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.     

Study of the L-transforming action on Brucella cells of tetracycline, streptomycin, rifampicin and their combinations with penicillin in in vitro experiments]

The in vitro study of the possible formation of L-forms of Brucella under the effect of tetracycline, streptomycin, rifampicin or their combinations with penicillin showed that passages of various Brucella species on media containing increasing concentrations of the antibiotics resulted in insignificant morphological changes in the cells. Passages of the same cultures on media containing combinations of the above antibiotics with penicillin resulted in more significant changes in the cell morphology, up to formation of spheroplasts. No L-transformation was observed under the experimental conditions.     

Electron Microscopy of Tissue Culture Cells Infected with Brucella abortus

Thin sections of hamster kidney tissue cultures were examined by electron microscopy over a 7-day period after infection with Brucella abortus 3183. Numerous bacteria and structures resembling L-forms were present both intracellularly and extracellularly after the first 24 hr of infection. Most intracellular microorganisms were enclosed by a cytoplasmic membrane, but in a few instances no limiting membrane was detected. After 4 to 7 days, fewer microorganisms were present, and most normal-appearing bacteria were intracellular, particularly in antibiotic-treated cultures. Structures typical of Brucella L-forms were extracellular at the latter time intervals. Several structures were observed in cells from infected cultures whose relationship to the infecting organisms is not known. These consisted of various membranous structures within cytoplasmic vacuoles, myelin-like structures surrounding occasional intracellular organisms, and small bodies present within vacuoles and extracellularly. The latter structures observed throughout the experimental period appeared to occur more frequently as the duration of the infection increased.     

The process of the persistence of Salmonella typhi L forms in the body of experimental animals studied by immunoradiometric analysis

The use of the immunoradiometric assay made it possible to reveal a prolonged persistence of the antigen of viable cultures of S. typhi stable L-forms, as well as to study the dynamics of its spread in the body of experimental animals. After both subconjunctival and intraperitoneal infection of guinea pigs the antigen of S. typhi L-forms spread slowly in the body of experimental animals with its localization first in the lymphoid formations in the pharynx and the intestine and subsequent undulatory accumulation in the marrow, spleen and bile. The persistence of the antigen of live S. typhi L-forms lasted as long as 6 months (the term of observation); killed L-forms could be detected for not more than 17 days. Regular inoculations of samples from different organs into media for the cultivation of S. typhi bacterial and L-forms yielded no positive results, which showed the difficulty of obtaining stable L-forms and evidenced the absence of their reversion in the body of experimental animals.     

Processes of bacterial L transformation and L form reversion in the body of argasid ticks

The experimental infection of tampan ticks (Ornithodoros moubata) with the bacterial cultures of Listeria monocytogenes and Salmonella typhimurium, as well as with their L-forms, was carried out. These experiments demonstrated that both the L-transformation of bacteria and the reversion of their L-forms into the initial bacterial culture could occur in the body of the ticks.     

Yersinia pestis L-forms in rodents and ectoparasites

Y. pestis L-forms and bacterial forms persist in the body of great gerbils for 40 days. L-forms are poorly phagocytized and can persist in phagocytes for a long time. In guinea pigs immunized with vaccine EV, Y. pestis antigen could be detected till day 160. An unstable L-form was isolated from Ornithodoros mites 3 years after their experimental infection with Y. pestis. Bacterial forms persist in mites for 1-3 years. For 5 years Y. pestis antigen is regularly detected in a high percentage of mites.     

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。     

3种革兰氏阴性细菌及其L型内毒素含量的测定

本文采用鲎试剂对大肠杆菌（ＡＴＣＣ２５９２２）、伤寒杆菌、绿脓杆菌（ＡＴＣＣ７８５３）及它们的Ｌ型内毒素的含量进行测定。结果显示细菌型与细菌Ｌ型均具有内毒素,但细菌Ｌ型内毒素含量较细菌型低（约为１／３￣１／２）。因此,认为细菌Ｌ型仍有一定的致病性。     

A comparative evaluation of the pharmacokinetics of different forms of benzathine benzylpenicillin]

Comparative randomized opened pharmacokinetic evaluation of benzathine benzylpenicillin in three dosage forms was performed. Benzathine benzylpenicillin was used as extencilline (2.4 million U or 1.2 million U, "Rh?ne-Poulenc Rorer", France) and as bicillin-5 (1.5 million U, "Synthesis" Russia). 33 patients were included in investigation (23 women and 10 men aged 16-60 years). 25 persons had verified rheumatism without blood circulation failure signs, 4--had chronic tonsillitis and 4 were healthy volunteers. Benzylpenicillin concentration was estimated by microbiology test in blood samples taken at 1, 3, 24 hours and 7, 14 and 21 days after intramuscular drug injection. After 2.4 million U extencilline injection (12 patients) its concentration, was at the inhibition level for beta-hemolytic streptococcus group A (25 ng/ml) for 3 weeks-period in 83.3 per cent of patients. After 1.2 million U extencilline injection (10 patients) or 1.5 million U bicillin-5 injection (12 patients) the above mentioned concentration was achieved on the 21st day in 30 and 0 per cent of patients respectively. Thus the treatment with benzathine benzylpenicillin at the 1.2 million U dose in the form of extencilline or bicillin-5 doesn't provide adequate antistreptococcal concentration in blood in prolonged period and is not suitable for correct rheumatism prophylaxis in adult patients.     

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.     

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.     

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.     

铜绿假单胞菌及L型诱导巨噬细胞凋亡的实验研究

目的研究铜绿假单胞菌（PA）及L型诱导巨噬细胞凋亡的能力,比较二者的差异。方法用生物素断端标记（TUNEL）法检测PA及L型感染巨噬细胞2、4、8、12、16和20h后各时间段的细胞凋亡率,Giemsa染色观察细胞凋亡情况,硝酸还原酶法检测培养液中一氧化氮（NO）的浓度变化。结果 PA及L型能诱导巨噬细胞发生凋亡,与对照组比较差异有统计学意义（P〈0.05）;L型诱导细胞的凋亡率弱于原菌（P〈0.05）;PA及L型感染组培养液NO浓度较对照组明显升高（P〈0.05）。结论 PA及L型可诱导巨噬细胞发生凋亡,L型较其原型诱导细胞凋亡的能力弱,NO可能在巨噬细胞凋亡中发挥一定作用。PA及L型可通过诱导巨噬细胞凋亡,发挥致病作用。     

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.     

大肠杆菌L型生物学特性及致病性的研究

我们对人工诱导的大肠杆菌L型从生物学特性、致病性以及耐药性R质粒传递等方面进行了初步的研究。结果表明:L型菌从菌体形态、菌落特征、生化反应等方面与原型菌有较大差别;原型大肠杆菌和相应的L型菌接合试验均阳性,但后者接合频率较前者明显低;将L型菌经膀胱和尾静脉感染小鼠,引起实验鼠脏器的间质性炎症,通过病理学和细菌学检查证明,L型菌可直接引起脏器感染,并非只有返祖后才能致病。     

Antimicrobial activities of daunorubicin and adriamycin derivatives on bacterial and protoplast type L-form cells of Bacillus subtilis 170, Escherichia coli B, and Proteus mirabilis VI. Structure--activity relationship

The antibacterial activity of ten N-alkylated derivatives of daunorubicin and adriamycin as well as of 5-iminodaunorubicin has been tested by using Bacillus subtilis 170, Escherichia coli B, and Proteus mirabilis VI and their stable protoplast type L-forms in an agar diffusion test. Eight of the substances showed similar activities against B. subtilis and the L-forms of all test organisms, but no activity against the bacterial forms of E. coli and P. mirabilis. The cell wall of these gram-negative bacteria is responsible for this resistance by not allowing the antibiotics to enter the cells. The piperidino compound N-(CH2)5 daunorubicin shows 2-4 times higher activity against B. subtilis and all L-forms in comparison to daunorubicin and the other derivatives. Five of the substances were inactive against all test strains. Their inactivity seems to be associated with the larger substituents at the C-3' position. Relations between molecular structure and activity are discussed considering data about the interaction with DNA and the antitumor activity. Stable protoplast type L-forms and their bacterial forms represent a suitable and effective test system to screen for more effective substances and to get more information about their mode of action.     

Immunoelectrophoretic analysis of the antigenic composition of Salmonella typhi in the process of L transformation and reversion

In the study of the antigenic composition of S. typhi L-forms and their revertants were determined. The stable L-forms were characterized by profound disturbances in the synthesis of Vi- and H-antigens. After reversion to bacterial forms all the revertants under study showed the complete restoration of their bacterial antigenic structure.     

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.     

血平板针尖样及云雾状菌落分离与鉴定

采集98份临床标本接种血平板培养,40份生长出针尖样及云雾状菌落。经分离鉴定38株为细菌L型,其中金黄色葡萄球菌（Staphyloeoccusaureus）居首位,占52．1％（20／38）．其返视性与L型平板分离相比较差。但对抗生素的敏感性则无差别。表明血平板可用于分离细菌L型,并保持了传统L型的特性。     

Validation and development of an immunofluorescence method for isolating and identifying mycobacteria L forms

The complement fixation test and the immunofluorescence test have demonstrated that the L-forms of mycobacteria retain their species-specific and genus-specific determinants and possess serological activity. The L-variants obtained by different methods differ in size, depending on the degree of the destruction of their cell wall. Specific antisera to the L-forms of mycobacteria, suitable for use in the indirect immunofluorescence test, have been obtained. These antisera are highly specific and permit not only the rapid detection, but also identification of the L-forms.