being registered in the kidney tissues and urine.     

Control of Salmonella enteritidis infections in poultry by polymyxin B and trimethoprim

Antimicrobial compounds were screened in vitro in Trypticase soy broth for antimicrobial activity against a virulent strain of Salmonella enteritidis. Of the several compounds tested, polymyxin B showed the strongest inhibition in vitro, preventing growth at a concentration of less than or equal to 10 micrograms/ml. Polymyxin B administered in the drinking water was effective in vivo for preventing infections in 1-day-old chickens but did not remove established infections in 1-week-old chickens. It was found that trimethoprim, which was not active in vitro, prevented colonization and removed existing infections in 1-day-old chickens when it was administered together with polymyxin B sulfate. Enrichment cultures in which selenite-cystine and tetrathionate broth media were used showed that chickens given a combination of 100 micrograms of polymyxin B sulfate per ml and 250 micrograms of trimethoprim per ml 24 h prior to oral inoculation with 10(8) to 10(9) CFU were negative for S. enteritidis after 7 days. Established infections (10(5) to 10(6) CFU/g of feces) in 1-week-old chickens were eliminated by treatment with the polymyxin-trimethoprim system. This antimicrobial agent treatment may be useful for preventing colonization in poultry and for eliminating S. enteritidis from infected flocks.     

Control of Salmonella enteritidis infections in poultry by polymyxin B and trimethoprim.

Antimicrobial compounds were screened in vitro in Trypticase soy broth for antimicrobial activity against a virulent strain of Salmonella enteritidis. Of the several compounds tested, polymyxin B showed the strongest inhibition in vitro, preventing growth at a concentration of less than or equal to 10 micrograms/ml. Polymyxin B administered in the drinking water was effective in vivo for preventing infections in 1-day-old chickens but did not remove established infections in 1-week-old chickens. It was found that trimethoprim, which was not active in vitro, prevented colonization and removed existing infections in 1-day-old chickens when it was administered together with polymyxin B sulfate. Enrichment cultures in which selenite-cystine and tetrathionate broth media were used showed that chickens given a combination of 100 micrograms of polymyxin B sulfate per ml and 250 micrograms of trimethoprim per ml 24 h prior to oral inoculation with 10(8) to 10(9) CFU were negative for S. enteritidis after 7 days. Established infections (10(5) to 10(6) CFU/g of feces) in 1-week-old chickens were eliminated by treatment with the polymyxin-trimethoprim system. This antimicrobial agent treatment may be useful for preventing colonization in poultry and for eliminating S. enteritidis from infected flocks.     

USE OF POLYMYXIN B IN A CAPTURE ENZYME IMMUNOASSAY FOR DETECTION OF SALMONELLAE SPP. LIPOPOLYSACCHARIDE

A capture enzyme immunoassay for detection of salmonellae sp. lipopolysaccharide was developed. The assay made use of polymyxin B sulfate, passively attached to a polystyrene matrix, to capture lipopolysaccharide. Bound lipopolysaccharride was then detected with a monoclonal antibody, specific for salmonellae spp. followed by goat antimouse antibody conjugated with horseradish peroxidase.
The analytical sensitivity of the assay was approximately 1 ng/ml of lipopolysaccharide. The results are comparable to those obtained with a competitive enzyme immunoassay previously developed. The sensitivity of the polymyxin B assay decreased to 4–5 ng/ml when the salmonellae spp. lipopolysaccharide was mixed with 1–100 μg/ml of Escherichia coli lipopolysaccharide, while this level of heterogeneous lipopolysaccharide, did not decrease the sensitivity of the competitive enzyme immunoassay.
The polymyxin B capture assay was advantageous in that polymyxin B is a standardized reagent that is relatively inexpensive and does not require extensive preparation or containment facilities. The assay is robust; however, because of the light sensitivity of polymyxin B, its stickiness to other reagents and interference by other lipopolysaccharides, this assay requires careful attention to detail and may therefore be an unsuitable assay for field use.     

In Vitro Effects of Carbenicillin Combined with Gentamicin or Polymyxin B Against Pseudomonas aeruginosa

Disodium carbenicillin and gentamicin sulfate have both shown promise in the treatment of infections caused by Pseudomonas aeruginosa. This study was designed to explore possible synergistic relationships among the new as well as the established antimicrobial agents used to treat such infections. With an agar dilution technique, minimum inhibitory concentrations of 27 strains of P. aeruginosa were determined in two-dimensional tests. Graphs of equal biological activity (isobolograms) demonstrated moderate synergistic effects of the carbenicillin-gentamicin combination over therapeutically feasible concentration ranges. In contrast, the combination of carbenicillin and polymyxin B showed only additive or slightly antagonistic effects. Tests of bacterial killing confirmed the presence of carbenicillin-gentamicin synergy in 3 of 6 strains of P. aeruginosa, but did not show true antagonism between carbenicillin and polymyxin B. Clinical trials of both drug combinations are advisable to determine whether therapeutic results can be improved, and whether the dosages of gentamicin or polymyxin B can thereby be reduced to lessen their toxic hazards.     

Production of Axenic Gonyaulax Cultures by Treatment with Antibiotics

The effects of amphotericin B, chloramphenicol, dihydrostreptomycin sulfate, neomycin sulfate, polymyxin B sulfate, potassium penicillin G, and streptomycin sulfate (used singularly and in various combinations at different concentrations) on the growth and development of four marine dinoflagellates of the genus Gonyaulax and associated bacteria were studied. The combination of amphotericin B, dihydrostreptomycin, neomycin, and penicillin G was highly effective in eliminating bacteria and fungi without reducing dinoflagellate growth and provided a useful method for obtaining axenic cultures of two Gonyaulax species, G. catenella and G. excavata.     

Positive antibacterial co-action between hop (Humulus lupulus) constituents and selected antibiotics

The research reported here deals with co-action of the hop (Humulus lupulus)-derived anti-bacterial compounds, lupulone and xanthohumol, with several antibiotics. Among the antibiotics investigated for their co-action, polymyxin B sulfate, tobramycin and ciprofloxacin had a positive co-action in inhibiting selected test bacteria. The disc/well-diffusion assay and the minimum inhibitory concentration test (MIC) were employed to determine co-action. Both Gram-positive and Gram-negative bacteria were used in the evaluation. There was some co-action against all Gram-positive bacteria tested. Surprisingly, there was some positive co-action even against certain Gram-negative bacteria but not against others. Particularly, there was no co-action against E.coli. An antibacterial cream with lupulone, neomycin and polymyxin B sulfate was prepared and showed co-action. Ideas for other practical applications of this effect are put forth. The mechanism of the synergistic effect is briefly discussed but no attempt was made to prove it experimentally.     

Succinate accumulation in pig large intestine during antibiotic-associated diarrhea and the constitution of succinate-producing flora

Succinate was the major organic acid detected in the hindgut content of pigs suffering from antibiotic-associated diarrhea. Antibiotic-associated diarrhea was induced by an oral dose of polymyxin B sulfate (3,000,000 units/day) or an intramuscular injection of enrofloxacin (0.6 g enrofloxacin/day). In the large intestine of enrofloxacin-treated pigs, Gram-negative facultative anaerobic rods phylogenetically related to Escherichia coli and Gram-positive facultative anaerobic non-spore-forming rods phylogenetically related to Lactobacilli were isolated as succinate producers. Succinate-producing Lactobacilli were only isolated as the succinate producer in polymyxin B sulfate-treated pigs. In contrast to antibiotic-associated diarrhea pigs, bacteria belonging to Bacteroidaceae, Fusobacteria, and Enterobacteriaceae were detected as succinate producers in a non-treated pig. In antibiotic-associated diarrhea conditions, antibiotic-resistant Enterobacteria, E. coli in particular, and Lactobacilli may contribute to an abnormal succinate accumulation and may affect water absorption in the hindgut that relates to an expression of antibiotic-associated diarrhea.     

Use of a topical antibiotic spray in vaginal surgery.

During vaginal hysterectomy with or without colporrhaphy a topical aerosol spray containing neomycin sulfate, polymyxin B sulfate and zinc bacitracin was used in 50 patients to decrease the change of postoperative pelvic infection; a placebo spray was used in another 50 patients. All patients were treated preoperatively with povidone iodine and postoperatively with nitrofurantoin and an antibacterial irrigating solution for the bladder if catheter drainage was necessary. The frequency of postoperative pelvic infection was 16% in the group sprayed with the antibiotic combination and 34% in the group sprayed with the placebo, a significant difference (P less than 0.05).     

Ultrastructural study of polymyxin-resistant isolates of Pseudomonas aeruginosa.

Upon exposure to 6,000 U of polymyxin B sulfate per ml, cells of the polymyxin-sensitive PAO 1 strain of Pseudomonas aeruginosa displayed in thin sections long projections arising from the outer membrane of the cell wall and extensive cytoplasmic degradation with accumulation of cytoplasmic membrane infoldings. Polymyxin-resistant isolates derived from the PAO 1 strain, however, grew well in the presence of 6,000 U of polymyxin per ml and exhibited none of these effects, having instead the appearance of a typically healthy cell. Freeze-etching of cells of the sensitive strain grown in basal medium without polymyxin revealed a concave cell wall layer studded with numerous particles. Freeze-etching of cells of the resistant isolates grown in basal medium containing 6,000 U of polymyxin per ml revealed a concave cell wall layer (i.e., the outer half of the outer membrane) in which most of these particles were absent. Thus, acquisition of resistance to polymyxin was correlated with an alteration in the architecture of the outer membrane. When the resistant isolates were grown in the basal medium lacking polymyxin and then freeze-etched, the particle distribution in the concave cell wall layer resembled that of the sensitive parent strain. The cells had regained sensitivity to polymyxin upon suspension in medium containing 6,000 U/ml as determined by their failure to grow and by internal damages seen in thin sections. These cells also had acquired increased sensitivity to ethylenediaminetetraacetate, whereas the polymyxin-resistant cells grown in the presence of polymyxin were resistant to lysis by ethylenediaminetetraacetate. The polymyxin-resistant isolates were not stable mutants but instead represented an adaptive response to the presence of polymyxin in the medium.     

Enumeration of Bacillus cereus in Foods

For the enumeration of vegetative cells and spores of Bacillus cereus in foods, a mannitol-egg yolk-phenol red-agar has been developed which exploits the failure of B. cereus to dissimilate mannitol, and the ability of most strains to produce phospholipase C. When a high degree of selectivity was required, polymyxin B sulfate in a concentration of 10 ppm appeared to be the most effective selective additive. Useful characteristics for the identification of presumptive isolates of B. cereus were found to be: morphology, dissimilation of glucose mostly to acetyl methyl carbinol under anaerobic conditions, hydrolysis of starch and gelatin, reduction of nitrate, and growth on 0.25% chloral hydrate agar.     

The PmrA-regulated pmrC gene mediates phosphoethanolamine modification of lipid A and polymyxin resistance in Salmonella enterica

The PmrA/PmrB regulatory system of Salmonella enterica controls the modification of lipid A with aminoarabinose and phosphoethanolamine. The aminoarabinose modification is required for resistance to the antibiotic polymyxin B, as mutations of the PmrA-activated pbg operon or ugd gene result in strains that lack aminoarabinose in their lipid A molecules and are more susceptible to polymyxin B. Additional PmrA-regulated genes appear to participate in polymyxin B resistance, as pbgP and ugd mutants are not as sensitive to polymyxin B as a pmrA mutant. Moreover, the role that the phosphoethanolamine modification of lipid A plays in the resistance to polymyxin B has remained unknown. Here we address both of these questions by establishing that the PmrA-activated pmrC gene encodes an inner membrane protein that is required for the incorporation of phosphoethanolamine into lipid A and for polymyxin B resistance. The PmrC protein consists of an N-terminal region with five transmembrane domains followed by a large periplasmic region harboring the putative enzymatic domain. A pbgP pmrC double mutant resembled a pmrA mutant both in its lipid A profile and in its susceptibility to polymyxin B, indicating that the PmrA-dependent modification of lipid A with aminoarabinose and phosphoethanolamine is responsible for PmrA-regulated polymyxin B resistance.     

Selective medium for Pseudomonas cepacia containing 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan and polymyxin B sulfate.

Contamination of solutions and lotions with Pseudomonas cepacia is a growing concern among health professionals. The identification of P. cepacia usually requires a long series of biochemical tests. In an effort to develop a more direct method, we evaluated plate count agar containing 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan and polymyxin B sulfate at respective concentrations of 1 and 75 micrograms/ml as a medium for selectively isolating P. cepacia. The medium inhibited the growth of all gram-negative bacilli and gram-positive cocci tested except P. cepacia and Serratia marcescens. These two microorganisms could easily be differentiated by their colony morphology and their reactions in the oxidase test. When nonsterilized water samples were inoculated with P. cepacia and spread or streaked on the selective medium, all P. cepacia organisms were recovered. These results demonstrate the usefulness of 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan and polymyxin B sulfate in the detection of P. cepacia. We believe that this selective medium could be useful in isolating P. cepacia from mixed bacterial flora that might be present in environmental water and water-related samples, such as solutions and lotions.     

Mechanism of Polymyxin B Resistance in Proteus mirabilis

The lipids from three types of organisms-a Proteus mirabilis wild type highly resistant to polymyxin B, a polymyxin B-sensitive mutant derived from the wild type, and the wild type grown in the presence of sulfadiazine resulting in phenotypic conversion to polymyxin B sensitivity-were examined to determine the nature of polymyxin B resistance. The phospholipid compositions were nearly identical; each organism contained similar small amounts of N-methyl phosphatidylethanolamine in addition to comparable quantities of phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. the fatty acid compositions were similar in the exponential phase of growth; in the stationary phase, sulfadiazine markedly inhibited the synthesis of cyclopropane fatty acids. Liposomes prepared from the dried lipids of the three types of organisms were extensively and similarly disrupted by the polymyxin. These findings suggest that polymyxin B resistance in P. mirabilis is determined by the cell envelope which prevents access of the antibiotic to the susceptible lipid target sites.     

Biosynthesis of polymyxin E by a cell-free enzyme system. IV. Acylation of enzyme-bound L-2,4-diaminobutyric acid

An enzyme fraction, which catalyzes the ATP-PPi exchange reaction dependent on the three constituent amino acids of polymyxin E, was partially purified from crude extracts of Aerobacillus polyaerogenes. The approximate molecular weight was estimated to be 640,000 by Sepharose 4B gel filtration. Incubation of the enzyme with octanoyl coenzyme A and diaminobutyric acid in the presence of ATP and an ammonium sulfate fraction yielded octanoyldiaminobutyric acid thioesterified to the enzyme protein. On mild alkali treatment, octanoyldiaminobutyric acid, identified by paper chromatography, was released from the enzyme protein. From its acid hydrolyzate, diaminobutyric acid and octanoic acid were recovered in a molar ratio of 1 to 0.7. An ammonium sulfate fraction was required as the source of an acyltransferase for acylation of the enzyme-bound diaminobutyric acid. When [14C]-threonine was incubated with L-2,4-diaminobutyric acid in the presence of octanoyl coenzyme A, octanoyldiaminobutyrylthreonine bound to the enzyme protein was formed. These results suggest that acyldiaminobutyric acid bound to the enzyme protein is a possible initiation complex in the biosynthesis of polymyxin E.     

Polymyxin B binding sites in Escherichia coli as revealed by polymyxin B-gold labeling

A complex of polymyxin B, bovine serum albumin, and colloidal gold was prepared and used for the ultrastructural localization of polymyxin B binding sites on thin sections of Epon-embedded Escherichia coli cells. Gold particles were found on the outer membrane of E. coli, which is consistent with reported biochemical findings. We concluded that gold labeling with polymyxin B is useful in localizing the binding sites of polymyxin.