Penicillin Staph, aureus

Bacterial resistance to antibiotics has been recognized since the first drugs were introduced for clinical use. The sulphonamides were introduced in 1935 and approximately 10 years later 20% of clinical isolates of Neisseria gonorrhoeae had become resistant. Similar increases in sulphonamide resistance were found in streptococci, coliforms and other bacteria. Penicillin was first used in 1941, when less than 1 % of Staphylococcus aureus strains were resistant to its action. By 1947,3 8% of hospital strains had acquired resistance and currently over 90% of Staph, aureus isolates are resistant to penicillin. Increasing resistance to antibiotics is a consequence of selective pressure, but the actual incidence of resistance varies between different bacterial species. For example, ampicillin resistance inEscherichia coli, presumably under similar selective pressure as Staph, aureus with penicillin, has remained at a level of 30-40% for mai years with a slow rate of increase. Streptococcus pyogenes, another major pathogen, has remained susceptible to penicillin since its introduction, with no reports of resistance in the scientific literature. Equally, it is well recognized that certain bacteria are unaffected by specific antibiotics. In other words, these bacteria have always been antibiotic-resistant. 

Penicilhns that are resistant to penicilhnase are the drug of choice for infections resistant to penicillin G, Staph, aureus, or coagulase-negative staphylococci. They are also effective for infections caused by nonenterococcus types of streptococci, such as streptococci groups A, B, C, and G, as weh as pneumococci. 

Staph, aureus is responsible for a variety of skin infections which require therapeutic approaches different from those of streptococcal infections. Staphylococcal cellulitis is indistinguishable clinically from streptococcal cellulitis and responds to flucloxacillin, but generally fails to respond to penicillin owing to penicillinase (P-lactamase) production. Staph, aureus is an important cause of superficial, localized skin sepsis which varies from small pustules to boils and occasionally to a more deeply invasive, suppurative skin abscess known as a carbuncle. Antibiotics are generally not indicated for these conditions. Pustules and boils settle with antiseptic soaps or creams and often discharge spontaneously, whereas carbuncles frequently require surgical drainage. Staph, aureus may also... 

The problem of (3-lactamases became critical in 1960 when the widespread use of penicillin G led to an alarming increase of Staph, aureus infections. These problem strains had gained the lactamase enzyme and had thus gained resistance to the drug. At one point, 80 per cent of all Staph, aureus infections in hospitals were due to virulent, penicillin-resistant strains. Alarmingly, these strains were also resistant to all other available antibiotics. 

Methicillin (Fig. 10.32) was the first semisynthetic penicillin unaffected by penicillinase and was developed just in time to treat the Staph, aureus problem already mentioned. 

More active than penicillin G versus some Gram-negative bacilli (Staph, aureus and E. colt). 

Soluble in water. Dextrorotatory. Inactivated by penicillinase as is penicillin G, but differs from the coihmon penicillin by its antibacterial activity and hydrophilic character. When an aq soln is kept at pH 2.7 and 3T for 2 hrs. there is a loss of antibacterial activity and an increase in dextrorotation. Active against Sarcina lutea, Proteus vulgaris. Salmonella typhimurium, Diplococcus pneumoniae. Shows practically no activity against B. subfilis and Staph, aureus. The toxicity is somewhat less than that of penicillin G. although penicillin N is excreted more slowly. 

Early workers [29] found that, like benzylpenicillin, vancomycin, ristocetin and bacitracin, novobiocin caused an excessive accumulation of cell wall precursor, uridine diphosphate-7V-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine-D-alanine-D-alanine (UDP-MurNAc-L-ala-D-glu-L-lys-D-ala-D-ala) in Staph, aureus and it was thus considered that novobiocin was a specific inhibitor of peptidoglycan synthesis with an effect similar to that of penicillin. However, subsequent studies led to the withdrawal of this hypothesis [26], since novobiocin caused the accumulation of other precursor-type compounds and also strongly inhibited both nucleic acid and protein synthesis in this organism. Thus, accumulation of particular precursors does not necessarily reflect the site of action of an antibacterial agent [30]. 

In many hospitals, over 90% of the nursing staff harbour penicillinase-containing, penicillin-resistant Staph, aureus in the nose, a state of affairs that is rare outside of hospitals. This has been attributed to the continual inhalation of traces of penicillin, which destroys the sensitive strains and thus liberates the nostrils as ideal culture-areas for growth of resistant strains (Gould, 1957). 

Cephalosporin C resembles penicillin in its ability to cause the accumulation of the uridine nucleotide (LVIII) in Staph, aureus and to bring about the lysis of growing organisms. Bacitracin behaves similarly, but it does not follow that this substance interferes with cell wall synthesis at the same point as the penicillinsi . 

Certain D-amino acids can be formed in bacteria by processes involving a racemase and D-amino acid transaminases. Amino acid activating systems have been found for D-alanine in Staph, aureus and a number of other bac-teria 34 is thus possible that free D-amino acids are sometimes incorporated as such into peptide antibiotics. On the other hand, the D-valine fragment of the penicillins arises from changes in an intermediate peptide that contains an L-valine residue. L-Valine in the culture fluid appears to be the precursor of the D-valine residue found in some of the actinomycins, but this relationship does not reveal the stage at which the inversion of configuration occurs. Differences in the permeability of cells or intracellular structures to l- and D-amino acids are liable to complicate the interpretation of experiments in this field. 

Sterilise stainless steel penicillin assay cylinders and immerse in groups of 20 in 20 ml of a forty-eight-hour culture of Salm. cholercesuis or a twenty-four-hou. culture of Staph, aureus, transfer to a filter paper in a petn dish and dry in an incubator for not more than sixty minutes. Drop one cylinder into each of 10 tubes containing 10 ml of the chosen... 

The entry of lysine into either Staph, aureus or Strep, faecalis is unaffected by the presence of common metabolic inhibitors such as cyanide, iodoacetate, fluoride, azide or 2,4-dinitrophenol. Cells in which the accumulation of glutamic acid has been prevented by growth in the presence of penicillin, are able to accumulate lysine to the normal extent (Gale and Taylor, 1947b). 

Fiq. 13. Effect of penicillin on the accumulation of free glutamic acid in Staph, aureus growing in rich medium 4 units benzyl penicillin/ml. medium added at arrow. Broken lines show growth of cells in media with and without penicillin. (From Gale and Taylor, 1947b.)... 

Bacitracin has an antibacterial spectrum similar to that of penicillin and Paine (1951) found that it also resembled penicillin in that, although it had no effect upon the ability of Staph, aureus to accumulate glutamic acid when tested in washed suspensions, it had a progressively inhibitory action on this ability if it was added to the growth medium before harvesting. Paine found that strains which were resistant to penicillin were also resistant to bacitracin. 

In Staph, aureus the accumulation of glutamic acid is activated by either manganese or magnesium, and the process can be inactivated by 8-hydroxyquinoline which appears to act by chelating the metal component. If penicillin is added to the growth medium some time before the cells are harvested, it is found to exert a progressive inhibition on the ability of the harvested cells to accumulate glutamic acid. The effect is probably a secondary one. Similar results are obtained with bacitracin. 

The addition of purines and pyrimidines accelerates the rate of protein synthesis in washed Staph, aureus as shown above. If penicillin in high concentrations is added to the incubation mixture, the additional protein synthesis due to the presence of purines and pyrimidines is abolished, although the basal protein synthesis is not significantly affected, as shown in Fig. 29. This effect is accompanied by a decrease in the purine-stimulated nucleic acid formation, but the concentration of penicillin which abolishes the additional protein synthesis does no more than halve (and frequently less than halve) the additional nucleic acid synthesis. Estimation of the proportions of the purine and pyrimidine bases in the nucleic acid fraction by the method of Smith and Markham (1950) modified by Wyatt (1951) shows that the changes lie in the ribonucleic acid fraction and that there is no significant alteration in the proportions of the various bases whether penicillin is present or not (Gale and Folkes, 1953b). 

Fig. 29. Action of penicillin on synthesis of (a) nucleic acid and (6) protein in washed suspensions of Staph, aureus incubated with glucose and a complete mixture of amino acids without (open histograms) and with a purine-pyrimidine mixture. Cross-hatched area of histograms indicates increase in nucleic acid or protein synthesis due to presence of purine-pyrimidine mixture. (Gale and Folkes, 1953b.)...

Krampitz and Workman (1947) showed that washed suspensions of Staph, aureus will oxidize sodium ribonucleate after a lag period and that the presence of high concentrations of penicillin (500-2000 units/ml.)... 

Hotchkiss (1949) found that suspensions of Staph, aureus incubated with mixtures of amino acids, purines, and pyrimidines in the presence of penicillin took up more uracil than in the absence of penicillin. It is possible that there is a connection between these various investigations. Since there is no obvious alteration in the uracil content of the staphylococcal nucleic acid formed in the presence of penicillin (Gale and Folkes, 1953a Park, private communication) it seems that the formation of the uridine-5 -pyrophosphate derivatives may represent a stimulation of a side reaction rather than a side-tracking of the incorporation of uracil into nucleic acid. On the other hand, should the incorporation of an essential base into nucleic acid be inhibited, it is probable that the whole nucleic acid synthesis would cease rather than that the relative proportions of the bases within the nucleic acid should change. The true answer must probably await the development of a method for the study of nucleic acid synthesis in detail. George and Pandalai (1948) have claimed that the action of penicillin can be reversed by the addition of nucleic acid to cultures. 

Penicillin has no effect on protein synthesis by washed Staph, aureus under the author s experimental conditions until the concentration is raised to a value several orders above the hmiting bactericidal level. It does, however, prevent the increase in rate of protein synthesis that normally follows the addition of purines and pyrimidines to the external medium. Its action is therefore somewhat similar to that of bacitracin except that it does not inactivate the protein-synthesizing mechanism of the cell which is not synthesizing nucleic acid. For the present its action is indicated in Fig. 33 as a blocking of the pre-protein template concerned with nucleic acid synthesis but the high concentrations and partial effects obtained would suggest that, if it does act in this way, it affects a specific part of the template system rather than the whole mechanism. This will be elaborated below. 

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