Biomedical subjects
Publications and source records attributed to R B Sykes.
A novel beta-lactamase with a broad spectrum of activity against penicillins and cephalosporins has been detected in a strain of Klebsiella aerogenes. Its synthesis is mediated by an R-plasmid of molecular weight 64 x 10(6).
The Pseudomonas plasmid RPL11 specifies a beta-lactamase that has properties distinguishing it from other plasmid-mediated beta-lactamases so far described, although it closely resembles plasmid Rms139 beta-lactamase.
Antibiotic G7063-2, isolated from a Streptomyces species, is a new nitrogen-containing analogue of phyllostine and terreic acid. In vitro G7063-2 is moderately active against both Gram-positive and Gram-negative bacteria and weakly active against fungi. It is toxic to mice.
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A cephalosporin, (6R,7R)-7-[(2R)-2-hydroxy-2-phenylacetamido]-3-(pyrid-2-yl-N-oxide) thiomethylceph-3-em-4-carboxylic acid (MCO), that could lead to a novel approach to the problem of beta-lactamase destruction is described. The compound is slightly more resistant to some beta-lactamases than is cephalothin, but it is still hydrolyzed by many to a varying degree. Hydrolysis of the beta-lactam bond of a cephalosporin releases the 3-substituent, which in MCO is itself an antibacterial agent, 2-mercaptopyridine-N-oxide. Thus, MCO has a dual mode of action, and bacteria that do not produce an effective amount of a beta-lactamase are inhibited by the intact cephalosporin, whereas those that do hydrolyze it are inhibited by the released antibacterial compound.
Cefuroxime is a new broad-spectrum cephalosporin antibiotic with increased stability to beta-lactamases. This stability, although no absolute in all cases, has the effect of widening the antibacterial spectrum of the compound so that many organisms resistant to the established cephalosporins are susceptible to cefuroxime. It is active against gram-positive organisms, including penicillinase-producing staphylococci, but it is less active against methicillin-resistant strains. In addition to its high activity against non-beta-lactamase-producing gram-negative bacteria, cefuroxime effectively inhibits the growth of many beta-lactamase-producing strains, including Enterobacter, Klebsiella, and indole-positive Proteus spp. It is highly active against Neisseria gonorrhoeae, Neisseria meningitidis, and also Haemophilus influenzae, including ampicillin-resistant strains. Cefuroxime is rapidly bactericidal and induces the formation and subsequent lysis of filamentous forms over a small concentration range.
Cefuroxime is a new broad spectrum cephalosporin antibiotic for administration by injection. It is stable to most beta-lactamases. It is active against gram-positive organisms, including penicillinase-producing staphylococci, and has wide activity against gram-negative bacilli including Enterobacter and many strains of indole-positive Proteus spp. The substance is also highly active against Haemophilus influenzae and Neisseria gonorrhoeae. Studies on human volunteers showed that it produced high, long-lasting blood levels with virtually complete recovery of unchanged antibiotic in the urine. No evidence of toxicity due to cefuroxime was found. Slight, short-lived pain followed intramuscular injection, and the compound was well tolerated intravenously.
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Production of beta-lactamase by 15 strains of Haemophilus influenzae has been investigated. All the strains produce a constitutive beta-lactamase, which readily hydrolyses penicillin G, ampicillin, and cephaloridine. The beta-lactamase produced by these strains is indistinguishable from the type-IIIa enzyme commonly found in strains of Escherichia coli. The beta-lactamase gene has been transferred from the enzyme-producing strains of Haemophilus to strains of H. parainfluenzae and a strain of E. coli.
The induction of beta-lactamase in Pseudomonas aeruginosa 1822s was studied using benzylpenicillin as inducer. The specific rate of beta-lactamase formation was constant throughout an induction experiment. Above a threshold (20 mug/ml), the specific activity increased linearly with the concentration of the inducer. Removal of the inducer resulted in a rapid cessation of beta-lactamase biosynthesis. Inhibition of protein synthesis by starvation for a required amino acid or by the addition of chloramphenicol also led to an instantaneous arrest in enzyme formation. In the absence of inducer, a basal beta-lactamase activity was formed. The basal and the induced enzymes seem to be identical since they had the same substrate profile, electrophoretic mobility, and molecular weight. In all these respects, induction of beta-lactamase in Pseudomonas aeruginosa is analogous to induction of the lac operon in Escherichia coli. However, there was a long, concentration-dependent lag before beta-lactamase was induced. This can be explained by the outer penetration barrier decreasing the rate of inducer uptake. The lag was significantly shorter for lysozyme-ethylenediaminetetraacetic acid-produced spheroplasts than for intact cells. Induction was obtained with all beta-lactam antibiotics tested, but not with other agents affecting the cell envelope.
Pseudomonas aeruginosa produces a low basal level of beta-lactamase (0.002 to 0.004 IU/mg of protein when benzylpenicillin is used as substrate). The beta-lactamase specific activity can be increased several hundredfold by growing the bacteria in the presence of beta-lactam antibiotics. This induction was studied in Pseudomonas aeruginosa 1822s. The single-cell resistance to benzylpenicillin was 750 mug/ml. In liquid culture all concentrations of benzylpenicillin tested (25 to 2,000 mug/ml) affected the bacteria similarly: beta-lactamase formation was induced, the cells became cholate sensitive, growth rate decreased, filaments were formed, and beta-lactamase was excreted. The effect appeared earlier the higher the concentration of the antibiotic. Most of the effects obtained are concerned with the functioning of the outer membrane. The excretion of beta-lactamase seems to be due to an opening of the periplasmic volume rather than to lysis of the cells. Carbenicillin gave the same effects as benzylpenicillin at the same concentrations; the 10-fold lower resistance to carbenicillin than to benzylpenicillin can be explained by the inability of the inducible beta-lactamase to hydrolyze carbenicillin. The induced beta-lactamase was first cell bound and to a great extent located in the periplasmic volume, but later it was excreted into the medium. This extracellular activity was responsible for the detoxification of the medium. This is analogous to the behavior of gram-positive bacteria rather than to that of Enterobacteriaceae.
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An outbreak of R-factor-mediated carbenicillin resistance in Pseudomonas aeruginosa in burned patients in March 1969 was followed by a second outbreak 6 months later. No R-factor-carrying P. aeruginosa strains were detected in the intervening period but R-factor-determined lactamase was commonly encountered, particularly in Klebsiella aerogenes strains. A comparison of the molecular properties of the R factors in pseudomonads from the first and second phases with those in the Klebsiella strains from the intervening period showed them to be very closely related. A single R-factor type therefore may have been maintained in the Burns Unit between the two Pseudomonas outbreaks as a plasmid conferring resistance to ampicillin in K. aerogenes.
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The product of beta-lactamase activity (a penicilloic acid in the case of a penicillin) is stoichiometrically oxidized by iodine. Hence, the beta-lactamase activity can be measured as decolorization of the blue starch-iodine complex. Since the decolorization is a slow process, the rate of decolorization gave an underestimate of the rate of penicillin hydrolysis until a steady state was obtained after 15 to 20 min. If the reaction mixture (3 ml) contained no more than 0.001 unit of enzyme, a correct determination of beta-lactamase activity was obtained from the microiodometric method described. The method is sensitive and some applications are mentioned.
RP1, a group of genes specifying resistance to carbenicillin, neomycin, kanamycin, and tetracycline and originating in a strain of Pseudomonas aeruginosa, was freely transmissible between strains of P. aeruginosa, Escherichia coli, and Proteus mirabilis. Acquisition of the multiple drug resistance specified by RP1 by these strains was accompanied by acquisition of an extrachromosomal satellite of covalently closed circular deoxyribonucleic acid of molecular weight about 40 million daltons and of buoyant density 1.719 g/cm(3) (60% guanine plus cytosine).