A new β-lactam antibiotic, named thienamycin, was discovered in culture broths of Streptomyces MA4297. The producing organism, subsequently determined to be a hitherto unrecognized species, is designated Streptomyces cattleya (NRRL 8057). The antibiotic was isolated by adsorption on Dowex 50, passage through Dowex 1, further chromatography on Dowex 50 and Bio-Gel P2, and final purification and desalting on XAD-2. Thienamycin is zwitterionic, has the elemental composition C11H16N2O4S (M. W.=272.18) and possesses a distinctive UV absorption (λmax=297 nm, ε=7, 900). Its β-lactam is unusually sensitive to hydrolysis above pH 8 and to reaction with nucleophiles such as hydroxylamine, cysteine and, to a lesser degree, the primary amine of the antibiotic itself. The latter reaction results in accelerated inactivation at high antibiotic concentrations.
A new antifungal antibiotic, named neo-enactin, was isolated from the cultured mycelia of Strain H 829-MY 10, identified as a new subspecies of Streptoverticillium and named Streptoverticillium olivoreticuli subsp. neoenacticus. The antibiotic was produced with a tetraene antifungal antibiotic, found mainly in the cultured mycelia of Strain H 829-MY 10 and it was extracted with methanol. The antibiotic is of a basic nature and it can be extracted with ethyl acetate at alkaline pH. Purification of neo-enactin was carried out by partition chromatography on cellulose and elution with ethyl acetate bufferized with phosphate buffer (pH 8.0). Neo-enactin shows strong antifungal activity and potentiates the antifungal activity of polyene antifungal antibiotics.
By using 2 neamine-negative mutants, MCRL 5003 and MCRL 5004, of Bacillus circulars, subunit assembly in the biosynthesis of butirosins was investigated. The two mutants were blocked at different steps in the biosynthetic pathway of butirosins, but could produce butirosins when the culture medium was supplemented with neamine. Mutant MCRL 5003 accumulated 2-deoxystreptamine (DOS) in the fermentation broth but could not utilize DOS for the biosynthesis of butirosins, whereas mutant MCRL 5004 could produce butirosins from DOS. Based upon the ability of these 2 mutants to incorporate a series of DOS-containing compounds considered as plausible biosynthetic intermediates of butirosins into butirosins or related antibiotics, the biosynthetic pathways for butirosins and 6'-deamino-6'-hydroxybutirosins were proposed. Feeding experiments with 3', 4'-dideoxy derivatives of neamine, ribostamycin and butirosins supported the pathway proposed for butirosins. A glucosamine auxotroph MCRL 5771 of B. circulars afforded some information as to the role of D-glucosamine in the biosynthesis of butirosins.
Piperacillin, a new semisynthetic penicillin, was found to have potent antimicrobial activity against nearly all (405) tested bacterial species. Piperacillin was consistently 4-16-fold more active than carbenicillin against the Enterobacteriaceae, 16-32-fold against Pseudomonas acruginosa and Pseudomonas species, and 16-fold against Streptococcus faecalis. Carbenicillin and piperacillin were equally effective against Staphylococcus aureus, but inactivated by beta-lactamase. A 38% overall synergy rate for the piperacillin-gentamicin combination was identified, a finding similar to that for carbenicillin-gentamicin. Highest incidences of synergy were found for both antibiotic pairs tested on gentamicin-resistant isolates (Ps. aeruginosa and Providencia). In vitro findings suggest that piperacillin alone or in combination with aminoglycosides may be highly efficacious in the treatment of most serious bacterial infections.
Pepleomycin (PEP), 3-[(S)-1'-phenylethylamino]propylaminobleomycin has potent activity and is less pulmonary toxic than bleomycin (BLM). Biological activity and toxicity of the following degradation products of PEP have been studied in detail: the product of carbamoyl migration (ISO), the product of decarbamylation (DC), the product of ring closure of the side chain on the pyrimidine moiety (RC), the depyruvamide product (DP) and the product of an enzymatic inactivation (DA). These degradation products showed much lower activity than PEP in vitro: antimicrobial and anti-HeLa activities, inhibition of DNA synthesis in AH66 cells and the DNA strand cleavage. Acute toxicity and pulmonary toxicity were tested in mice. Results indicated much lower acute toxicity corresponding to the decreased in vitro activity when compared to PEP. DP and RC did not cause lung fibrosis in mice, while ISO and DC showed 1/2.6 and 1/5.7 degree of pulmonary toxicity, respectively, in comparison with PEP.
The activity of purified prolyl hydroxylase (proline, 2-oxoglutarate dioxygenase, EC 1. 14. 11. 2) was enhanced about 3-fold by addition of bleomycin in the assay mixture. Various members of the bleomycin family, their derivatives and degradation products were investigated for activities against prolyl hydroxylase together with their activities of DNA chain breakage to determine relationships between the structure of bleomycin and its various actions. All the bleomycins with various terminal amine parts and desamide bleomycin stimulated the enzymatic activity but did not exhibit an effect on DNA chain breakage. The stimulatory activity of bleomycin was not decreased by hydrolysis with 0.3 N H2SO4 at 80°C for 6 hours, conditions which liberates the sugar moiety, but was eliminated by hydrolysis with 6 N HCl at 105°C for 24 hours. In contrast both treatments decreased the DNA chain breakage activity of bleomycin. Optical spectral studies revealed that all the bleomycins and their hydrolysates which stimulated the prolyl hydroxylase activity made complexes with ferrous ion, one of the cofactors of this enzyme.
Aclacinomycin A was observed to effect the thermal denaturation of DNA and to increase Tm. The visible absorption spectrum of the antibiotic showed bathochromic and hypochromic shifts upon reaction with native and heat-denatured DNA. [14C]Aclacinomycin A was demonstrated by equilibrium dialysis to bind to DNA. Native calf thymus DNA appeared to possess one binding site per ca. 6 nucleotides for the antibiotic with an apparent association constant of ca. 1.2×106 M-1. Heat-denatured DNA showed much less affinity for the antibiotic: one binding site per ca. 6 nucleotides with an apparent binding constant of ca. 3.5×104 M-1. The difference of association constants between double- and single-stranded DNAs suggested that the antibiotic may be intercalated between base pairs of the DNA double helix. [14C]Aclacinomycin A exhibited higher affinity for poly(dAdT) than for poly(dIdC). The antibiotic showed a significant difference spectrum with porcine tubulin, indicating an interaction with tubulin. The binding to tubulin was also demonstrated by equilibrium dialysis of [14C]aclacinomycin A and tubulin.
During the sudden decrease in RNA synthesis in Streptomyces aureofaciens, i.e. around the 6th hour of cultivation, synthesis of adenosine and guanosine tetraphosphates and pentaphosphates begins. The synthesis of these nucleotides is highest during the onset of chlortetracycline production, around the 20th hour of cultivation and continues. During this phase of growth of S. aureofaciens, RNA and protein synthesis are reduced by about one order of magnitude as compared to the rate which can be observed at the beginning of cultivation, but the synthesis is not inhibited by exogenous CTC.
Two penicillins and 5 cephalosporins were evaluated for their ability to pass through the outer-membranes of Proteus morganii, Citrobacter freundii and Escherichia coli. Cefazolin, ceftezole and cephaloridine showed high permeability through the outer-membranes of these Gram-negative bacteria. Benzylpenicillin and cephalothin, on the contrary, showed low permeability. The outer-membrane permeability of ampicillin and cephalexin varied from species to species. C. freundii was found to have the highest barrier against both the penicillins and the cephalosporins, and E. coil appeared to have a low barrier against the cephalosporins. The hydrophobic character of the β-lactam antibiotics, which was estimated by a reversed-phase thin-layer chromatography was closely related to the outer-membrane permeability. In general, the more hydrophilic antibiotic showed the higher outer-membrane permeability. However, cephaloridine, the most lipophilic compound among the antibiotics tested, showed good permeability.
Treatment of outer membranes of Serratia marcesceus with polymyxin B results in the formation of blebs. This effect is thought to be due to the action of the antibiotic on the lipopolysaccharides, proteins, phospholipids or a combination thereof. It is unclear whether this effect is dissociative, degradative or due to an inhibition of the assembly of outer membrane components. Prior studies showed that lipopolysaccharides and polymyxin B form complexes, but direct visualization of the in situ action of polymyxin B had not been accomplished. Isolated outer membranes normally exhibit a periodicity of the polysaccharide molecules when stained by the thiosemicarbazide-silver technique. Polymyxin B treated outer membranes display a change in their basic morphology. This effect is very drastic in the sensitive strain as demonstrated by the large gaps in the deposition of the granules in the modified outer membrane structure. Thus it appears that the polysaccharide molecules (probably the lipopolysaccharide) either alone or in association with protein or phospholipids are the primary targets of the antibiotic.
The lysis of Staphylococcus aureus FDA 209P induced by benzylpenicillin was completely inhibited by cellular lipoteichoic acid isolated from an homologous organism. The cells prevented from penicillin-induced lysis were in static state and did not lose viability. The lipoteichoic acid inhibited either extracellular autolysin activity in culture supernatant or autolysis of whole cells in exponential phase of S. aureus. These results indicate that the prevention of penicillin-induced lysis of S. aureus by the lipoteichoic acid was brought about by the inhibition of autolytic activity of the organism.