Article

L forms of Staphylococcus aureus

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Abstract

All but four of twenty-five strains of Staphylococcus aureus exposed to concentrations of 60 μg. benzylpenicillin/ml. (or 100–500 μg. methicillin/ml. for penicillinase-producing strains) on nutrient agar media containing 3.5 % (w/v) sodium chloride and 10 % (v/v) horse serum, gave rise to typical L colonies. The L forms were subcultured on nutrient agar and in broth containing 3.5 % sodium chloride and were found to be completely resistant to the penicillins, cycloserine, ristocetin, vancomycin and cephalosporin, but to be sensitive to other antibiotics, often in slightly lower concentrations than the parent cocci. The L forms produced coagulase and, in two of three strains tested, were lysogenic although resistant to phage lysis. In studies of the transformation of staphylococci to L forms, it was noted that a variable but often very large proportion of the early L microcolonies failed to develop into typical colonies.

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... Smith and Willis (19) have reported that L-forms of S. aureus resembled their original coccal forms in the qualitative production of catalase, coagulase, deoxyribonuclease, gelatinase, lipase, and colonial pigmentation; fibrinolysin and hemolysin could not be observed. Several authors also report that coagulase production is maintained in the L-forms (11,22). As regards antibiotic sensitivity, L VOL. 20, 1970 873 on December 19, 2020 by guest http://aem.asm.org/ ...
... As regards antibiotic sensitivity, L VOL. 20, 1970 873 on December 19, 2020 by guest http://aem.asm.org/ Downloaded from colonies of staphylococci are reported to have the same sensitivity pattern as the original cocci when the antibiotic does not interfere with the cell wall synthesis; but usually when these antibiotics are investigated, the L colonies are found to have a lower minimal inhibitory concentration value than the original cocci (22). The L-forms, however, are resistant to antibiotics which only influence the cell wall synthesis (22). ...
... Downloaded from colonies of staphylococci are reported to have the same sensitivity pattern as the original cocci when the antibiotic does not interfere with the cell wall synthesis; but usually when these antibiotics are investigated, the L colonies are found to have a lower minimal inhibitory concentration value than the original cocci (22). The L-forms, however, are resistant to antibiotics which only influence the cell wall synthesis (22). Coccal forms reverted from L-forms are reported by Marston (10) to show the same sensitivity pattern as the original coccal strains. ...
Article
Penicillinase-producing strains of Staphylococcus aureus are transformed into stable L colonies by 70 to 100 subcultures on methicillin-containing medium with a suitable high osmolarity. During transformation, the penicillinase activity is lost. This loss in activity is not the result of only the penicillinase-negative mutants transforming to L colonies. If unstable L colonies are filtered through 0.45-μm membrane filters immediately after transformation, still no penicillinase activity is seen; this is also the case if the filtrated L colonies are reverted into coccal forms. The mechanism responsible for the loss of penicillinase activity is discussed. A loss of the penicillinase plasmid is proposed as the most reasonable explanation.
... (L100) were purchased from the American Type Culture Collection (ATCC; Manassas VA). L-form cells (L3751) were prepared from L100 by exposure to 100 U of penicillin in Enterococcal Brain Heart Infusion/S (EBH/S) supplemented with 5% NaCl, 5% sucrose, and 10% horse serum as previously described [5,26]. L-forms were then cultured on similarly supplemented brain heart infusion agar containing no antibiotic. ...
... Primary screening was based on the differential activity assay versus S. aureus and its L-form. L-forms are protoplast-type cells derived from S. aureus that are able to replicate in appropriate osmotic conditions despite the lack of a functional cell wall [5,7,26]. As previously shown in [7], L-forms are equally or more sensitive than parental cells to those antibiotics acting on molecular targets other than cell wall biosynthesis. ...
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With the increasing need of effective antibiotics against multi-drug resistant pathogens, lantibiotics are an attractive option of a new class of molecules. They are ribosomally synthetized and posttranslationally modified peptides possessing potent antimicrobial activity against aerobic and anaerobic Gram-positive pathogens, including those increasingly resistant to β-lactams and glycopeptides. Some of them (actagardine, mersacidin, planosporicin, and microbisporicin) inhibit cell wall biosynthesis in pathogens and their effect is not antagonized by vancomycin. Hereby, we apply an efficient strategy for lantibiotic screening to 240 members of a newly described genus of filamentous actinomycetes, named Actinoallomurus, that is considered a yet-poorly-exploited promising source for novel bioactive metabolites. By combining antimicrobial differential assay against Staphylococcus aureus and its L-form (also in the presence of a β-lactamase cocktail or Ac-Lys-D-alanyl-D-alanine tripeptide), with LC-UV-MS dereplication coupled with bioautography, a novel producer of the potent microbisporicin complex was rapidly identified. Under the commercial name of NAI-107, it is currently in late preclinical phase for the treatment of multi-drug resistant Gram-positive pathogens. To our knowledge, this is the first report on a lantibiotic produced by an Actinoallomurus sp. and on a microbisporicin producer not belonging to the Microbispora genus.
... Although much biochemical information is available about stabilized, osmotically fragile bacterial L-forms of Staphylococcus aureus, nothing is known of their adaptation and structural or physiological changes as they might occur when these organisms are adapted to grow in the ordinary bacteriological media. Williams (40) succeeded in adapting a staphylococcal L-form to a medium containing only 0.2 M (1.2%) NaCl, but he could not adapt it to media of lower osmolality. In addition, he used serum-containing media throughout the experiment. ...
... DISCUSSION L-forms are considered to be fragile and to require osmotic protection for growth. Staphylococcal L-forms, however, have been adapted to media of low osmolality (32,40). This investigation is the first successful attempt to adapt an osmotically fragile L-forms of halotolerant S. aureus to survival and growth at near physiological isotonicity. ...
Article
An L-form derived from halotolerant Staphylococcus aureus Tasaki was adapted to growth in a brain heart infusion medium without any supplemental osmotically protective solutes (360 mOsm/kg). This L-form had no chemically detectable peptidoglycan residues on its surface. Electron microscopic observations confirmed morphologically the absence of the structures and also of other osmotically protective polymers within or exterior to the cytoplasmic membrane. The osmotic stability and susceptibility to bacitracin, D-cycloserine, and vancomycin of the L-form adapted to growth in 360 mOsm osmotically unprotective medium was higher than that of the L-form grown in 1,950 mOsm supplemented with 4.5% NaCl. The adapted L-form tended to be more sensitive to almost all of the antibiotics examined, other than the inhibitors for cell wall-synthesis, than the original L-form strain requiring osmotic protection for growth. Chemical analysis of the membrane of the adapted L-form indicated 16.3% total lipids and 20.6% proteins by dry weight of the membrane, and it contained larger amounts of lipid phosphorus (20.0 microgram/mg).
... Gooder and Maxted (18) found that group A streptococcal L-colony growth on agar was enhanced when a filter-paper covering was placed over the inoculated agar surface of a medium which did not otherwise support good growth of L-forms. Williams found no effect due to a filter-paper covering on colony formation by an L-phase variant of S. aureus (35). van Boven and Hijams et al. (3,4) reported that, dependent upon the experimental conditions, the filtration end point of group A streptococcal L-phase variants AED and GL8 varied between 0. 25 These elements, because of their size, had escaped microscopic examination, and, because of adverse conditions imposed by the covering filter, had been unable to develop as recognizable colonies. ...
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Membrane filters (Millipore Corp.; pore sizes 1.2 to 0.22 mum) were placed on the surface of L-phase growth medium solidified with agar. The filter and the surrounding medium were inoculated with either protoplasts or stable broth-grown L-phase variants obtained from Streptococcus faecium strain F24. The L-phase inoculum gave rise to viable L-colonies on the filters and on the medium, whereas protoplasts gave colony formation only on the medium. However, when the Millipore filters were covered by a layer of solid L-phase medium, 75 mum or greater in depth, before inoculation with protoplasts, colony formation resulted but with atypical morphology. In contrast, inoculation of protoplasts on Nuclepore and Sartorius membrane filters did give rise to L-colonies on the surface and underneath the filters after 2 days of incubation at 37 C. Submicroscopic, viable L-phase elements produced during colony formation were capable of passing through membrane filters with pore channels as small as 0.22 mum; these elements required transfer from underneath the filters to fresh agar medium in order to develop into L-phase colonies. Membrane filters were also placed on the surface of L-phase growth medium solidified with gelatin. Inoculation of the filters and surrounding medium with a lysozyme-prepared protoplast suspension gave rise to streptococci on the surface of the filters and on the medium. However, inoculation with the stable broth-grown L-phase variants gave rise to atypical colonies on the medium and only small patches of abortive growth on the filters.
... If the test is carried out at 370C, then the addition of sodium chloride to the medium in a final concentration of 5Y% also results in a greater number of resistant strains detected (Churcher, 1968). The additional sodium chloride is thought to prevent lysis of the surviving cocci, which are deficient in cell-wall material (Williams, 1963; Pratt, 1966), by increasing the osmotic strength of the medium (Barber, 1964). Because cephalosporins also exert their main effect on the cell-walls of bacteria it was thought that the use of low incubation temperatures and osmotic support may have a similar effect on the rate of detection of resistant staphylococci. ...
Article
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The use of polyvinylpyrrolidone, an inert polymer resembling plasma proteins in its colligative effects, in the testing of micrococcaceae for sensitivity to methicillin and cephradine is described. Generally results are quite comparable with those of conventional methods. The absence of any inhibitory effect of the polymer compared to sodium chloride, and its physiological inertia compared to sucrose, along with its suitability for sterilisation by autoclaving are seen as advantages. It is suggested that the use of this substance may give results which are more applicable to the in vivo situation. This may apply particularly in the case of cephradine.
... Depending on the phage strain, the reactions ranged from phages that showed productive lysis to a phage that even failed to adsorb. Besides this dependence on phage particularities, alterations or absence of phage receptors are most likely the reason for the lack of phage typability described for three strains of S. aureus (22), in contrast to the lytic action of two typing phages reported in the present study. Also, a transforming activity of free phage DNA has to be considered. ...
Article
Lysogenicity and phage typability of Staphylococcus aureus L-form cells are described. Spontaneously produced phages were found in thin sections of S. aureus L colonies. The dimensions of the tail and head resemble those of the morphological group BIII2 (T. Krzywy, I. Durlakowa, A. Kucharewcz-Krukowska, S. Krynski, and S. Slopek, Zentralbl. Bakteriol. Mikrobiol. Hyg. Abt. 1 Orig. Reihe A 250:287-295, 1981). Phages 3A and 3C of the international typing set lysed both the bacillary and L form of S. aureus.
... The incorporation of serum lipids to cell wall-removed S. aureus is not surprising, as this phenomenon has been observed in S. aureus L-forms. Bacterial L-forms are derived from typical bacteria, often through treatment with cell wall-active antibiotics, and lack an organized cell wall, yet they can proliferate in suitable media (52). Supplementation of medium with serum is often used to grow L-forms. ...
Article
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Comprehensive lipidomics of S. aureus grown in the presence of human serum suggests that human serum lipids can associate with the cell envelope without being truly integrated into the lipid membrane. However, fatty acids derived from human serum lipids, including unsaturated fatty acids, can be incorporated into lipid classes that can be biosynthesized by S. aureus itself. Cholesteryl esters and triglycerides are found to be the major source of incorporated fatty acids upon hydrolysis by lipases. These findings have significant implications for the nature of the S. aureus cell surface when grown in vivo . Changes in phospholipid and glycolipid abundances and fatty acid composition could affect membrane biophysics and function and the activity of membrane-targeting antimicrobials. Finally, the association of serum lipids with the cell envelope has implications for the physicochemical nature of the cell surface and its interaction with host defense systems.
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Bacitracin is produced by strains of Bacillus licheniformis. The commercial product contains a main component, bacitracin A (Fig. 1), and at least nine additional closely related polypeptides (Regna, 1959). In neutral or slightly alkaline solution, bacitracin A is slowly transformed into bacitracin F (Fig. 2) (Regna, 1959) which has very little antibacterial activity (Hickey, 1964).
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A method of induction of L colonies of Staphylococcus aureus by cycloserine, the propagation of wall-defective staphylococci (WDS), and the development and antimicrobial susceptibility of stable WDS are presented. The growth and morphology of the cycloserine-induced L colonies and WDS were indistinguishable from those induced by methicillin or lysostaphin. The antimicrobial susceptibility of the stable cycloserine-induced WDS was similar to that of the stable methicillin-induced and lysostaphin-induced WDS by both broth dilution and agar diffusion techniques. A survey of the English literature reveals that wall-defective bacteria are always resistent to antimicrobial agents which inhibit cell wall synthesis (cycloserine, penicillins, and cephalosporins). They are virtually all susceptible to antimicrobial agents which have other modes of action. The susceptibility to vancomycin and bacitracin varied from study to study.
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L colonies of Staphylococcus aureus were induced with vancomycin and propagated on agar. The yield of L colonies induced by vancomycin was very low in comparison with other inducers.
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The ability of 258 strains of Staphylococcus aureus and 132 strains of S. epidermidis to produce L-form colonies in a standard environment was investigated. Production of these colonies by S. aureus was correlated with their phage type. Phage type 7 strains produced L-form colonies in larger numbers and more consistently than those of phage type 80/81. No L-form colonies were produced by the S. epidermidis strains tested.
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Since their discovery in 1945 (Johnson et al., 1945), the bacitracin peptides have been extensively studied by microbiologists, biochemists, and chemists. Although the major effort has been directed toward elucidation of the mechanism for the antimicrobial activity of bacitracins, the peptides have also served as useful tools for studying various biochemical processes and the chemistry of lipid peptide interactions. The primary goal of this article is to review the literature concerning the mechanism of action of bacitracin. Although this problem has been studied for over twenty years, the mechanism for bacitracin’s biological activities has not been unambiguously defined. Indeed, the bacitracins apparently affect a number of biochemical processes and it is not clear that the peptides inhibit bacterial growth by virtue of a single effect on bacterial metabolism. The timing of this review was dictated by the rapid proliferation of research in this area, and the existence of several different proposals in the literature for the mechanism of action of bacitracin.
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Vancomycin is a unique glycopeptide structurally unrelated to any currently available antibiotic. It also has a unique mode of action inhibiting the second stage of cell wall synthesis of susceptible bacteria. There is also evidence that vancomycin alters the permeability of the cell membrane and selectively inhibits ribonucleic acid synthesis. Induction of bacterial L-phase variants from susceptible organisms with vancomycin is extremely difficult, and such variants are unstable. Stable L-phase variants induced by other agents are susceptible to vancomycin. Vancomycin is active against a large number of species of Gram-positive bacteria, such as Staphylococcus aureus (including methicillin-resistant strains), Staph. epidermidis (including multiple-resistant strains), Streptococcus pneumoniae (including multiple-resistant strains), Str. pyogenes, Str. agalactiae, Str. bovis, Str. mutans, viridans streptococci, enterococci, Clostridium species, diphtheroids, Listeria monocytogenes, Actinomyces species and Lactobacillus species. There has been no increase in resistance to vancomycin during the past three decades. Enhancement of antimicrobial activity has been demonstrated with the combination of vancomycin and an aminoglycoside against Staph. aureus, Str. bovis, enterococci and viridans streptococci. The combination of vancomycin and rifampicin are antagonistic to most strains of Staph. aureus, though indifference and occasionally synergism have been shown, but is synergistic against strains of Staph. epidermidis. It shows indifference against enterococci. Vancomycin and fusidic acid are indifferent against Staph. aureus.
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L-forms of Staphylococcus aureus H do not synthesize the cell wall material mucopeptide. Isolated membranes prepared from these L-forms, however, have been shown to make important cell wall intermediates when supplied with appropriate substrates.
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Cell membranes of Mycoplasma were isolated either by osmotic lysis or by ultrasonic disruption of the organisms. The membranes were dissolved in phenol-acetic acid-water (2:1:0.5, w/v/v), and membrane proteins were separated electrophoretically in polyacrylamide gels containing 5 m urea and 35% (v/v) acetic acid. The electrophoretic patterns of membrane proteins were highly specific for the different Mycoplasma strains examined. The use of this method to prove the identity or dissimilarity of Mycoplasma strains is suggested.
Article
Penicillinase-producing strains of Staphylococcus aureus are transformed into stable L colonies by 70 to 100 subcultures on methicillin-containing medium with a suitable high osmolarity. During transformation, the penicillinase activity is lost. This loss in activity is not the result of only the penicillinase-negative mutants transforming to L colonies. If unstable L colonies are filtered through 0.45-mum membrane filters immediately after transformation, still no penicillinase activity is seen; this is also the case if the filtrated L colonies are reverted into coccal forms. The mechanism responsible for the loss of penicillinase activity is discussed. A loss of the penicillinase plasmid is proposed as the most reasonable explanation.
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Novobiocin (NB) reduced lysis and prevented spheroplast formation in penicillin-treated E. coli. It caused a slow lysis of preformed spheroplasts when these were suspended in an environment conducive to their growth, and prevented reversion of spheroplasts to rods. It did not lyse “resting” protoplasts of Bacillus megaterium, but inhibited the growth of protoplasts, and brought about slow lysis of these forms.
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SUMMARY Three strains of penicillin-induced and one strain of D-cycloserineinduced L-forms of Staphylococcus aureus were investigated for components of the normal bacterial cell wall. None of the 4 strains was found to contain more than 0.008 yo of its dry weight as muramic acid, indicating the lack of mucopeptide. These forms also lacked the cell-wall ribitol teichoic acid as shown by the failure to yield any material containing ribitol. Neither muramic acid nor ribitol was detected as acid-soluble nucleotides in the L-forms, and teichoic acid was not released into the supernatant medium. Serological evidence for polysaccharide A was not found, but there was serological identity between extracts of membranes obtained from staphylococci and extracts of membranes isolated from the L-forms.
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Staphylococcus aureus can incorporate exogenous straight-chain unsaturated and saturated fatty acids (SCUFAs and SCFAs, respectively) to replace some of the normally biosynthesized branched-chain fatty acids and SCFAs. In this study, the impact of human serum on the S. aureus lipidome and cell envelope structure was comprehensively characterized. When grown in the presence of 20% human serum, typical human serum lipids, such as cholesterol, sphingomyelin, phosphatidylethanolamines, and phosphatidylcholines, were present in the total lipid extracts. Mass spectrometry showed that SCUFAs were incorporated into all major S. aureus lipid classes, i.e ., phosphatidylglycerols, lysyl-phosphatidylglycerols, cardiolipins, and diglucosyldiacylglycerols. Heat-killed S. aureus retained much fewer serum lipids and failed to incorporate SCUFAs, suggesting that association and incorporation of serum lipids with S. aureus requires a living or non-denatured cell. Cytoplasmic membranes isolated from lysostaphin-produced protoplasts of serum-grown cells retained serum lipids, but washing cells with Triton X-100 removed most of them. Furthermore, electron microscopy studies showed that serum-grown cells had thicker cell envelopes and associated material on the surface, which was partially removed by Triton X-100 washing. To investigate which serum lipids were preferentially hydrolyzed by S. aureus lipases for incorporation, we incubated individual serum lipid classes with S. aureus and found that cholesteryl esters (CEs) and triglycerides (TGs) are the major donors of the incorporated fatty acids. Further experiments using purified Geh lipase confirmed CEs and TGs being the substrates of this enzyme. Thus, growth in the presence of serum altered the nature of the cell surface with implications for interactions with the host. IMPORTANCE Comprehensive lipidomics of S. aureus grown in the presence of human serum suggests human serum lipids can associate with the cell envelope without being truly integrated into the lipid membrane. However, fatty acids-derived from human serum lipids, including unsaturated fatty acids, can be incorporated into lipid classes that can be biosynthesized by S. aureus itself. Cholesteryl esters and triglycerides are found to be the major source of incorporated fatty acids upon hydrolysis by lipases. These findings have significant implications for the nature of the S. aureus cell surface when grown in vivo . Changes in phospholipid and glycolipid abundances and fatty acid composition could affect membrane biophysics and function and the activity of membrane-targeting antimicrobials. Finally, the association of serum lipids with the cell envelope has implications for the physicochemical nature of the cell surface and its interaction with host defense systems.
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Biochemical capabilities of bacterial and L-phase organisms of 15 bacterial strains were examined by using a variety of modified routine diagnostic biochemical tests. The results demonstrated that 13 of the tests examined were suitable for use with L-phase variants, that L-phase variants and revertant bacterial phase organisms retained diagnostically significant capabilities of the respective bacterial or L-phase organisms from which they were derived, and that the 13 biochemical tests could be usefully employed to relate a given L-phase variant to a given bacterial phase organism, to distinguish L-phase variants of different species, and to aid in the identification of nonreverting L-phase variants.
Chapter
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A lysogenic strain of Staphylococcus aureus was transformed into its protoplast L form. After stabilization and subcultivation in the L-form state for more than 200 subcultivations, it was tested for maintenance and inducibility of the temperent phage found in the parent strain.In the parent strain and L form, optimal phage production was inducible by 1 and 2 μg of mitomycin-C/ml.Besides regular phages with heads of approximately 35 × 85 nm, some L-form cells also produced polyheads of up to more than 1000 nm in length.RésuméUne souche lysogénique de Staphylococcus aureus a été transformée dans sa forme L. Après stabilisation et plus de 200 subcultures dans l'état de la forme L, la persistance d'un phage tempéré, trouvé dans la forme bactérienne d'origine, a été étudiée.Dans la forme L ainsi que dans la bactérie d'origine, une production optimale des phages a été obtenue par 1 et 2 μg de mitomycine-C/ml. L'étude de leur ultrastructure permet de classer ces phages dans la type B2 (1) ou B1112 (10) avec une tête oblongue, mesurant 35 × 85 nm.On observe également la production de particules anormales, munies de ≪ polytêtes ≫ qui peuvent atteindre 12 fois la longueur habituelle.
Article
Staphylococcus aureus is a prominent human pathogen and is known to form L-forms in vitro and in vivo during infection. However, the conditions of L-form formation are not optimal and the mechanisms of L-form formation in this organism are unknown. Here we optimized the conditions of S. aureus unstable L-form formation and constructed a transposon mutant library and screened for mutants defective in unstable L-form formation. Our results revealed that 20% sucrose, 3.5% sodium chloride, 750-1000 units of penicillin and 33 oC were optimal conditions for L-form formation. Stationary phase cultures of S. aureus formed L-forms better than log phase cultures. The S. aureus L-form colonies showed typical "fried egg" morphology and the cells had deficient cell wall, morphological diversity, and stained Gram-negative. The mutant library screens identified 15 mutants deficient in L-form formation and sequencing analysis identified mutations in 8 genes and 3 intergenic regions. Real-time PCR analysis indicated that except gntK, 7 genes including glpF, glpK, NWMN_0623, NWMN_0843, NWMN_0333, NWMN_0872, and NWMN_1269 were preferentially expressed in L-forms compared to normal cell walled form (P<0.05). The identified genes involved in L-form growth mapped in the pathways for energy production, iron homeostasis, transporters, DNA repair, membrane biogenesis, and biosynthesis. Our findings shed new insight into the molecular basis of S. aureus unstable L-form formation and may have implications for development of novel drugs targeting S. aureus L-forms for improved treatment.
Chapter
The initial observations on the effect of benzylpenicillin on the bacterial wall came in the early 1940s shortly after its introduction as a therapeutic agent. Gardner [74] found that concentrations of penicillin, much lower than those required to kill a range of different bacteria, caused considerable morphological changes to the organisms with apparent damage to the cell envelope. In certain organisms, cell division also appeared inhibited. These observations were extended by Duguid [58] who showed that growth was a necessary requirement for penicillin action, and that killing of the bacteria was apparently associated with cell lysis. He suggested that ‘penicillin at these concentrations interferes specifically with the formation of the outer cell wall, while otherwise allowing growth to proceed until the organism finally bursts its defective envelope and undergoes cell lysis’. Many Gram-positive bacteria, particularly staphylococci, are extremely sensitive to the inhibitory action of penicillin, suggesting that its action is highly specific. This observation prompted the first biochemical studies of Park and Johnson [208] who described the accumulation of a labile phosphate compound by Staphylococcus aureus when treated with penicillin. The accumulated material was subsequently shown to be composed of several nucleotide compounds, all of which contained an unknown amino sugar, and some, the uncommon D-isomers of glutamic acid and alanine too [201–203] ; the function of these compounds was not known. However, when it was discovered the wall of S. aureus contained a structure analogous to part of the accumulated nucleotidepeptide, it became clear that the compounds were in fact the biosynthetic precursors of the wall [209]. At this time, considerable advances were being made in the determination of bacterial wall structure, many of which pointed to its being unique. These findings, taken in conjunction with the other studies on the mode of action of penicillin, pointed to the wall as a structure necessary for the survival of the organism and in consequence a site for the selective action of antibiotics. The biosynthesis of peptidoglycan has been discussed in detail in the previous chapter and a number of antibiotics have been mentioned where information relating to biosynthesis has resulted from investigation of their mode of action. The mode of action of these and other antibiotics will now be described in greater detail at a molecular level. This consideration will be related to their site of action in the synthesis of peptidoglycan, which may be at the level of synthesis of the nucleotide precursors, the membrane-bound stages involving the lipid carrier or the terminal stages involving transfer of the newly-synthesized unit to the growing peptidoglycan.
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The third review written by South Korean researchers discusses the recent trends in the research and production of violacein, which is a purple pigment produced by both natural and genetically modified bacterial strains. The bisindole violacein is formed by the condensation of two tryptophan molecules through the action of five proteins. The genes required for its production, vioABCDE, and the regulatory mechanisms employed have been studied within a small number of violacein producing strains. As a compound, violacein is known to have diverse biological activities, including as an anticancer agent and as an antibiotic against Staphylococcus aureus and other Gram-positive pathogens. Identifying the biological roles of this pigmented molecule is of particular interest, and understanding violacein’s function and mechanism of action has relevance to those unmasking any of its commercial or therapeutic benefits. As usually happens with specialized metabolites, the production of violacein and its related derivatives is strictly regulated and its production is limited. To face this production bottleneck, various groups are seeking to improve the fermentative yields of violacein through genetic engineering and synthetic biology. The two research papers completing the issue are brilliant examples of what was anticipated within the reviews as critical steps in the discovery and development of novel specialized metabolites. Interestingly, both of them are on lantibiotics, which represent an attractive option of a new class of molecules that might overcome arising resistance. Lantibiotics are ribosomally synthetized and posttranslationally modified peptides possessing potent antimicrobial activity against aerobic and anaerobic Gram-positive pathogens, including those increasingly resistant to β-lactams and glycopeptides. For some of them, a specific mode of action inhibiting cell wall biosynthesis (not antagonized by vancomycin) has been demonstrated, explaining the renewed interest for such chemical class of antibacterial peptides. Finally, the last paper by a research group from China highlights the need to improve the fermentation conditions to sustain sublancin 168 production by a strain of Bacillus subtilis. Fermentation is the favourite way to produce this antimicrobial peptide, but the authors claim that the low yield of this stable lantibiotic, that has a broad spectrum of antimicrobial activity, has constrained its commercial application. In this specific case, the authors first screen carbon and nitrogen sources to identify key medium ingredients and then develop an experiment design approach to optimize chemical composition of the cultivation medium and temperature of incubation. The volumetric antimicrobial peptide productivity was double and the study envisages further increments that might be achieved following the developed model.
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L-phase variants and small colony (G-phase) variants derived from penicillinase-producing Staphylococcus aureus strains were tested for penicillinase (beta lactamase) production. A refined variation of the modified Gots test for penicillinase was used to demonstrate penicillinase synthesis. Penicillinase synthesis was reduced in L-phase variants and G-phase variants when compared to parental strains. After reversion of variants to vegetative stages had been induced, revertants were tested for production of penicillinase, coagulase, and alpha hemolysin, mannitol fermentation, and pigment production, and comparisons were made between parent and reverted vegetative forms. All revertants of G-phase variants retained penicillinase activity. Most revertants of L-phase variants showed reduction or loss of penicillinase activity. Retention of coagulase activity, alpha hemolysin production, mannitol fermentation, pigmentation, and phage type varied among revertants.
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SUMMARY At minimal bacteriostatic concentrations, bacitracin had four distinct actions on Staphylococcus aureus: (a) prevention of growth; (b) induction of lysis; (c) suppression of induced enzyme synthesis; (d) stimulation of the reduction of 2,3,5-triphenyltetrazolium chloride. In contrast, peni- cillin and cycloserine had activities (a) and (b) but neither (c) nor (d), and chloramphenicol had activities (a) and (c) but neither (b) nor (d). With bacitracin, properties (a) and (b) but neither (c) nor (d) were enhanced by Zn2+; other metal ions were inactive. Sequential inactivation of bacitracin by autoclaving revealed that the bacteriostatic property was quite labile whereas the other three activities were more heat resistant. Thus, although several distinct mechanisms have been demonstrated whereby bacitracin can injure bacterial cells, the present data do not indicate which of these, if any, is associated with the primary biochemical lesion that results in suppression of growth.
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Staphylococcal L forms revealed characteristics not observed previously. The ability to produce coagulase was retained by the variant. In the presence of blood, many L forms were found within red cells, comparable with the intraerythrocytic stages reported for streptococci. Agar lysis was a unique characteristic of the mature L form.Inhibitors were not required to initiate the L stage and the variant and classical form grew commensally on enriched media. As nutrition was simplified, the L stage was supported, whereas the classical form did not reproduce. A small inoculum resulted in exclusive growth of L forms even in media containing special growth factors. The relative ease with which the L forms are produced experimentally suggests that this phase of growth may occur in vivo.
Corse for their technical assistance during this work. I am also grateful to Messrs Eli Lilly and Co. for supplies of cycloserine , to Leo Laboratories Ltd. for fucidin, and to Glaxo Research Ltd
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I wish to thank Miss S. Harding and Miss J. Corse for their technical assistance during this work. I am also grateful to Messrs Eli Lilly and Co. for supplies of cycloserine, to Leo Laboratories Ltd. for fucidin, and to Glaxo Research Ltd. for cephalosporin.
Phage typing of staphylococci The role of high electrolyte concentration in the
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