In vitro and in vivo antibacterial activities of the fluoroquinolone WIN 49375 (amifloxacin).

WIN 49375 (amifloxacin) is a synthetic antibacterial agent of the quinolone class. It is similar in chemical structure to pefloxacin but differs by containing a methylamino, rather than an ethyl, substituent at the 1-N position. The activity of WIN 49375 in vitro was comparable to those of norfloxacin and pefloxacin against Enterobacteriaceae and generally greater than those of tobramycin and cefotaxime. WIN 49375 was more active in vitro than carbenicillin and mezlocillin against Pseudomonas aeruginosa isolates and showed moderate activity against Staphylococcus aureus, with MICs of less than or equal to 2 micrograms/ml. The in vitro activity of WIN 49375 was not markedly affected by the presence of human serum, the size of the bacterial inoculum, or changes in pH between 6 and 8. Against systemic, gram-negative bacterial infections in mice, WIN 49375 was generally less active than cefotaxime but more active than gentamicin. WIN 49548, the major piperazinyl-N-desmethyl metabolite of WIN 49375, was aa effective as the parent drug against experimental infections in mice when given parenterally. When administered orally, however, this metabolite was less potent than WIN 49375. WIN 49375 was highly active by the oral route, with 50% effective doses within two- to threefold of those obtained with parenteral medication.

Novel amino-substituted 3-quinolinecarboxylic acid antibacterial agents: synthesis and structure-activity relationships.

A series of novel 3-quinolinecarboxylic acid derivatives have been prepared and their antibacterial activity evaluated. These derivatives are characterized by fluorine attached to the 6-position and substituted amino groups appended to the 1- and 7-positions. Structure-activity relationship studies indicate that antibacterial potency is greatest when the 1-substituent is methylamino and the 7-substituent is either 4-methyl-1-piperazinyl, 16, or 1-piperazinyl, 21. Derivatives 16 and 21, the 1-methylamino analogues of pefloxacin and norfloxacin, respectively, show comparable in vitro and in vivo antibacterial potency to these two known agents. The activity (vs. Escherichia coli Vogel) of 16 (amifloxacin) is the following: in vitro MIC (microgram/mL) = 0.25; in vivo (mice) PD50 (mg/kg) = 1.0 (po), 0.6 (sc).

Lipids and lipoteichoic acid of autolysis-defective Streptococcus faecium strains.

Two of four previously isolated autolysis-defective mutants of Streptococcus faecium (Streptococcus faecalis ATCC 9790) incorporated substantially more [14C]glycerol into lipids and lipoteichoic acid than did the parent strain. Consistent with increased accumulation of lipids and lipoteichoic acid, significantly higher levels of phosphorus were found in the corresponding fractions of the two mutant strains than in the wild type. Although the autolysis-defective mutant strains contained the same assortment of lipids as the wild type, the relative amount of [14C]glycerol incorporated into diphosphatidylglycerol increased, accompanied by a decreased fraction of phosphatidylglycerol. These results suggested that increased cellular content of two types of substances, acylated lipoteichoic acid and lipids (notably diphosphatidylglycerol), which previously had been shown to be potent inhibitors of the N-acetylmuramoylhydrolase of this species, contributed to the autolysis-defective phenotype of these mutants. Consistent with this interpretation are observations that (i) cerulenin inhibition of fatty acid synthesis increased the rates of benzylpenicillin-induced cellular lysis and that (ii) Triton X-100 or Zwittergent 3-14 treatment could reveal the presence of otherwise cryptic but substantial levels of the active form of the autolysin in cells of three of four mutants and of the proteinase-activable latent form in all four mutants.

Does penicillin kill bacteria?.

The thesis is presented that the bactericidal action of penicillin and of other inhibitors of cell wall peptidoglycan synthesis, such as vancomycin and cycloserine, is secondary or tertiary to their ability inhibit specific reactions in the assembly of an osmotically protective cell wall. Examples are given of the inhibition of these reactions, which results in inhibition of cell growth (bacteriostatic action) in the absence of either cellular lysis or rapid loss of viability. Thus, in some instances, inhibitory concentrations of these drugs are, in effect, sublethal; this is true, for example, for Streptococcus mutans, a species of bacteria that is part of the normal flora of the oropharynx and that can cause subacute bacterial endocarditis. On the other hand, the damaging effects of the subminimal inhibitory concentrations of penicillin G on Streptococcus faecalis, a species with an active autolytic enzyme system, can be uncovered and converted to a lytic (and lethal) response by partial inhibition of fatty acid synthesis with low concentrations of cerulenin. Some theoretical and practical implications of the occurrence and inhibition of these secondary lethal consequences are discussed.

Release of autolytic enzyme from Streptococcus, faecium cell walls by treatment with dilute alkali.

The autolytic enzyme (endo-beta-1,4-N-acetylmuramoylhydrolase) of Streptococcus faecium (S. faecalis ATCC 9790) was released in a soluble form from insoluble cell wall-autolytic enzyme complexes by treatment with dilute NaOH at 0 degree C. Treatment of cell wall-enzyme complexes, obtained from either exponential- or stationary-phase cells, with 0.008 to 0.01 N NaOH gave maximum yields of autolytic enzyme activity. At a fixed concentration of NaOH, the yield of autolysin increased with increasing wall densities and was accompanied by the release of methylpentose and phosphorus in amounts proportional to the autolysin. Since extraction of wall-enzyme complexes with 4.5 M LiCl at 0 degree C also removed methylpentose and phosphorus, release of enzyme with NaOH did not appear to result from hydrolysis of covalent linkages. The autolytic enzyme activity released from intact cells, or cell walls, was predominantly in the later (proteinase activable) form which could be activated by trypsin or a proteinase present in commerical bovine plasma albumin.

Morphological and physiological study of autolytic-defective Streptococcus faecium strains.

Three autolytic-defective mutants of Streptococcus faecium (S. faecalis ATCC 9790) were isolated. All three autolytic-defective mutants exhibited the following properties relative to the parental strain: (i) slower growth rates, especially in chemically defined medium; (ii) decreased rates of cellular autolysis and increased survival after exposure to antibiotics which block cell wall biosynthesis; (iii) decreased rates of cellular autolysis when treated with detergents, suspended in autolysis buffers, or grown in medium lacking essential cell wall precursors; (iv) a reduction in the total level of cellular autolytic enzyme (active plus latent forms of the enzyme); (v) an increased ratio of latent to active forms of autolysin; and (vi) increased levels of both cellular lipoteichoic acid and lipids.

Cellular lysis of Streptococcus faecalis induced with triton X-100.

Lysis of exponential-phase cultures of Streptococcus faecalis ATCC 9790 was induced by exposure to both anionic (sodium dodecyl sulfate) and nonionic (Triton X-100) surfactants. Lysis in response to sodium dodecyl sulfate was effective only over a limited range of concentrations, whereas Triton X-100-induced lysis occurred over a broad range of surfactant concentrations. The data presented indicate that the bacteriolytic response of growing cells to Triton X-100: (i) was related to the ratio of surfactant to cells and not the surfactant concentration per se; (ii) required the expression of the cellular autolytic enzyme system; and (iii) was most likely due to an effect of the surfactant on components of the autolytic system that are associated with the cytoplasmic membrane. The possibility that Triton X-100 may induce cellular lysis by releasing a lipid inhibitor of the cellular autolytic enzyme is discussed.

Autolytic defective mutant of Streptococcus faecalis.

Properties of a variant of Streptococcus faecalis ATCC 9790 with defective cellular autolysis are described. The mutant strain was selected as a survivor from a mutagenized cell population simultaneously challenged with two antibiotics which inhibit cell wall biosynthesis, penicillin G and cycloserine. Compared to the parental strain, the mutant strain exhibited: (i) a thermosensitive pattern of cellular autolysis; (ii) an autolytic enzyme activity that had only a slightly increased thermolability when tested in solution in the absence of wall substrate; and (iii) an isolated autolysin that had hydrolytic activity on isolated S. faecalis wall substrate indistinguishable from that of the parental strain, but that was inactive when tested on walls of Micrococcus lysodeikticus as a substrate. These data indicate an alteration in the substrate specificity of the autolytic enzyme of the mutant which appears to result from the synthesis of an altered form of autolytic enzyme.

Bacteriophage SPO1-induced macromolecular synthesis in minicells of Bacillus subtilis.

SPO1 bacteriophage injects its DNA into minicells produced by Bacillus subtilis CU403 divIVB1. The injected DNA is partially degraded to small trichloracetic acid-precipitable material and trichloroacetic acid-soluble material. The injected DNA is not replicated; however, it serves as a template for RNA and protein synthesis. The RNA produced specifically hybridizes to SPO1 DNA, and the amount of RNA hybridized can be reduced by competition with RNA isolated at all stages of the phage cycle from infected nucleate cells of the B. subtilis CU403 divIVB1. An unrelated phage, SPP1, also induces phage-specific RNA in infected minicells. Translation occurs in SPO1-infected minicells resulting in at least eight proteins which have been separated by gel electrophoresis, and two of these proteins have mobilities similar to proteins found only in infected B. subtilis CU403 divIVB1 nucleate cells. A large proportion of the polypeptide material synthesized in infected minicells is very small and heterogeneous in size.

Spackle and immunity functions of bacteriophage T4.

Cells of Escherichia coli B infected with the immunity-negative (imm2) mutant of bacteriophage T4 are able to develop a substantial level of immunity to superinfecting phage ghosts if the ghost challenge is made late in infection. This background immunity is not seen in infections with phage carrying the spackle (s) mutation in addition to the imm2 lesion. The level of immunity in s(-) infections is intermediate between that of imm(-) and wild-type infections under standard assay conditions. With respect to genetic exclusion of superinfecting phage, cells infected with imm(-) phage are completely deficient, whereas infections with the s(-) phage are only partially deficient compared to wild-type infections. Whereas s(-)-infected cells are unable to resist lysis from without by a high multiplicity of infection (MOI) of superinfecting phage, cells infected with imm(-) phage show less than wild-type levels of resistance and the majority of cells remaining intact are unable to incorporate leucine or form infective centers. Under conditions of superinfection by low MOI of homologous phage, imm(-)-infected cells are lysis inhibited, whereas s(-)-infected cells do not show this property. Superinfecting phage inject their DNA into imm(-)-infected cells with the same efficiency as seen in wild-type infections, but this efficiency is reduced when the cells are first infected with s(-) phage. The s function of T4 appears not only to affect the host cell wall as previously postulated by Emrich, but may also affect the junctures of cell wall and membrane with consequences similar to those of the imm function.