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Inhibition of cell-wall-muco-peptide formation in Escherichia coli by benzylpenicillin and 6-[D(-)-alpha-aminophenylacetamido]penicillanic acid (ampicillin)
... phase as an index of activity, reported that the side-chain structure markedly influenced activity. They found, in agreement with other workers (Rolinson & Stevens, 1961 ; Rogers & Mandelstam, 1962), that D-cc-aminobenzylpenicih (D-ampicillin) was considerably more active than benzylpenicillin (penicillin G). The concentration which produced lysis in one generation (' LIOG ' value) was about one-tenth as great for D-ampicillin as for benzylpenicillin. ...
... 911 the penicillin derivatives tested had some toxicity for the Erwinia strain used (Table 1). In accordance with the results of others (Rolinson & Stevens, 1961; Rogers & Mandelstam, 1962; Boman & Eriksson, 1963) we observed that the aminobenzylpenicillins were more toxic than benzylpenicillin. The difference between DL-a-aminobenzylpenicillin ( D : L = 6 : 4) and the pure D-isomer were not such as to indicate a 3-fold higher activity of the pure D-compound, as reported by Boman & Eriksson (1963). ...
... The possibility that side-chain structure may affect the intrinsic activity of the molecule must be recognized. The greater toxicity of the aminobenzylpenicillins than benzylpenicillin, originally observed with some Gram-negative bacteria (Rolinson & Stevens, 1961), may be an example; this higher activity also applies to specific enzyme systems (Rogers & Mandelstam, 1962). In our case, the relatively poor annulment by pantoyl lactone of the inhibitions by the aminobenzylpenicillins could be interpreted as indicating increased intrinsic activity toward the Erwinia resulting from the presence of a primary a-amino group in the side chain. ...
By using growth and division-inhibition at 16 hr as indices of activity, eight penicillins, differing in the side chain, were tested against an Erwinia species, and compared with benzylpenicillin. DL-α-Aminobenzylpenicillin, DL-3-chloro-α-aminobenzylpenicillin, D-α-aminobenzylpenicillin, and methicillin were more toxic for growth than was benzylpenicillin. 6-Aminopenicillanic acid and cloxacillin were about equal to benzylpenicillin in toxicity; triphenylmethylpenicillin and 2-ethoxy-1-naphthylpenicillin were much less toxic. All the compounds inhibited division of the bacteria but, at 50% of normal growth, methicillin, cloxacillin and 2-ethoxy-1-naphthylpenicillin resulted in significantly longer organisms than the others. Pantoyl lactone, when present in the medium from the time of inoculation, in all cases decreased the length of the organisms, increased growth (with a highly toxic concentration of penicillin), and decreased the accumulation of keto acids and ultraviolet-absorbing materials in the medium. It was concluded that the side chain of these penicillins is not essential for the inhibition of growth and division of this Erwinia sp.; quantitative differences in activity associated with the side chain appear to result from the influence of the side chain on factors such as penetration, or strength of binding at the active site.
... However, the synthetic process could still be inhibited fully by methicillin, and this result matched the observation that methicillin also inhibited the growth of such benzylpenicillin-resistant strains (22). Rogers and Mandelstam (132) found that ampicillin 6-(D-a-aminophenylacetamido)-penicillanic acid, known to be more antibiotically active than benzylpenicillin against some gram-negative strains, also inhibited the synthesis of mucopeptide in E. coli at a lower concentration than did benzylpenicillin. Thus, in general, the parallel between anti-biotic activity and inhibition of mucopeptide synthesis is well established. ...
... Available information suggests that the relationship between the Park nucleotide and the wall mucopeptide is different in E. coli. Although mucopeptide synthesis in this organism is inhibited by penicillin (105,132), no simultaneous accumulation of uridine nucleotides of the Park type has so far been demonstrated. However, Comb, Chin, and Roseman (37) isolated a series of nucleotides of the composition shown in Table 2 from normal cells of E. coli K235. ...
... is likely to be killed during the viable count determination, which makes the death rate seem faster than it really is. Secondly, penicillin is known to give rise to large elongated organisms (see review by Rogers, 1962) and, if such organisms started to divide, the number of viable units might increase more rapidly than in normal growth. ...
... The penicillin dependence of the lysis ratio is also a useful method for the characterization of penicillin-resistant mutants. The most commonly accepted explanation for penicillin action (see reviews by Strominger, 1960; Rogers, 1962) is that the compound blocks cell-wall synthesis by inhibiting an enzyme which joins the Park & Strominger mucopeptide with the acceptor of the wall structure (Park & Strominger, 1957). Protein synthesis and presumably a large part of the cell metabolism is, however, unaffected by penicillin (Mandelstam & Rogers, 1959; Hancock & Park, 1958) and as the result of continued increase in cell mass the osmotic pressure is increased until lysis occurs. ...
SUMMARY The paper is a survey of the action of a-aminobenzylpenicillin (ampicillin) on Escherichia coli. The rate at which lysis was induced in exponentially growing organisms was studied for different concentrations of ampicillin, using pure D- and L-forms as well as a 6 : 4 mixture. The interpolated con- centration of penicillin which gives lysis in one generation, the' LIOG value ', has been used for characterization of penicillin derivatives with twelve different side chains. These LIOG values were also used for characterization of some penicillin-resistant mutants. The lysis rate has been recorded for cultures of concentrations between 4 x 105 and lo8 rods/ml. and found to be independent of the population density. The use of five different media showed that for a given ampicillin concentration the time to lysis was proportional to the growth rate. The addition of penicillinase to a culture growing with penicillin rescued the organisms as late as a few minutes before lysis. Synergistic effects on the lysis rates were found with 6-amino- penicillanic acid and two amino acids, which as residues are side chains, in two of the penicillins tested. Different models for penicillin action in E. coli are discussed.
... The aminopenicillin ampicillin (available since 1961) and the third-generation cephalosporin ceftiofur (available since 1988) are two of the most used b-lactam antibiotics on Washington State dairies (8). Penicillin is also widely used, but it can have 10-fold lower activity against E. coli compared with ampicillin (26) and therefore was not included in this study. Ampicillin is an injectable antibiotic that is used to treat metritis and pneumonia in cattle, and it is mostly excreted unchanged. ...
We studied the relative fitness benefits of a cephalosporin resistance enzyme (CTX-M-15) that is displacing a similar enzyme (CMY-2), which is extant in E. coli from dairy cattle in Washington State. In vitro experiments demonstrated that CTX-M-15 provides a significant fitness advantage, but only in the presence of very high concentrations of antibiotic that are only found when the antibiotic ampicillin, and to a lesser extent ceftiofur, is excreted in urine from treated animals. As such, the increasing prevalence of bacteria with bla CTX-M-15 is likely occurring ex vivo .
... Cell septation can be inhibited without gross inhibition of cell extension, not only by interfering with DNA replication but also by partially inhibiting mucopeptide synthesis. Penicillins inhibit mucopeptide synthesis in gram-positive and gram-negative bacteria (79,89,113). At concentrations causing about 20 to 70% inhibition of synthesis, penicillin added to growing cultures of E. coli causes the cells to extend in length and to bulge. ...
... Cell septation can be inhibited without gross inhibition of cell extension, not only by interfering with DNA replication but also by partially inhibiting mucopeptide synthesis. Penicillins inhibit mucopeptide synthesis in gram-positive and gram-negative bacteria (79,89,113 anionic substance suramin or certain other substances were added to cultures, the length of the chains of cocci was greatly increased. These chains could be broken up by the addition of either supernatant fluid from young cultures which contained cell lysin or of small amounts of lysozyme; suramin was shown to inhibit the action of lysozyme. ...
... All the findings previously obtained with Bacillus subtilis (Hahn et al. 1954;Leonard & Housewright, 1963) and those given here for B. anthracis show that tetracycline and/or chloramphenicol permits polymerization of glutamic acid although inhibiting growth. Polymerization, unlike protein synthesis, evidently does not depend on the assembly of activated amino acids on ribosomes and, to this extent, it resembles the synthesis of other peptides like the antibiotics gramicidin (Winnick & Winnick, 1961) and tyrocidine (Mach, Reich & Tatum, 1963;Mach & Tatum, 1964), and those which occur in cell walls (Rogers & Mandelstam, 1962). Another sign that the amino acid sequence of these peptides is not specified like that of protein is that the composition of tyrocidin is partly determined by the constituents of the culture medium (Mach & Tatum, 1964). ...
... Perhaps a difference in virulence might be observed if animals with intermediate levels of antibody were tested. It is well established that penicillin interferes with the synthesis of the mucopeptide portion of the cell wall (Park & Strominger, 1957; Rogers & Mandelstam, 1962; Izaki, Matsuhashi & Strominger, 1966). We have derived serum-sensitive mutants by selection with cephalosoporin and bacitracin, as well as with penicillin. ...
Serum-sensitive mutants have been derived from serum-resistant smooth virulent Salmonella typhimurium and S. enteritidis strains by selection for resistance to cephalosporin or penicillin. Chemical analyses of the lipopolysaccharides of these mutants reveal that they belong to at least three different rough or semi-rough classes. Partial or total loss from the lipopolysaccharide of the sugars responsible for O antigenicity resulted in loss of virulence, as well as increased sensitivity to the bactericidal effect of antibody plus complement. However, such loss is not necessary for serum sensitivity because two serum-sensitive mutants possessed lipopolysaccharides indistinguishable from the smooth serum-resistant parents and were nearly as virulent.
Under chaotropic conditions, DNA released from lysed cells causes the aggregation of paramagnetic beads in a rotating magnetic field in a manner that is independent of the presence of other cellular components. The extent of aggregation correlates with the mass of DNA in a quantitative manner (JACS, 134:5689-96) and from this, the number of DNA-containing cells in the sample is enumerated. Microbial growth testing is demonstrated by monitoring bead aggregation with E. coli in the presence of ampicillin. Without the need for fluorescent labeling or Coulter Counting, the white blood cell count can be defined directly from a microliter of crude whole blood. Specificity is brought to the process by coupling bead-based immunocapture with DNA-bead aggregation allowing for the enumeration of CD4+ T-cells from human blood samples. The results DNA-induced bead aggregation had a 95% correlation with those generated by flow cytometry. With the process requiring only inexpensive, widely-available benchtop laboratory hardware, a digital camera and a simple algorithm, this provided a highly-accessible alternative to more expensive cell counting techniques.
The chapter describes the approaches that have been made to understand the control of bacterial surface area during the cell cycle. Such investigations have shown that the apparently casual timing of division is a consequence of variation in cell size at division. Other theoretical investigations suggest the ways in which the average cell length can be determined by the kinetics of surface growth. Hypotheses make predictions of the time of insertion of surface elongation sites and of their mode of operation during the cell cycle. These can be tested by analysis of the variation in cell size under different conditions and by topographic studies of sites of surface growth. The control of surface extension can be analyzed by studying the variation in cell size and shape under different conditions of steadystate growth, and to some extent by the effect of specific inhibitors of protein, DNA, and peptidoglycan synthesis. The large variety of mutants known to have abnormal morphology must all be considered relevant to the study of surface extension as each class of mutant reveals some aspect of the biological influences determining morphology. Thus, the proportion of septa1 to peripheral wall can be changed in some mutants, whilst another mutant has been described in which a constraint on length extension leads to increased width. There are also mutants in which length extension and septum formation are no longer coordinated with chromosome termination.
The chapter discusses the biogenesis of the wall in bacterial morphogenesis. The characteristic shapes of bacterial cells can only be maintained by a contribution from all of the polymers in the walls, rather than by any one of them. Among the simplest and most easily defined changes in the shape of microorganisms is one from rods to spherical-shaped bacteria or from spherical protoplasts to rod-shaped bacilli. Several mutations and physiological conditions can bring about this change in different species of bacteria. Wall composition, biosynthetic mechanisms, and the physical properties of walls all refer to large populations of whole bacteria or broken preparations made from them. Changes in surface location of teichoic acids, or even more of the D-alanine substituent on it, within the wall of growing organisms could possibly have profound effects upon the morphology of the wall and upon cell shape. A complication of work with morphological mutants is the plethora of molecular and physiological changes that arise from single mutations. This difficulty can be overcome by finding two or more different sorts of mutations that have some of the biochemical changes in common, but which result in the same morphology.
Résumé Divers aspects de l'action de la cloxacilline et de la nafcilline surEscherichia coli ont été étudiés. La cloxacilline s'est montrée plus efficace que la nafcilline quand il s'agissait d'arrêter la croissance et d'amener la lyse et la formation de sphéroplastes; par contre, elle s'est montrée moins efficace que plusieurs autres pénicillines (y compris l'acide 6-aminopénicillanique) à ces égards. Ces résultats tendent à démontrer que la cloxacilline et la nafcilline possèdent un mode d'action qui est typique des pénicillines sur cet organisme.
The gateway to the preparation of a virtually limitless list of “semisynthetic” penicillins was not fully opened until isolation of the penicillin “nucleus” 6-aminopenicillanic acid (6-APA). As this fermentation-produced chemical became available in quantity, efforts were directed toward producing structurally modified penicillins having (a) a greater degree of intrinsic activity and a wider spectrum than that possessed by penicillin G, along with acid stability and oral absorbability comparable to that of penicillin V; (b) a low degree of deleterious binding to serum proteins; (c) reduced allergenicity; and (d) resistance to microbial p-lactamases. This chapter considers the extent of success achieved in each of these areas. The chapter also presents, in an effort to better emphasize the significance of some of the improvements attained, a brief review of the history of penicillin over the more than 30-year period from its discovery by Alexander Fleming up to the point where the production of new semisynthetic penicillins has become a routine procedure.
The development of resistance to ampicillin (aminophenylacetamidopenicillanic acid) and penicillin G was investigated by selecting resistant variants of strains of Escherichia coli. Resistance occurred in stepwise manner. The parent strains contained minute amounts of penicillinase and production of the enzyme by resistant variants increased in relation to increase in resistance. Evidence was obtained that production of penicillinase was responsible for this increase in resistance. By using resistant variants and naturally occurring ampicillin-resistant organisms, it was found that production of only small amounts of penicillinase was sufficient to confer a high degree of resistance to the Gram-negative bacteria examined. In contrast, Gram-positive bacteria required much greater quantities of penicillinase for high grade resistance. It is suggested that an important reason for this difference is the complex lipid-containing cell wall of Gram-negative bacteria, which slows penetration by penicillin and enables small amounts of penicillinase to protect the cell. The penicillinase of all the Gram-negative organisms investigated was found to be a β-lactamase and to be more active against penicillin G than against ampicillin. This provides one explanation for the greater activity of ampicillin against Gram-negative bacteria.
This chapter describes correlations that exist between the chemistry of microstructures or organelles and the chemistry of biocides, which act selectively on, or in a particular microstructure. It is noted that such comparisons can provide a more direct approach to the discovery of useful new biocides. In specific, the chapter discusses bacteria, fungi, viruses, and protozoa. In context to microstructures of bacteria, bacteria possess many structural peculiarities that differentiate it from higher plants and animals. In context to biocides, many substances are known which block the synthesis of new cell wall. This inhibition does not harm the bacterium until it begins to grow, where upon the high osmotic pressure ruptures the unprotected cytoplasmic membrane. The mode of action of the antibiotic oxamycin is known in considerable detail. Oxamycin is a structural analog of D-alanine and can displace this amino acid from two enzymes in true competition.
1. The biosynthesis of teichoic acid in cell suspensions of two strains of Staphylococcus aureus is partially inhibited by the same low concentrations of penicillin that inhibit mucopeptide synthesis by 90-100%. Further increase in the concentration of the antibiotic by several hundred-fold still fails to cause any greater inhibition of teichoic acid synthesis. 2. Other conditions, such as amino acid deficiency or the presence of cycloserine or 5-fluorouracil, that inhibit mucopeptide synthesis also inhibit teichoic acid formation. 3. The degree of inhibition of teichoic acid synthesis caused by relatively high concentrations (10mug./ml.) of benzylpenicillin depends critically on the age of the culture from which the cell suspensions have been prepared. 4. No significant amounts of soluble teichoic acid have been found in the fluid from cells incubated in the presence of penicillin. 5. A high proportion of the teichoic acid formed in the presence of penicillin can be removed from wall preparations at room temperature by 0.1n-ammonia. This is not true of the teichoic acid formed in the absence of penicillin. 6. The teichoic acid extracted with ammonia from preparations of cell walls made from cells treated with penicillin is excluded from Sephadex G-25, has a low molar ratio of glucosamine to phosphorus and contains muramic acid, alanine, glutamic acid, glycine and lysine. 7. The implications of these results for the mechanism of action of penicillin are discussed.
Roantree, Robert J. (Stanford University School of Medicine, Stanford, Calif.), and John P. Steward. Mutations to penicillin resistance in the Enterobacteriaceae that affect sensitivity to serum and virulence for the mouse. J. Bacteriol
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630–639. 1965.—Series of mutants resistant to benzylpenicillin or α-aminobenzylpenicillin were derived from serum-resistant strains of Escherichia coli and Salmonella by the gradientplate technique. Serum-sensitive mutants were detected in series derived from 16 of the 19 strains used, and these retained the parental O type. Most series were characterized by a mutational step to a high degree of sensitivity to serum. Penicillin-resistant mutants of virulent S. typhimurium and S. enteritidis were less virulent than the parent strains; those which were very sensitive to serum usually showed the greatest loss of virulence. One class of mutants from S. enteritidis was sensitive to human serum but virulent for mice. We found that the mice lack bactericidal antibody against this strain and that immunization with it leads to a high degree of protection.
Some general properties of the cell walls of four Gram-positive and two Gram-negative bacteria have been studied. The rate of hydrolysis of cell walls of several bacteria, by 2 N HCl at 100° C, has been followed by estimation of liberated reducing substances and hexosamine. Several extraction procedures have been used in a study of the lipid content of the cell walls of E. coli and B. subtilis. Cell wall preparations have been analysed for their N and P contents, reducing substances and hexosamine liberated on acid hydrolysis and total lipid contents. The lipid content of the cell walls of the Gram-negative bacteria, E. coli and Salmonella pullorum is substantially higher than that of any of the Gram-positive cell wall preparations. The products of acid hydrolysis of cell walls have been examined by paper chromatography. The amino acids of the cell walls have been identified. The amino acids of Strep. pyogenes wall have been examined before and after removal of the type-specific M-protein with trypsin. Cell walls of some Gram-positive show very limited amino-acid constitutions, being devoid of aromatic and sulphur-containing amino acids. A much more complete range of amino acids including aromatic, certain sulphur-containing amino acids, arginine and proline, was found in the cell walls of the two Gram-negative species. The recently isolated amino acid, diaminopimelic acid, has been identified in the walls of certain bacteria. Three unknown ninhydrin-reacting substances have been appeared on chromatograms of cell wall hydrolysates. The sugar components of the cell walls have been identified. The presence of group specific substances in the cell wall of Strep. pyogenes has been demonstrated.
A BIOCHEMICAL explanation for the antimicrobial activity of penicillin has been proposed1. In this hypothesis the structure of penicillin was related to that of intermediates known to be involved in the synthesis of cell wall material2. It was suggested that penicillin interferes with a transpeptidation reaction responsible for the cross-linking of mucopeptide chains in the cell wall polymer. The reaction is represented as follows: The ability of penicillin to interfere with this reaction was attributed to its putative structural resemblance to the D-alanyl-D-alanine portion of the peptide chain.
Hugo, W. B. (University of Nottingham, Nottingham, England), and A. D. Russell. Action of penicillin on Aerobacter cloacae. J. Bacteriol.
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411–417. 1961.—A study has been made of the action of penicillin on a penicillinase-producing strain of Aerobacter cloacae. It has been found that in culture media rendered hypertonic by the addition of sucrose (0.33 to 0.66 m), provided a source of magnesium ions is present, cell wall lesions occur, giving rise to osmotically fragile spheroplasts. Evidence is also presented of a lethal effect not associated with cell wall damage. Penicillinase production is not a complete challenge to the action of penicillin and in certain experimental situations renders the cells more susceptible to penicillin action.
THERE is abundant evidence to support the theory that at least one way in which penicillin exerts its antibacterial effect is to prevent the synthesis, during cell division, of a rigid component of the cell wall. If this action takes place in normal culture medium, the result is lysis ; if in a hypertonic environment, the cell changes into a spherical body or spheroplast1. We have found that the parent amine, 6-aminopenicillanic acid2, from which the natural penicillins may be considered to be derived by acylation, induces morphological changes similar to those produced by benzylpenicillin, 6-(alpha-phenoxypropionamido)penicillanic acid (`Broxil'), and phenoxymethylpenicillin (penicillin V).
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