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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.

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A tetracycline-susceptible strain of Listeria monocytogenes type 4b was converted to stable L-forms by penicillin. L-form variants resistant to tetracycline were then selected from a predominantly tetracycline-susceptible L-form population on plates containing penicillin and increasing concentrations of tetracycline. The origin of tetracycline-resistant L-forms from the parent Listeria strain was confirmed biochemically, by immunofluorescence, and by polyacrylamide gel electrophoresis. Scanning and transmission electron microscopy confirmed the typical L-form structure and the complete lack of cell wall in both L-form strains. The level of [3H]tetracycline uptake was lower in tetracycline-resistant than in susceptible cells.

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In previous studies the in vivo conversion of Brucella suis to L-form state was put in evidence. The L forms isolated from mouse spleen had original structural aspects in common: the absence of the cell wall layer and the extracellular multilayer "membranous" structures. The biological characterization of these L forms and the preliminary identification of specific chemical markers of the bacterial envelope is reported in the present study, performed with the stable L forms well-growing in the liquid media. The electron microscopy confirmed the absence of cell wall and the presence of numerous dense multilayer membranous structures in the L forms cultivated for a long time on appropriate media. This aspect was changed in the L forms adapted to growth on the ordinary medium for brucella: numerous small dense bodies limited by unit membrane were observed. The chemical analysis of stable L forms showed the absence of diaminopimelic acid, confirming the lack of peptidoglycan. The result of chemical determination in L forms of the Na-2-keto-3-deoxyoctonate was negative. However, biological assays suggested that outer membrane components such as LPS and receptors for the bacteriophage Weybridge remained in the L forms, albeit in reduced amount as compared to parental brucella.

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Previous attempts to obtain in vitro wall-deficient stable L-forms of various strains of Brucella have failed because the obtained spheroplasts revert quickly to bacterial form. Here, we report the isolation of L-forms from mice infected with a B. suis strain type 1 and treated with penicillin. In defined experimental conditions, L-type microcolonies associated with tissue debris were observed in primary spleen cultures, even on antibiotic free media. After several transfers on penicillin-containing medium. typical, tissue-free L colonies were obtained. At first, when cultivated on antibiotic-free medium, these colonies reverted to the bacterial form (identified as B suis, biotype 1). Later, after approximately fifteen transfers on penicillin-supplemented medium, they no longer reverted even after several subcultures on antibiotic-free medium. The L-forms' ultrastructural features included many giant empty bodies, considerable variation in size, shape and density of the wall-deficient cells, and many multilayered membranes. The stabilized L-forms were propagated in vitro and inoculated into mice, and then recovered from their spleens as tissue associated L-microcolonies. An occasional in vivo revertant was identified as B. suis, biotype 1. These data provide one possible explanation for earlier failures to detect the presence of atypical bacteria in clinical or experimental Brucella infections.

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A group B streptococcus strain carrying plasmid DNA determining resistance to several drugs was converted by penicillin to cell wall (CW) defective and then to CW deficient variants (L-forms). The stable CW deficient variants became susceptible to antibiotics in study. Dye-buoyant density analysis of the DNA of CW deficient variants showed that the loss of antibiotic resistance was associated with the loss of extrachromosomal DNA.

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Human diploid cells in culture, infected with a balanced amount of living group A streptococci, were able to survive the infection and could be divided and propagated normally thereafter. The streptococci were rapidly phagocytized by the tissue culture cells. At the beginning, they kept their typical appearance, as well as their ability to fix dyes and group-specific immunoglobulins. After 1 to 2 days, the number of detectable streptococci decreased and they underwent important morphological changes. After some subsequent divisions of the cell line, streptococci persisted in cells only as large, isolated, swollen cocci, and no longer grew on suitable media. After six to eight divisions, a noticeable percentage of the tissue culture cells were very similar in appearance to the same cell line experimentally infected with "stable" L-variants. Cultures on L-phase media of supernatant fraction and cells, made 24 to 48 hr after inoculation, showed typical L-colonies. These grew well on media without antibiotics, as well as on media containing penicillin or vancomycin. They could be propagated on media with penicillin for months and were able to revert to group A streptococci after several subcultures on antibiotic-free media. Controls of uninoculated tissue culture cells never showed the presence of any microorganism. Group A streptococci inoculated into Eagle's basal medium, which was used for the tissue cultures, did not grow and never gave rise to L-colonies, even though the medium contained penicillin. Previous data suggest a biochemical explanation for this conversion, which otherwise is an occasional phenomenon.

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L forms induced from two strains of group A Streptococcus were inoculated intraperitoneally and intravenously into conventional and germ-free mice. After intravenous injection, streptococcal L forms disappeared very quickly from the blood, whereas, after intraperitoneal injection, it was possible to isolate them as long as 25 days after inoculation. Observations suggest that a certain degree of reversion to a bacterial form may occur spontaneously in animals.

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