Influence of Serum and Calcium on the Bactericidal Activity of Gentamicin and Carbenicillin on Pseudomonas aeruginosa

Because calcium was found to be antagonistic in vitro to the activity of colistin and polymyxin B on Pseudomonas aeruginosa, the effects of calcium and serum on gentamicin and carbenicillin were also examined. Serum was antagonistic to gentamicin in antibiotic tube dilution tests on five strains of P. aeruginosa. Serum was not antagonistic to carbenicillin in tube dilution tests. Physiologic concentrations of calcium antagonized the activity of gentamicin but not carbenicillin. The antagonism observed with gentamicin was less than that previously seen with colistin. The antagonistic effect of calcium and serum was removed by a chelating agent. Gentamicin and carbenicillin may be more active in vivo against P. aeruginosa than colistin or polymyxin B.     

Antibiotic Susceptibilities of Serratia marcescens and Enterobacter liquefaciens

Production of 5'-nucleotides by Serratia marcescens and Enterobacter liquefaciens correlates with deoxyribonuclease production, indicating the close relationship between these two organisms. To determine further relationships, susceptibilities of 279 strains of the tribe Klebsielleae were determined by the high-potency disc method, agar-dilution method, or both, by using 14 antibiotics. Ninety-seven per cent of S. marcescens (201 of 207 strains) and 100% of E. liquefaciens (17 strains) had minimum inhibitory concentration (MIC) of 100 mug/ml or greater with colistin and polymyxin B. With these two antibiotics, 93% of other Enterobacter species (28 strains) had MIC values of less than 1.6 mug/ml, and 100% of Klebsiella (27 strains) had MIC values less than 1.6 mug/ml. Consistent patterns were not noted with the other antibiotics tested, but the results with colistin and polymyxin B provide additional evidence of the close relationship of S. marcescens and E. liquefaciens.     

Novel polymyxin derivatives are less cytotoxic than polymyxin B to renal proximal tubular cells

The emergence of very multiresistant Gram-negative bacterial strains has reinstated polymyxins (polymyxin B, colistin), pentacationic lipopeptides, in the therapy, in spite of their nephrotoxicity. Extensive tubular reabsorption concentrates polymyxin in proximal tubular cells. The novel polymyxin derivatives NAB739, NAB7061 and NAB741 have their cyclic part identical to that of polymyxin B, but their side chain consists of uncharged octanoyl-threonyl-d-serinyl, octanoyl-threonyl-aminobutyryl, and acetyl-threonyl-D-serinyl respectively. In this study, we compared the toxicities of NAB739, NAB7061 and NAB741 with that of polymyxin B by using the porcine renal proximal tubular cell line LLC-PK1 electroporated or incubated with the selected compound. Both the ability to cause cell necrosis (quantified as the leakage of lactate dehydrogenase) and the ability to cause apoptosis (as quantified by counting apoptotic nuclei) were assessed. In electroporated cells, polymyxin B induced total (>85%) necrosis of the cells at 0.016 mM, whereas an approx. 8-fold concentration of NAB739 and NAB7961 and an approx. 32-fold concentration of NAB741 was required for the same effect. In cells treated without electroporation (incubated), polymyxin B elicited a marked degree (approx. 50%) of necrosis at 0.5mM, whereas the NAB compounds were inert even at 1mM. Neither polymyxin B nor the NAB compounds induced apoptosis.     

Enzymatic Degradation of Colistin Isolation and Identification of α-N-Acyl α,γ-Diaminobutyric Acid and Colistin Nonapeptide

Colistin, a fatty acyl peptide antibiotic, was attacked by proteolytic enzymes such as papain, ficin and bromelain, and as degradation product, a peptide portion retaining the ring structure of colistin was liberated. In contrast, an analogous antibiotic polymyxin B showed a characteristic resistance to the catalytic activity of papain.

Colistin nonapeptide and α-N-fatty acyl α,γ-diaminobutyric acid were obtained as products from the above enzymatic hydrolyzates of colistin and their chemical and physicochemical properties were investigated.

Contrary to colistin, this colistin nonapeptide was inactive to Escherichia coli. NIHJ and to many other strains even at a concentration of 800 mcg/ml by the agar dilution method. As α-N-fatty acyl α,γ-diaminobutyric acid which is rest part of colistin was added to colistin nonapeptide, antimicrobial activity of colistin nonapeptide did not increase.     

Elaboration of a rapid method of determining the sensitivity of Brucella to antibiotics]

Optimal conditions were developed for determination of antibiotic sensitivity in Brucella by using enzyme immunoassay directly in the primary cultures of the material tested. The Brucella concentration in the material tested should be not lower than 1.10(6) microbial cells/ml and the time of culture incubation be 24 hours at 37 degrees C. The obligatory condition is to use a liquid medium, i.e. the Albimi broth with 1% glucose. To inhibit the foreign microflora it is recommended to use polymyxin B and amphoglucamine in a concentration of 3 microgram/ml. The use of enzyme immunoassay was shown that it was possible to determine the antibiotic sensitivity of Brucella in practice.     

Susceptibility of Recent Isolates of Pseudomonas aeruginosa to Gentamicin, Polymyxin, and Five Penicillins, with Observations on the Pyocin and Immunotypes of the Strains

The susceptibilities of recently isolated strains of Pseudomonas aeruginosa to gentamicin, polymyxin B, carbenicillin, ampicillin, penicillin G, and two newer penicillins were tested with the inocula-replicating technique by using undiluted and 10(-3) dilutions of the cultures. With either inoculum, polymyxin B was the most active agent, and a comparison with previous data from this laboratory showed that the susceptibility of P. aeruginosa to this antibiotic had not changed over the past 20 years. Gentamicin was nearly as active as polymyxin, all but 2 of the 141 strains tested with the diluted inoculum being inhibited by 6.25 mug/ml or less. AB-2288, an agent resembling carbenicillin, was four times more active than carbenicillin or BLP-1654; the last two were equally active against the 10(-3) inoculum. A more marked inoculum effect was noted with the penicillin analogues tested, the increase in minimum inhibiting concentration with the undiluted culture being eight-fold for carbenicillin and at least 16-fold for AB-2288 and BLP-1654. Pyocin typing and serotyping failed to demonstrate any clearly predominating types.     

Mitogen-activated protein kinases-dependent induction of hepatocyte growth factor production in human dermal fibroblasts by the antibiotic polymyxin B

Hepatocyte growth factor (HGF) stimulates migration and proliferation of keratinocytes and has been suggested to be involved in wound healing. The cationic antibiotic polymyxin B (PMB) is commonly used as a topical antibiotic for wound care. If PMB possesses an HGF-inducing activity, the antibiotic is potentially beneficial for wound healing in addition to minimizing chances of infection. In this study, we found that PMB markedly induced HGF production from various types of cells including human dermal fibroblasts. Its effect was stronger than the effects of epidermal growth factor and cholera toxin and was comparable to the effect of 8-bromo-cAMP. Among the polymyxin family and polymyxin derivatives, colistin was also effective, whereas colistin methanesulfonate had only a marginal effect and PMB nonapeptide was ineffective. The stimulatory effect of PMB was accompanied by upregulation of HGF gene expression. Increase in phosphorylation of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) was observed from 0.25h to 6h after the addition of PMB, while increase in phosphorylation of p38 mitogen-activated protein kinase (MAPK) was detected from 24h to 60h after PMB addition. The MAPK/ERK kinase inhibitor PD98059, the JNK inhibitor SP600125 and the p38 MAPK inhibitor SB203580 all potently inhibited PMB-induced HGF production. Lastly, proliferation of human dermal fibroblasts was significantly stimulated by PMB. These results indicate that PMB-induced HGF production and proliferation of human dermal fibroblasts and suggest that activation of MAPKs is involved in the induction of HGF production.     

Survival of Haemophilus equigenitalis in different antibiotic-containing semen extenders.

The ability of Haemophilus equigenitalis, the causal agent of contagious equine metritis 1977, to survive in various antibiotic-containing semen extenders was studied at different environmental temperatures. Gentamicin sulphate was found to be markedly superior to ampicillin or a combination of sodium benzyl penicillin and polymyxin B sulphate, Semen treated with the former antibiotic was either sterile at cultural examination or else yielded appreciably fewer colonies of H. equigenitalis than the untreated semen control. Ampicillin had no observable effect on the survival of this organism. Gentamicin was most effective when semen-extender mixtures were held at room temperature rather than at 37 or 4 degrees C. No detrimental effects on sperm motility were observed following the use of the different antibiotic-containing semen extenders in the presence or absence of H. equigenitalis.     

Stimulation of adhesion molecule expression in human vascular endothelium by Bacteroides fragilis toxins--influence of polymyxin B

The aim of this study was to examine the influence of polymyxin B on the level of expression of adhesion molecules E-selectin, ICAM-1, and VCAM-1 on human vascular endothelium activated with B. fragilis endotoxins or enterotoxin. Lipopolysaccharides were extracted by phenol-water method from one nonenterotoxigenic (NTBF) and three enterotoxigenic (ETBF) B. fragilis strains. LPS preparations were purified with nucleolytic enzymes and ultracentrifugation. Enteotoxin (BFT) was prepared from the supernatant of reference B. fragilis ATCC 43858 culture by precipitation with ammonium sulphate. BFT preparations were purified with the application of ion-exchange chromatography and hydrophobic chromatography. Adhesion molecule expression on the surface of human vascular endothelial cells (HMEC-1 cell line) was determined after simultaneous stimulation with bacterial compounds at the concentration of 10 micrograms/ml and polymyxin B at the concentration of 20 micrograms/ml. Endothelial cells were activated for 4 hours (E-selectin expression) or for 24 hours (ICAM-1 and VCAM-1 expression). Adhesion molecules were detected in immunoenzymatic test (ELISA) with the use of mouse, monoclonal antibodies against human ICAM-1, VCAM-1, and E-selectin. The results of performed experiments suggest, that polymyxin B changes the level of adhesion molecule expression on human vascular endothelium. This antibiotic causes changes in the expression of endothelial ICAM-1, VCAM-1, and E-selectin during simultaneous stimulation of endothelium with B. fragilis endotoxins or enterotoxin. In the majority of cases the addition of polymyxin B leads to the up-regulation of examined adhesion molecules.     

Selective Increase in Hydrogen Peroxide Resistance of a Coagulase-positive Staphylococcus

Staphylococcus culture 105B was serially treated with 0.05% hydrogen peroxide at 54.4 C or without hydrogen peroxide at this temperature to determine changes in resistance to these conditions and in catalase activity of the surviving populations. Resistance of the final surviving populations to H(2)O(2) treatment and to heat treatment without H(2)O(2) was 5.6 and 4.5 times greater, respectively, than the parent culture. Catalase activities of the cell-free extracts of survivors of the H(2)O(2) treatments and of the heat treatments were 33.56 and 2.69 times greater, respectively, than the control. The untreated control cultures grew in Peptonized Milk (Difco), but addition of sodium pyruvate to the medium was necessary to support growth of survivors.     

Increased susceptibility of injured spores of Bacillus subtilis to cationic and other stressing agents

Sublethally heated spores of Bacillus subtilis NCTC 8236 were susceptible to posttreatment concentrations in agar of polymyxin B sulphate, sodium hydroxide, cetylpyridinium chloride and sodium lauryl sulphate that did not prevent colony formation to untreated spores. The non-ionic surfactants polysorbates 20 and 80 were not inhibitory when used at high concentrations against both heated and unheated spores. The method has been developed for detecting sublethal injury in biocide-exposed spores, since iodine-treated spores became highly susceptible to polymyxin contained in recovery agar.     

Use of analogs of primary metabolites in the selection of the producer of polymyxin B]

A number of amino acids were found to have effects on the growth of the polymyxin B-producing culture and biosynthesis of the antibiotic by it. Of special importance was the stimulating effect by methionine. Four selection stages were carried out with using structural analogs of purines and amino acids as selective factors. There were no stable variants with increased antibiotic productivity among the mutants resistant to the analogs of purines and leucine. The levels of polymyxin B accumulation by the variants resistant to 4-fluorophenylalanine were 30 to 50 per cent higher than those in the controls and the variants were characterized by low morphological and antibiotic production variation in the subcultures. The mechanisms of the methionine physiological effect and the prospects of using analogs of the primary metabolites in improvement of the culture producing polymyxin B are discussed.     

Plasmodium berghei: the antimalarial action of artemisinin and sodium artelinate in vivo and in vitro, studied by flow cytometry

Sodium artelinate, a new water-soluble and relatively stable derivative of artemisinin, and its parent compound were tested for their antimalarial action. Experiments were done in vitro with synchronous cultures of Plasmodium berghei. The inhibition of growth by different concentrations of sodium artelinate and artemisinin was determined using flow cytometry. In vivo testing was done by subcutaneous injection of each drug in mice infected with P. berghei. Sodium artelinate, being stable in aqueous solution, was also administered orally to infected mice by its addition to their drinking water. Comparison of the parent compound and the derivative showed that sodium artelinate was slightly less active than artemisinin both in culture and in vivo. However, after oral administration of sodium artelinate, parasites were cleared from the blood with one-half to one-tenth of the dose used in the experiments with subcutaneous injection. The number of mice which were cured by oral administration of sodium artelinate was greater than after subcutaneous injection, even with a total oral dose lower than the injected dose.     

The nature and site of biocide-induced sublethal injury in Bacillus subtilis spores

Spores of Bacillus subtilis NCTC 8236 exposed at 22 degrees C to test biocides (alkaline glutaraldehyde, an iodophor, Lugol's solution, sodium hypochlorite and sodium dichloroisocyanurate) demonstrated varying degrees of injury to stressing agents (sodium hydroxide, sodium lauryl sulphate, polymyxin B sulphate or cetylpyridinium chloride) incorporated into a recovery agar medium. This injury to stressing agents was expressed mainly during outgrowth.     

Increased Antibiotic Sensitivity in Pseudomonas aeruginosa Following Passage in Carbenicillin-containing Media

Twelve dissimilar clinical isolates and 4 type cultures of Pseudomonas aeruginosa have been repeatedly passaged on agar containing 200 μg carbenicillin/ml. Passaged variants were compared with control organisms for their sensitivities to a range of antibiotics initially by a multodisk test and subsequently by serial dilution in agar. Two of the variants, both derived from clinical isolates, showed pronounced increases in sensitivity to several antibiotics, particularly kanamycin, neomycin, gentamicin and colistin sulphate. In some instances the minimum inhibitory concentration (MIC) for the passaged variants was 32–64 times lower than that for the control organisms. These potentiations contrast with previous results obtained by other workers for P. aeruginosa . In addition, several other of our passaged variants developed a more moderate degree of enhanced sensitivity to a limited number of antibiotics. Eight (67%) of the clinical isolates and one type culture did not become more sensitive to any of the antibiotics tested following carbenicillin passage. Onset of increased antibiotic-sensitivity varied with the strain, particular antibiotic and medium employed for passage. Although the addition of sucrose (0·5 M) and magnesium sulphate (0·01 M) to the passage medium appeared to delay development of antibiotic-sensitivity their presence eventually encouraged larger potentiations in antibiotic activity. The significance of the conversion of P. aeruginosa into forms with increased susceptibility to several antibiotics during chemotherapy with carbenicillin is discussed.     

Defensin Susceptibility and Colonization in the Mouse Model of AJ100, a Polymyxin B-Resistant, Brucella abortus RB51 Isolate

Intracellular pathogens selected for increased susceptibility to polycations are commonly attenuated, yet the effect of decreased susceptibility to polycations on pathogenicity has not been researched. The polymyxin-resistant mutant Brucella abortus AJ100 was characterized by comparing its susceptibility to the polycationic antibiotic polymyxin B, defensins, and lactoferricin, and its colonization and clearance in the mouse model to the parent strain RB51. MIC (minimum inhibitory concentration) values determined by Etest for AJ100 and RB51 were 1.5 and 0.25 μg/ml, respectively. Though AJ100 is less susceptible to polymyxin B than RB51, it was more susceptible than its parent strain to the cationic defensins melittin, magainin 2, and cecropin P1. In the mouse model, initial colonization of the spleen was lower for AJ100 than RB51, and the rate of clearance from the spleen was faster for AJ100 than RB51. However, initial colonization and clearance rates of AJ100 from the liver were indistinguishable from those of RB51. This study suggests that the susceptibility profile of Brucella to polycationic defensins rather than polymyxin B may be indicative of differential survival in the spleen and liver in the mouse and is indicative of spleen and liver residential macrophages’ differing ability to inactivate Brucella.     

Resistance of Pseudomonas to Quaternary Ammonium Compounds: II. Cross-Resistance Characteristics of a Mutant of Pseudomonas aeruginosa

Tube dilution experiments showed that benzalkonium chloride (BC)-resistant mutants of Pseudomonas aeruginosa grown in the presence of 1,000 mug of BC per ml were at least 20 times more sensitive to polymyxin B and colistin sulfate than the BC-sensitive (BCS) parent strain. BCS cells selected for resistance to 500 mug of polymyxin B per ml remained sensitive to BC. There was little difference in the amount of carbenicillin, gentamicin sulfate, or rifampin needed to prevent growth of either the BCS or BC-resistant (BCR) strains. Growth of BCR cells was inhibited by ethylenediaminetetraacetate at a concentration of 400 mug/ml or less, whereas the BCS strain grew at ethylenediaminetetraacetate levels of 10,000 mug/ml. Phenylmercuric acetate and thimerosal inhibited growth of BCR and BCS cells at concentrations of 10 mug/ml or less. BCR cells were cross-resistant to >1,000 mug/ml concentrations of five other quaternary ammonium compounds, including three with C(16) alkyls and two with alkyl groups of shorter length. The BCS strain was also resistant to >1,000 mug/ml concentrations of the three quaternary ammonium compounds with C(16) alkyl groups but, in addition to BC, was inhibited by 200 mug/ml levels or less of the two quaternary ammonium compounds containing alkyl groups of less than 16 carbon atoms.     

Polymyxin B biosynthesis and tricarboxylic acid cycle enzymatic activity]

Activity of pyruvate dehydrogenase (PDG), isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase was found in the extracts of the cells of Bac. polymyxa 153, an organism producing polymyxin B. Dependence of the activity of the above enzymes on the carbon source in the medium, aeration conditions, strain features and culture age was shown. A low level of polymyxin B biosynthesis was observed at high activity of PDG and dehydrogenases of the tricarbonic acid cycle. Increased antibiotic production was recorded against the background of decreases values of the above enzyme activities.     

Mode of Synergism between Colistin and Sulfisomezole in Inhibiting the Growth of Proteus Organism

When either colistin at 1,000 μg/ml or sulfisomezole at 125 μg/ml was used separately, growth of a strain of Proteus mirabilis was not inhibited. However, when 1 μg/ml of colistin and 25 μg/ml of sulfisomezole were used together in agar media, growth was inhibited. The synergistic action of colistin and sulfisomezole was also demonstrated in broth culture, when a smaller inoculum such as 10⁶ cells/ml was used. The lethal and lytic effect of this synergism parallels the characteristic effect of colistin towards colistin-sensitive gram-negative organisms. When the mode of this synergistic action was analyzed by adding each compound in sequence to a growing culture of Proteus, it was found that growth of organism for about 4 generations in the presence of sulfisomezole was a prerequisite for revealing the lethal and lytic effects of colistin. In cultures where these two compounds were present at the beginning of incubation, the synergistic effect was abolished by the addition of p-aminobenzoic acid (PABA) at an early stage of incubation, but not at a late stage. Methionine, serine, and betaine, when used together, had the same effect as PABA. An insufficiency of the three compounds induced by sulfisomezole, was considered to afford the receptor site of colistin to Proteus.     

Polymyxins as Novel and Safe Mucosal Adjuvants to Induce Humoral Immune Responses in Mice

There is currently an urgent need to develop safe and effective adjuvants for enhancing vaccine-induced antigen-specific immune responses. We demonstrate here that intranasal immunization with clinically used polypeptide antibiotics, polymyxin B (PMB) and colistin (CL), along with ovalbumin (OVA), increases OVA-specific humoral immune responses in a dose-dependently manner at both mucosal and systemic compartments. Enhanced immunity by boosting was found to persist during 8 months of observation. Moreover, mice intranasally immunized with OVA plus various doses of PMB or CL showed neither inflammatory responses in the nasal cavity and olfactory bulbs nor renal damages, compared to those given OVA alone. These data suggest that polymyxins may serve as novel and safe mucosal adjuvants to induce humoral immune responses. The polymyxin adjuvanticity was found to be independent of endotoxins liberated by its bactericidal activity, as indicated by similar enhancing effects of PMB in lipopolysaccharide (LPS)-hyporesponsive and LPS-susceptible mice. However, despite the presence of preexisting anti-PMB antibodies, we observed no reduction in the adjuvant function of polymyxins when they were given intranasally. Furthermore, the titers of OVA-specific Abs in mice intranasally immunized with OVA plus PMB or CL were significantly higher than those in mice administered with polymyxin analogues, such as polymyxin B nonapeptide and colistin methanesulfonate. The levels of released β-hexosaminidase and histamine in mast cell culture supernatants stimulated by PMB or CL were also significantly higher than those stimulated by their analogues. These results suggest that both the hydrophobic carbon chain and hydrophilic cationic cyclic peptide contribute to the mucosal adjuvanticity of PMB and CL.