Growth and adhesion of Enterococcus faecium L-forms

Abstract Comparisons of growth and surface colonisation of Enterococcus faecium L-forms and their cell-walled forms were undertaken to produce information about their ability to form sessile cells. The growth of L-forms in liquid culture was slower than that of the parent. This was reflected in their longer lag phase and slower specific growth rates: 0.16 h⁻¹ for the L-form and 0.81 h⁻¹ for the parent. Although E. faecium L-forms attached to a silastic rubber surface, the attached population density was 10–100-fold less than that of the parent. Confluent biofilms on the silastic surfaces were not observed for either bacterial form. Comparison of the attachment of E. faecium L-form and parent may provide important information on how bacteria overcome host defence mechanisms and antibiotic treatment.     

Ultrastructure of LLR-mutants of E. coli K12]

The author studied the ultrastructure of two spherical E. coli K12 mutants (llr) obtained under the effect of N-nitroso-N-methylurea. Seven morphological types of cells differing from one another by shape, size and cytoarchitectonics were distinguished. Superficial structures of the majority of the cells were represented by the membranes of the cell wall and the cytoplasmic membrane of common structure. Some of the cells had only one membrane coat and a high electron optic density of the cytoplasm. Transitional forms of cells were also encountered. The ultrastructure of each morphological type in the population of the llr-mutants was described in detail. The capacity of the mutants to vacuolization, to the intra- and extracellular budding, and also the ability to form multiple membrane structures resembled analogous structures of stable L-forms of the Gram-negative microbes. The problems of morphological differentiation of the L-forms and of the llr-mutants, and also problems connected with the formation of the multiple membrane structures and small elemental bodies in the cells of the llr-mutants are discussed.     

The chemical composition of the membranes of protoplasts and L-forms of Staphylococcus

Membrane fractions were prepared from Staphylococcus aureus H and 100 after dissolution of the cell walls by a lytic enzyme from Streptomyces griseus. Membranes were also prepared from the L-forms derived from the same strains. The membranes were analysed for protein, lipid, carbohydrate and RNA contents, and the fatty acid composition of the lipids was determined. A branched-chain saturated C(15) acid was the major component in all samples, and the correspondence between L-forms and parent bacteria was fairly close. The lipids were separated into non-polar-lipid, glycolipid and phospholipid fractions; the L-forms contained a little more neutral lipid and much more glycolipid than the parent bacteria. In all membranes the glycolipid, which accounted for all the carbohydrate present, was a diglucosyl diglyceride. The major phospholipids of the protoplast membranes were phosphatidylglycerol and some lipoamino acids (lysine and a little alanine). On the other hand, diphosphatidylglycerol was the chief phospholipid found in L-form membranes.     

Inactivation of penicillin-induced staphylococcal L-forms by human serum high density lipoprotein

In penicillin-susceptible bacteria, penicillin causes growth of a small fraction of cells as wall-deficient forms if an appropriate osmoprotection is provided (unstable L-forms). A subfraction of human serum high density lipoprotein (HDL₃) was shown to have the ability to inactivate unstable L-forms of Staphylococcus aureus. The active principle was distinguishable from the well-documented trypanosome lytic factor 1 with respect to density, size, and other properties. This L-form cytotoxicity therefore seems to represent a novel antimicrobial entity in human serum.     

Submicroscopic structure of the streptococcal L forms isolated from a living body]

The authors studied in the L-forms of streptococcus induced in the living organism. Submicroscopic structure of the L-forms under study was analogous to the L-forms of the museum streptococcus strain and to the L-forms of some other bacteria. As revealed on the ultra-thin sections of the protoplast-like cells the intracytoplasmic membrane structures were located close to the cytoplasmic membrane and also passed through the whole cell in the form of a band. The latter was in contact with the nucleotide. The elemental bodies were found in the vesicular and the protoplastic cells, and also in the space between the cells; sometimes they formed groups surrounded by a membrane.     

Physical separation of hemopoietic stem cells from cells forming colonies in culture

Mouse bone marrow cells in suspension were separated into a number of fractions on the basis of cell density by equilibrium density gradient centrifugation, or on the basis of cell size by velocity sedimentation. After each type of separation, the cells from the various fractions were assayed for their ability to form macroscopic spleen colonies in irradiated recipient mice, and for their ability to form colonies in a cell culture system. The results from either separation technique demonstrate that cells in some fractions formed more colonies in vivo than in the culture system, while cells in other fractions formed more colonies in culture than in the spleen. The results of control experiments indicate that this separation of the two types of colony-forming cells was not an artifact of the separation procedures. From these experiments it was concluded that the population of cells which form colonies in culture under the conditions used is not identical to the population of cells detected by the spleen colony assay.     

人体病理组织中细菌L型的电镜观察

对9例细菌L型感染的人体病理组织进行透射电镜观察。结果显示:(1)L型可分布于组织间质或进入吞噬细胞、上皮细胞和癌细胞等细胞胞质;(2)L型具有多形态、大小不等、胞壁缺陷、电子密度低等特点,细胞胞质内的细菌L型尚有A、B两种形态区别;(3)L型在组织中形成了不完全箍缩分裂;(4)宿主细胞超微结构仅出现轻微改变。本结果与文献报道基本一致,提出了透射电镜下病理组织中细菌L型的形态特征及分布;并对L型感染与慢性炎症的关系等问题进行了讨论。     

Electron microscopic findings on reversion of Listeria L-forms

Reversion in L-forms of Listeria monocytogenes was studied with the use of electron microscopy. In the culture undergoing the process of reversion cells differing in size, form and the electron density of the cytoplasm were present. The process of reversion was characterized by the increase of cytoplasmic density, the decrease of nucleoid, the appearance of fibrillary material of medium electron density on the membrane surface and the longitudinal elongation of the cell. The cell wall of the revertants was thinner and its outer layer with greater electron density was less pronounced than in the initial culture.     

Ultrastructure of Brucella abortus L-forms induced by penicillin in a liquid and in a semisolid medium

Brucella abortus L-forms were induced by 5.0 or 10.0 mug of penicillin/ml in a broth medium containing 0.3 m sucrose, and in a semisolid medium containing 10% calf serum and 20.0, 40.0, or 60.0 mug of penicillin/ml. After 96 hr of incubation, L-forms of various sizes and shapes were observed. Basic structures of the L-forms were similar whether induced in liquid or semisolid medium. L-forms had two "unit" membranes, each consisting of two outer dense layers separated by a lucent layer. A few large, irregularly shaped organisms in penicillin-treated broth cultures had additional surface material and were referred to as "transitional" forms. In contrast with L-forms, the bacterial cells were fairly uniform in size and shape, were smaller, and had a more complex cell wall structure. Small bodies limited by a "unit" membrane were present within and around numerous L-forms from liquid and semisolid medium cultures. Other internal membranous structures were also seen in some L-forms. Most Brucella L-forms described in this paper reverted to bacteria in the absence of penicillin and were structurally characteristic of unstable L-forms.     

咽部活组织中细菌L型的检出及意义

应用病原微生物培养、电镜、组织切片细菌学检查及Ｌ型抗体免疫组化染色等方法,检测６４例慢性咽炎组织的细菌Ｌ型。结果有４２例培养出细菌Ｌ型,其阳性率为６５．６％;它与切片革兰氏染色Ｌ型检出阳性率（６７．２％）无显著性差异,Ｐ＞０．０５。透射电镜在慢性咽炎组织的间质及上皮细胞、巨噬细胞等细胞内见皮细菌Ｌ型;且Ｌ型抗体免疫组化染色亦证实组织中有细菌Ｌ型抗原。提示,细菌Ｌ型感染与慢性咽炎关系密切,Ｌ型侵入组织并在宿主细胞内生长的特征,可能是慢性咽炎反复发作,迁延不愈的重要原因。     

Study of the sensitivity of the L-forms of group A Streptococcus to antibiotics in artificial nutrient media and in human cell cultures

Sensitivity of L-forms of group A streptococci to 5 antibiotics such as erythromycin, lincomycin, tetracycline, gentamicin and chloramphenicol was studied in an artificial nutrient medium and cell cultures i.e. human fibroblast diploid cells and transplantable human heart cells (Girardi). In vitro investigation of the antibiotic effect on the streptococcal L-forms revealed their sensitivity to erythromycin (MIC, 0.4 micrograms/ml), lincomycin (MIC, 0.08 microgram/ml) and tetracycline (MIC, 2 micrograms/ml). The streptococcal L-forms were slightly sensitive to gentamicin (MIC, 6 micrograms/ml) and chloramphenicol (MIC, 30 micrograms/ml). Complete inhibition of the growth of the L-forms in the Girardi cells on the 1st day of the experiment after the antibiotics administration in single doses was induced by lincomycin, 5 micrograms/ml, erythromycin, 10 micrograms/ml, and tetracycline, 100 micrograms/ml. In the diploid cells, the respective figures were 50, 100 and 200 micrograms/ml. Chloramphenicol and gentamicin had an inhibitory effect on the growth of the L-forms but produced no sanative effect.     

Mitogenic Activity of Cytoplasmic Membranes Isolated from L-Forms of Staphylococcus aureus

Cytoplasmic membranes of L-forms of Staphylococcus aureus exerted a strong mitogenic effect on splenocytes of athymic nude mice as well as normal mice, while a cytoplasmic fraction of the same bacteria did not show definite mitogenicity. The mitogenic principle(s) of the membrane fraction was resistant to treatment with trypsin and was heat stable (at 100 C for 10 min). The active principle(s) in the insoluble residue of the membrane fraction digested with trypsin was not extracted with cold acetone, but could be solubilized by extraction with a cold chloroform-methanol mixture (2:1, v/v). The mitogenic principle(s) in the extract was fractionated by silicic acid column chromatography. Among five fractions separated by chromatography, fractions eluted with chloroform-methanol mixtures (1:1 and 1:20, v/v) were found to be strongly mitogenic. The cytoplasmic membranes of the L-forms also exerted a definite mitogenic effect on guinea pig splenocytes, but not on the thymocytes.     

Different subsets of T cells in the adult mouse bone marrow and spleen induce or suppress acute graft-versus-host disease.

Fractionation of normal adult mouse spleen and bone marrow cells (C57BL/Ka) was performed by discontinuous Percoll density gradients. The fractionated low density (1.050-1.060 g/ml) C57BL/Ka spleen cells completely suppressed acute lethal graft vs host disease (GVHD) when coinjected with unfractionated C57BL/Ka spleen cells into sublethally irradiated (400 rad) BALB/c mice. In dose response experiments, as few as 0.5 x 10(6) low density cells from the spleen fractions suppressed acute GVHD induced by 2.5 x 10(6) unfractionated allogeneic spleen cells. Although the low density spleen fractions inhibited acute GVHD, the high density (1.075-1.090 g/ml) spleen fractions induced acute GVHD in sublethally irradiated BALB/c recipients. Fractionation of C57BL/Ka bone marrow cells showed that none of the high or low density fractions or unfractionated cells induced lethal GVHD. When these fractions were tested for their capacity to suppress GVHD by coinjection with C57BL/Ka unfractionated spleen cells, all fractions protected the BALB/c recipients. Unfractionated bone marrow cells showed modest protection. Evaluation of the dose response characteristics of the suppressive activity of the low and middle density (1.060-1.068 g/ml) bone marrow cell fraction showed that reproducible protection could be achieved at a 5:1 ratio of inducing to suppressing cells. The low density fractions of both bone marrow and spleen cells had a marked depletion of typical TCR(+)-alpha beta CD4+ or CD8+ T cells, and a predominant population of TCR(+)-alpha beta CD4- CD8- T cells. Purified populations of the latter cells suppressed GVHD. Recipients given unfractionated C57BL/Ka spleen cells and protected with low-density bone marrow or spleen cells were chimeras.     

A novel method for differentiating L-form bacteria from their parental form using the Hucker Gram staining technique

L-forms of Enterococcus faecium, Bacillus subtilis and Pseudomonas syringae pv. phaseolicola were differentiated from their parent, cell-walled forms by a modified Gram staining technique. The addition of glutaraldehyde to the culture medium fixed the cells to prevent lysis of the L-forms. The cell-walled forms exhibited typical Gram staining reactions whereas the L-forms remained red due to the counterstain. L-forms were easily differentiated from cell-walled forms by their size and morphology which was made more obvious by the staining procedure. This is a very rapid and easy technique which distinguishes L-form bacteria from cell-walled organisms.     

Cell fusion between L-forms and protoplasts of Staphylococcus aureus

Mixtures of various combinations of Lysostaphin protoplasts and stable L-forms of Staphylococcus aureus, which have different markers for drug resistance, were treated with polyethylene glycol (PEG) to examine the development of doubly resistant fusion products (fusants). To recover doubly resistant colonies as L-forms, they were incubated in 4.5% NaCl-brain heart infusion (BHI) broth containing penicillin G (PCG) for enrichment culture and cultured in PCG-4.5% NaCl-BHI agar medium (method 1), while to recover doubly resistant fusants as L-forms and coccal forms, they were grown on reversion medium (R medium) which causes reversion of protoplasts or fusants to parent type cells, and then cultured on assay media, i.e., R medium, BHI agar medium or PCG-4.5% NaCl-BHI agar medium (method 2). Under both experimental conditions, doubly resistant fusants developed as L-form cells by PEG treatment of pairs of protoplasts carrying the chloramphenicol (CP)-resistance plasmid and L-forms having chromosomal resistance to streptomycin (SM). In the reverse combinations, i.e., protoplasts showing chromosomal SM-resistance and L-form cells carrying the CP-resistance plasmid, the first method gave no doubly resistant colonies. By the second method, without enrichment culture on R medium, the latter combination gave doubly resistant fusants as L-form, coccal-type and mixed-type colonial forms, while when the PEG-treated mixture was enriched on R medium, fusants were obtained exclusively as the coccal type on either R medium or BHI agar assay medium. Neither of the methods yielded colonies of doubly resistant fusants on PEG-treatment of pairs of protoplasts and L-forms both of which were chromosomal, but with different drug resistances. These results show that PEG-induced cell fusion between protoplasts and L-forms of S. aureus, unlike the fusion between protoplasts or between L-forms, resulted in transfer of the drug resistance controlled by the plasmid to the fusion products. The fusants obtained were L-forms in method 1, and coccal type in the method 2.     

Cross-reacting antigens of human thymus myoid cells and stable L-forms of group A streptococci

Indirect immunofluorescence has shown a similarity between the antigen components of group A streptococcus L-forms and human thymus myoid cells. An analogous antigen (or antigens) is present in the cytoplasmic membrane of human myocardial cell fibers. The depletion of antiserum to the streptococcal L-forms both by the culture of L-forms grown in meat or casein media and by the homogenate of the cardiac muscle leads to the inhibition of immunofluorescence. The depletion of serum by the homogenate of other tissues (liver) or by L-form culture does not virtually affect the immunofluorescence intensity. According to the authors' opinion, the similarity of antigens of group A streptococcus L-forms to the antigenic components of organ tissues is likely to be responsible for long-term persistence of the microorganisms under consideration and to favour, in some cases, the occurrence of autoantibodies. The latter circumstance might lead to pathological changes in organs containing cross-reacting antigens.     

Electron microscopic study of the interrelation of the L forms of beta-hemolytic Streptococcus group A with mouse peritoneal macrophages

Interrelations between the L-forms of group A beta-hemolytic streptococci and mouse peritoneal macrophages have been studied by electron microscopy. The macrophages have been shown to actively phagocytize L-form cells in great amounts. Most of the phagocytized L-forms are destroyed and become nonviable, but a few of them survive as elementary bodies within 48 hours.     

Polyacrylamide Gel Identification of Bacterial L-Forms and Mycoplasma Species of Human Origin

Polyacrylamide gel electrophoretic patterns of acidified phenol extracts prepared from whole cells can be used for the identification of bacterial L-forms and Mycoplasma species of human origin. Ten human Mycoplasma serotypes and eight L-forms belonging to five different genera were studied. The gel patterns were sufficiently distinct and reproducible that it was possible not only to identify L-forms at the genus level (group with streptococci) and different Mycoplasma serotypes but also to differentiate between the two of them. The parentage of L-forms of Streptobacillus moniliformis L1, Listeria monocytogenes, Streptococcus MG, and Staphylococcus aureus Smith strain was established by relating their gel patterns directly to parent bacteria. It was found that an L-form designated S. moniliformis An (ATCC 14220) was actually an L-form of Proteus. In addition, it was shown electrophoretically that no relationship existed between the Streptococcus MG L-form and M. pneumoniae. The applicability of this method as a diagnostic and taxonomic tool for the differentiation of L-forms and mycoplasmas is discussed.     

Density and age of cells in the population of the methanol-assimilating yeast Candida boidinii

A population of the yeast Candida boidinii growing in the chemostat in a medium with methanol was separated in dextran solutions of different density. The density of cells was found to vary from 1.04 to 1.11 g/cm3, being 1.08 g/cm3 on the average. Changes in the cell density correlated with variations in the content of water and dry matter in the cells and its density. Cell fractions of different density contained cells which mainly belonged to a certain phase in the cell cycle. However, the fractions were not homogeneous. The authors propose a scheme to account for changes in the cell density during the cell cycle. According to this scheme, within the cycle, the density of the cell rises twice (when the cell prepares for the budding and in the budding phase II) and drops down twice (in the budding phases I and III).     

嗜酸乳杆菌发酵上清液对常见肠道致病菌及其L型抑菌作用的实验研究

目的 探讨嗜酸乳杆菌（L. acidophilus）对肠道致病菌及其L型菌的抑菌作用,为临床开发L. acidophilus活菌制剂提供实验依据。方法 采用肉汤稀释法,检测L. acidophilus 36 h发酵上清液对肠道致病菌及其L型菌吸光度值的影响,计算抑菌率,探讨乳杆菌的抑菌活性。结果 3个浓度的L. acidophilus发酵上清液对3种常见肠道致病菌（致病性大肠埃希菌、伤寒沙门菌、痢疾志贺菌）及其L型菌均有不同程度抑菌作用,其中原液浓度抑菌作用最强。结论 L. acidophilus是一种可用于治疗多种肠道致病菌及其L型菌感染引起肠道疾病的微生态制剂,其代谢产物发挥抑菌作用是其机制之一。