Electron microscopical investigations on in vitro and in vivo interactions of normal Staphylococcus aureus cells with rat peritoneal macrophages showed that these bacteria were rapidly endocytosed and digested even in the absence of specific antibodies. In contrast to the parental strains oxacilin-induced and stable variante lacking a cell wall (L-forms) were ingested without subsequent formation of phagolysomes and digestive vacuoles. The intracytoplasmic L-form bodies retained their characteristic ultrastructure, i.e. no visible alterations occurred. Some morphological aspects of the L-forms and their persistence in macrophages 7 days after intraperitoneal administration of L-form to rats, suggest the possibility of their intracellular survival.
Although lysozyme and penicillin are different in their molecular action on cell wall murein they produce similar morphological changes in Erysipelothrix rhusiopathiae grown on agar media. 2,000-5,000 pg/mllysozyme and 0.1 -1 IU/ml penicillin induce filament formation. Filaments are able to divide in rods, which shows that only cross wall formation and separation are inhibited. Higher doses of lysozyme (10,000 pg/ml) and penicillin (>1 IU/ml) inhibit cell wall synthesis and induce L-form growth. The propagation of this protoplast type L-form was investigated by microphotographic series in phase contrast microscope during L-form induction and in the stable L-form state. I n both cases L-form cells propagate by formation and growth of small granular elements of about 0.2-0.6 pm in diameter, which spread in different directions in the agar medium. The multiplication process may be explained by the plasticity and flexibility of the L-form cell and its cytoplasmic membrane and by the structural and functional interaction between the "folded chromosome" and the surrounding cytoplasm.Erysipelothrix rhusiopathiae is a small gram-positive rod-shaped bacterium which plays an important role as the agent of swine erysipelas. This organism is sensitive to penicillin, lysozyme, and other antimicrobial agents interfering with cell wall biosynthesis, and it can be converted into the L-form state (MADOFF and DIENES 1967, PACHAS and CURRID 1974, TOSHKOV et al. 1975). The Erysipelothrix L-form is a characteristic protoplast type L-form ( GUMPERT et al. 1978).
The comparative fatty acid analysis of extractable and non-extractable lipids of Escherichia coli W 1655 F+ and it.s stable protoplast, type L-form shows quantitative as well a.s qualitative differences. From 10 different fatty acids obtained 16:0, 17:0, and 18:O are present a t about the same quantities in the lipid fractions of the bacterial and L-form. The absence of larger amounts of 12:0, 14:0, and 14:pOH fatt,y acids in non-extract,able L-form lipids ref1ect.s the loss of the cell wall in L-form cells. 16: 1 fatty acid was found in L-form lipids only. This qualitative difference and the 2-3 times higher content of 18: 1 in L-form lipids and the 7 times lower content of cyc 19:O in extractable lipids of t h e L-form may be interpreted as alterat.ions characteristic for the changed composition of the cytoplasmic membrane in L-form cells.
The stable L-form of Erysipelothrix rhuaiopathiae is a typical protoplast type L-form. Cell8 a m surrounded by a trilamellar cytoplasmic membrane only. They grow in form of 8ggregatiom in liquid media and their diameters vary between 0.1 and 2 pm. Always a large portion of cells undergoes lysis. It seems to be characteristic for L-form cultures of E . rhusiopathiae that always many artifact structures are formed. The artifacts are spherical particles with diameters of 0.1 pm to more than 3 pm. They can be differentiated from L-form cells only by electron microscopy. The artifacts consist of electron dense amorphous material and their surface is irregular without a clear boundary line. Obviously, these artifacts are produced from protein components of the medium and from cytoplasmatic components of the lysing L-form cells.Erysipelothrix rhusiopathiae is a small gram positive bacterium which causes the swine erysipelas. This is a dangerous disease and it can produce large losses in animal stocks. E . rhusiopathiae shows an enigmatic pathology and, therefore, studies were I n this paper the morphological and ultrastructural properties of the stable L-form cells of E . rhusiopathiae grown in liquid media and of the accompanying artifact structures will be presented. Materials and methodsThe L-form strains used were produced from a strain of E. rhuaiopathiae isolated from the liver of 8 swine suffered from erysipelas. The L-forms were induced by treatment of the bacteria with 500-1000 U/ml penicillin G on Brain Heart Infusion Agar (BHI) from OXOID supplemented with 3% NaCI, 10% inactivated horse serum, and 1% yeast extract (OXOID). The L-forms grow in form of typical fried egg colonies. They could be adapted to grow in liquid media by transferring agar blocks with abundant L-colony growth in BHI broth supplemented with 3% NaCI, 10% horse serum, and 1% yeast extract. Further cultivation of the L-forms was carried out in BHI broth or TODD HEWITT broth (OXOTD) both supplemented with 10% horse serum, 1% yeast extract, and 0.9% NaCl only.For electron microscopic investigations samples were embedded in nutrient agar (lye), fixed with glutaraldehyde (5%; 5 hrs) and osmiumtetroxide (1%; 18 hrs), dehydrated in acetone, embedded in Vestopal, and cut with an ultramicrotome (Ultrotome 111, LKB). The sections were stained with lead citrate according to REYNOLDS (1963) and examined in a SIEMENS Elmiskop I electron microscope operated a t 80 kV. Results and discussionThe L-form cells grow in BHI broth or TODD HEWITT broth during the first 24 hrs a t 37 "C without shaking as small spherical elements forming an equal light turbidity.
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