Lipid composition of Staphylococcus aureus and its derived L-forms.

Two strains of Staphylococcus aureus (Newman and Tazaki) and their derived L-forms were cultured in serum-containing broth and the differences in their lipid compositions were analyzed. Cardiolipin accounted for more than 50% of the total phospholipid phosphorus in L-forms, but for less than 25% in parent bacteria. The cardiolipin content of L-forms was very high through all growth phases, although it increased gradually as growth proceeded. Significant amounts of cholesterol and its esters were present in parent strains and L-forms, all of which incorporated serum cholesterol into the cell membrane. On the other hand, they could be detected in the L-forms but not in the parent strains when they were cultured in serum-free broth. To examine the ability of L-forms to synthesize cholesterol, the cholesterol content of L-forms cultured in serum-free broth was compared with that of the medium. The results indicated that staphylococcal L-forms could synthesize cholesterol and its esters. These differences in lipid composition suggested that modification of membrane lipids may occur as an adaptational change in response to the disappearance of the cell wall.     

Presence and synthesis of cholesterol in stable staphylococcal L-forms.

The sterol which was present in two strains of a stable staphylococcal L-form was analyzed by gas-liquid chromatography and combined gas-liquid chromatography-mass spectrometry. The retention time of the sterol on gas-liquid chromatography was the same as that of authentic cholesterol. Analysis of the sterol by mass spectrometry showed a molecular ion at an m/e of 386 and the same patterns of major ions above an m/e of 145 as those of authentic cholesterol. As a result, the sterol in staphylococcal L-form was identified as cholesterol. A parent strain and its L-forms were cultured in medium containing [14C]acetate, and the synthesis of cholesterol was examined. In the L-forms, 0.52% of the total lipid radioactivity was found in cholesterol fraction, whereas no significant radioactivity was detected in the cholesterol fraction of the parent strain, indicating that staphylococcal L-forms have acquired the capacity to synthesize cholesterol.     

Studies on the Minimum Reproducible Unit of Staphylococcal L-Forms

The minimum size of a reproducible unit of staphylococcal L-forms was determined by filtration and electron microscopic methods. Ultrathin sections of an induced strain of Staphylococcal L-forms (STA-EMT-1) in liquid medium revealed several types of structures, all of which were bound by a single membrane and most of which possessed ribosome-like granules. Many of the small granules were less than 0.3 μm and were attached to the membrane of the large bodies. Using a serial filtration method, it was observed that viable L-forms were still detected in 0.22 μm filtrate, but the viable cell count of L-forms decreased in number with the decrease in pore size of membrane filters. A fractionation technique, using L-forms filtered through a membrane filter with a 0.45 μm pore size, revealed that there were three classes of small bodies but only the first class with ribosome-like granules over approximately 0.2 μm in diameter seems to be able to reproduce.     

L-forms ofHaemophilus influenzae: Growth and reversion as influenced by a strain ofNeisseria perflava

Factors excreted into the medium by various bacteria and fungi exert a significant effect on the growth and revertibility of bacterial L-forms. Studies with L-forms ofHaemophilus influenzae have been facilitated by a strain ofNeisseria perflava, which not only promotes the growth of the L-forms on media of normal osmolarity (horse blood agar), but also enhances the revertibility of the L-forms to the parent bacterium. L-forms derived fromN. perflava also exhibit this growth-enhacing property, an effect that does not appear to be related to X and V factors. Preliminary characterization of the active component produced byNeisseria perflava indicates that it is present in broth filtrate, diffusible through agar, heat labile, and of large molecular size.     

Reversion to the Streptococcal State of Enterococcal Protoplasts, Spheroplasts, and L-Forms

A method is described for predetermining whether lysozyme-damaged enterococci grow as either the parent strain or as L-forms. Organisms treated with lysozyme grew as L-forms on media solidified with low concentrations of agar, or the damaged cells grew as streptococci on media solidified with high concentrations of gelatin. After induction, some of the L-forms reverted to the parent strain, but most did not during three routine subcultures. Continued spontaneous reversion occurred through approximately 30 subcultures after induction. However, subsequent progeny did not revert, even when subjected to conditions such as the gelatin medium which strongly favors growth in the streptococcal phase.     

Induction, growth and antibiotic production of Streptomyces viridifaciens L-form bacteria

AIMS: To induce, cultivate and investigate the characteristics of L-form bacteria derived from the filamentous actinomycete Streptomyces viridifaciens. METHODS AND RESULTS: L-forms were induced in a liquid medium supplemented with lysozyme and penicillin. A stable culture which no longer required inducing agents but could still revert, was obtained by the twelfth subculture. The specific growth rate of stable L-forms was faster (0.751) than unstable L-forms (0.361). After the exponential growth phase, the cell diameter continued to increase, as did the percentage of vacuoles. Morphologically, the L-forms appeared as spherical bodies with no signs of differentiation and were sensitive to osmotic stress, indicating removal of the cell wall. The L-forms produced secondary metabolites although much lower levels of antibiotic were assayed in the L-forms compared with the cell walled forms. CONCLUSION: Stable L-form bacteria were induced from S. viridifaciens and their growth characterized. The L-forms produced secondary metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: Stable Streptomyces L-forms were induced and have potential as biocontrol agents.     

Bacterial L-forms require peptidoglycan synthesis for cell division

Cell-wall-less bacterial variants, or L-forms, have been described in many bacterial species under laboratory conditions, in infected eukaryotic cell cultures and inside animals. Of special interest for human health is the formation of L-forms as a consequence of specific antibiotic treatments, and the potential involvement of L-forms in persistent and relapsing infections. An old enigma about L-forms is how they can divide in the absence of cell wall synthesis, since septum formation is an essential requisite for cell division. However, the classical definition of L-forms as cell-wall-less bacterial variants may need a revision to accommodate recent observations by Richard d'Ari and coworkers: genetic and biochemical evidence indicates that E. coli L-forms induced by beta-lactam antibiotics do contain small amounts of peptidoglycan, essential for their growth and probably required for septum formation. If these observations are extrapolated to all known L-forms, the very concept of cell-wall-less bacteria may need revision, and be restricted to mycoplasmas and their relatives.     

Regularities and cellular mechanism of spontaneous mutations in enterobacteria

The regularities and a possible mechanism of the formation of spontaneous mutations in enterobacteria were studied. Possible causes and the mechanism of these processes were analyzed. It was shown that the mechanism of formation of spontaneous mutations is not related to the replication process and DNA polymerase errors. Mutations arise abruptly within 10-20 min due to a cosmophysical factor of unknown origin. It is assumed that cosmophysical radiation makes cell membranes excitable. Upon membrane pulsation, different volumes (portions) of cell substance are ejected through expanding pores. From this substance, viable heteromorphous mutant forms of colonies and cells (bio- and serum variants, L-forms, "parastrains", and new ecoforms of bacteria) are formed.     

细菌L型与喉癌关系的研究

用组织切片革兰氏染色、免疫组织化学染色等方法,对８５例喉癌重新切片,进行细菌Ｌ型检查,结果发现有６５例革兰氏染色Ｌ型菌阳性,其阳性率为７６．５％。５３例（６４．７％）Ｌ型抗体免疫组化染色和革兰氏染色Ｌ型菌均阳性,两者总符合率为８３．５％。细菌Ｌ型呈多形性,分布于癌巢、癌间质,常聚集成堆,也可呈散在性分布。５８例（６８．２％）癌细胞胞浆内也见到Ｌ型菌。提示细菌Ｌ型感染与喉癌关系密切。     

Alterations of the L-forms of a sporebearing bacillus

A large pleomorphic gram-negative bacillus developed as a contaminant on blood-agar. Spores were formed in one culture. L-forms were produced with penicillin on blood-agar with 2.5% NaCl; they grew well when transplanted to agar with 0.5% NaCl. After several transplants and long incubation of the L-forms without penicillin, in three transplants small gram-negative pleomorphic bacilli grew, but no L-forms. This occurred once on blood-agar and twice on 30% gelatin. The growth obtained from these small bacilli was similar in morphology and in the physical properties of the organisms to the altered L-forms of Proteus and Salmonella. Multiplication of the pleomorphic organisms and development of branching filaments from the round forms was apparent. The original large gram-negative bacillus was regularly recovered from the L-forms, and was recovered several times from the descendants of the small bacilli. These observations are essentially similar to those made with L-forms of Proteus and with an L-form studied in 1952, indicating alterations in L-forms of bacteria which do not produce B type L-forms.     

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.     

鼻咽癌与细菌L型的关系

大量研究表明,鼻咽癌与ＥＢ病毒有关。通过对９８例鼻咽癌组织的切片革兰氏染色Ｌ型检查、电镜和Ｌ型抗体免疫组化染色等研究,发现鼻咽癌组织中细菌Ｌ型亦甚常见,切片革兰氏染色有７８例查见细菌Ｌ型,其阳性率为７９．６％;免疫细化染色Ｌ型抗原检出阳性率为６２．２％,电镜不仅在细胞间质见到细菌Ｌ型,而且在癌细胞、巨噬胞细等细胞胞质内也见到细菌Ｌ型。提示,细菌Ｌ型与鼻咽癌关系十分密切,很可能是鼻咽癌致癌因子之一。     

L-Form Induction, Morphology, and Development in Two Related Strains of Erysipelothrix rhusiopathiae

Two related strains of Erysipelothrix rhusiopathiae, one the parent and the other an L-form revertant, were studied for their propensity or ability to produce L-forms under the influence of penicillin. The parent strain produced L-forms in nutrient solid media in an osmolarity range between 0.85 and 5.0% NaCl concentration whereas the revertant strain did so between 0.5 and 3.0% NaCl concentration. When various hyperosmolar media were tried without penicillin, recovery of L-forms from the revertant strain was optimal at a salt concentration of 2.0%, whereas the parent strain occasionally produced a few L-forms on 3.0% salt medium only. The process of penicillin-induced transformation from bacteria to L-form followed an unusual morphological sequence, beginning with beading of the bacterial body, followed by disintegration into granules from which the L-form colony derived. No large bodies were seen during the initial process of L-form induction, but they evolved later from the original granules and had the potential to reproduce L-type growth. The spontaneous development of L-forms in hyperosmolar media had a different morphological sequence starting with elongation of the bacteria into filaments which later developed polar and central dilatations from which granules and L-type growth developed. The differences in biological behavior between these related bacterial strains suggest that the revertant strain developed new properties, probably of genetic origin. Consequently, the assumption that L-forms revert to the "parent" bacteria may not always be justified. It can be made only after the biological properties of the parent and the revertant organisms have been properly identified.     

从医院使用的新洁尔灭溶液中分离出细菌L型的报告

本文报道,作者采用高渗和等渗牛肉汤试管及平板培养法,从某医院正在使用的新洁尔灭器械浸泡液中分离出4株细菌L型,其中金黄色葡萄球菌L型1株,表皮葡萄球菌L型2株,类白喉杆菌L型1株。上述细菌经形态观察,细胞壁染色,返祖鉴定等一系列细菌L型鉴定程序;并通过电镜观察,菌体细胞图像分析。其结果均提示,细菌L型与原菌之间存在明显差异。作者认为,新洁尔灭器械浸泡液,消毒灭菌的不彻底性,是造成术后感染的重要因素。     

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.     

Stable L-forms of Clostridium perfringens and their growth on glass surfaces.

L-forms of Clostridium perfringens were induced in brain heart infusion broth containing 10% sucrose and 2 units of penicillin. After a few hours of growth, spheroplasts, granules, and elongated bacilli were apparent. At 24-h intervals, serial subcultures were made in the above medium which resulted in a culture composed entirely of spheroplasts (or protoplasts) and granules. Upon the withdrawal of penicillin these L-form cultures grew well and, after 100 passages, there was no reversion to the bacillary form. Sucrose could also be withdrawn from the medium. The effects of centrifugation, osmotic stabilizer, ultraviolet light, temperature, pH, and lyophilization upon stable L-forms were examined. L-forms were found to attach to the walls of culture tubes during trowth and sheets of L-form growth were obtained on cover slips in Leighton tubes and on the sides of medicine bottles.     

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.     

Filterability of Streptococcal L-Forms

The filterability of the broth-grown stable L-form derived from Streptococcus faecium F24 was tested by filtration under the influence of varying amounts of applied pressure. A decrease in the pore size of the filter resulted in a corresponding decrease in viable count, but no major effect was noted due to the different pressures applied. Serial filtration of a deoxyribonuclease-treated L-form culture in the mid-logarithmic phase of growth resulted in recovery of viable L-forms from the 0.45-mum filtrate but not from the 0.22-mum filtrate. It is possible that disruption of the L-form bodies with release of small viable elements had occurred. Protoplasts, diluted in an osmotic stabilizer, were filtered similarly; L-forms could be grown from the filtrate passing through the filters of 0.45 mum or greater. Filtration of the parent streptococci gave rise to streptococcal colonies from the 1.2-mum filtrate only.     

Induction of Enterococcal L-Forms by the Action of Lysozyme

Suspensions of enterococci were treated with lysozyme in the presence of osmotic stabilizers. The resulting osmotically fragile bodies prepared from Streptococcus faecium strain F24 and S. faecalis strain E1 gave rise to L-forms under optimal osmotic and nutritional conditions for treatment and subsequent growth. The most critical component of the growth medium, to obtain maximum yields, was the nature and concentration of the added salt. The two most effective salts were sodium chloride and ammonium chloride in the range of 2 to 3% (w/v) added to a suitable agar base. Ammonium chloride was more versatile, because it could be used with either sucrose or polyethylene glycol 4000 as the osmotic stabilizer for preparation and dilution of the osmotically fragile bodies. Sodium chloride would not consistently support growth of S. faecium F24 as L-forms when polyethylene glycol 4000 was used as the osmotic stabilizer during lysozyme treatment. Time-course studies of concurrent cell wall removal and L-form induction suggested that maximal induction required only cell wall damage rather than complete wall removal. This method for induction of L-forms from a suspension of enterococci is a significant improvement over other presently known methods.     

金黄色萄葡球菌L型特异性抗体的免疫组化试验在妇科感染性疾病诊断上的应用

本文对49例慢性子宫内膜炎、慢性宫颈炎等妇科疾病的病理切片,用金黄色葡萄球菌细菌型及L型的特异性杭体作酶免疫组化染色(PAP法)证明在金黄色葡萄球菌引起的各种妇科感染中,细菌型及L型混合感染占60％,单独细菌型感染占28.9％,单独L型感染占11.1％。本研究证明了金葡菌变异为L型后,有特异性杭原出现,并证明细菌L型的特异性抗原有一定的免疫学诊断价值。