Determination of antibodies to Streptococcus group A polysaccharide by an immunodiffusion method in human sera in various pathological processes of streptococcal etiology

A method for the assay of antibodies to the specific antigenic determinant of group A streptococcal polysaccharide (A-polysaccharide) in human sera was developed. The sera were tested in the precipitation test in agar gel with different doses of A-polysaccharide. The presence of a high level of the above-mentioned antibodies is indicative of infection caused by group A streptococcus, but not streptococci of other groups or by the L-forms of streptococci. In 87.5% of patients with primary rheumatism a high level of antibodies to the specific antigenic determinant of A-polysaccharide was detected during the first day of the disease, which confirms most convincingly the etiological role of group A streptococcus in rheumatism. Considerable differences in the level of antibodies to A-polysaccharide in the active and non-active phases of rheumatism have been established, which makes it possible to use the presence of a high level of these antibodies as an indicator of the rheumatic process activity. A considerable percentage of sera with a high level of antibodies to A-polysaccharide was also detected in erysipelas and acute glomerulonephritis patients.     

Further study of the L forms of Streptococci in blood cultures from the patients with rheumatic fever depending on the course of the disease and the methods of cultivation]

Cultures of blood from patients with rheumatism during the active and inactive phases of the disease, treated and untreated with antibiotics were studied. The following results were obtained in blood cultures (on Klodnisky's medium with a 0.01% cystine) of 105 patients with rheumatism: L-forms of streptococcus were isolated in the 88 cases, irrespective of the severity of the disease or the treatment, streptococcus--in 8, and the result proved to be negative in 9 cases. Hemoculture on broth with a 1% glucose solution proved to be positive (streptococcus) in one patient only. The constancy with which the L-forms of streptococcus were isolated in hemocultures from the patients with rheumatism and also the peculiarity of their pathogenic properties modelling the rheumatism under experimental conditions pointed to their role in the pathogenesis of rheumatism. A method of isolation of hemocultures on Klodnitsky's medium with a 0.01% cystine can be recommended for the diagnosis of a septic condition caused by the L-forms of streptococcus in patients suffering from rheumatism.     

Ultrastructure of the L forms of Streptococcus group B

The submicroscopic organization of the L-forms of beta-hemolytic streptococci of group B has been studied in the course of their cultivation. The L-forms of group B streptococci differ from those of group A streptococci by a higher growth rate. On the submicroscopic level, the activity of ATP-ase has been revealed on the internal side of the cytoplasmic membrane. Regularities in the localization of intramembranous particles sized 6-18 nm in the hydrophobic area of the membrane have been established by means of freezing-etching. With the adequate methods of fixation, the continuous three-layer structure of the cytoplasmic membrane can be determined in all elements of the L-form population.     

Identification with the aid of various serological reactions of L forms of streptococci isolated from the live organism]

Growth inhibition, agglutination, precipitation, and passive hemagglutination tests were used for the identification of the L-forms of streptococci isolated from the organism of experimental rabbits both after the infection with the L-forms of streptococci and with the streptococci of group A. The tests were positive not only with the antiserum of homologous, but also of heterologous strains of the L-form of streptococcus, group A. The L-form cultures isolated from the experimental animals failed to differ from the laboratory strain of the L-forms of streptococcus, group A, by serological properties.     

Comparative biological characteristics of the L-form of Streptococcus group A and B

The comparative study of the biological and serological properties of the L-forms of streptococci, groups A and B, has been made. Their morphological similarity on the level of light microscopy has been demonstrated. The use of ring precipitation, gel diffusion, passive hemagglutination, aggregate hemagglutination, as well as the immunoferritin technique, has made it possible to establish the presence of specific antigens in the L-forms of streptococci, groups A and B. Serological cross reactions are negligible. The future development of a diagnosticum for the specific indication of these antigens is proposed. The fact of the presence of specific antigens in the L-forms of streptococci in comparison with the initial streptococcal strains has been confirmed.     

Microbial associations in chronic respiratory tract diseases

In the peripheral blood of patients with chronic bronchitis the presence of Mycobacterium pneumoniae antigens has been registered in 20.0% of cases and the presence of group A Streptococcus haemolyticus antigens, in 24.0% of cases, the transformation of streptococci into the L-form being observed in 12.0% of cases. The presence of M. pneumoniae, streptococci and their L-forms, as well as associations of these microorganisms, is characteristic of patients with chronic obstructive bronchitis, which is, probably, one of the reasons for the maintenance of bronchial obstruction. In patients with chronic pneumonia and fibrosing alveolitis the antigens of the bacterial form of streptococcus and its L-forms have been detected only in a few cases.     

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.     

Antigenic characteristics of the L forms of Streptococcus group B

Stable L-forms of group B streptococci (GBS) have been obtained and their antigenic features have been studied by the serological methods (the passive hemagglutination test, the aggregate agglutination test, the gel diffusion test), as well as by using ferritin and peroxidase labels with the subsequent electron microscopy. The use of the serological methods has made it possible to reveal the antigenic differences between the stable L-forms of GBS and their bacterial forms. Specific antigenic substances can be found in the supernatant fluid obtained after the sedimentation of the ultrasonically disintegrated cellular mass of streptococcal L-forms and bacterial cultures. The use of ferritin and peroxidase labels has revealed the specificity of GBS L-form antigen and its localization on the cytoplasmic membrane of all L-form structural elements.     

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.     

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.     

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.     

Preliminary results of a bacteriologic study of the blood and urine of children with nephritis]

Single examination of the urine and blood by the method described in this paper was carried out in 14 children suffering from glomerulonephritis. L-form cultures were isolated in 11 cases. The cultures were isolated both at the active phase and during the remission. Three L-form cultures reverted into streptococci spontaneously. The revertants showed a sharp difference from the L-forms by the cell morphology and also by the colour and morphology of the colonies.     

L-forms of bacteria in the urine of patients with chronic pyelonephritis]

The authors present the data concerning the study of 200 patients suffering from chronic pyelonephritis; in 28 of these L-forms of bacteria were revealed in the urine. Of 46 L-cultures isolated from these patients 13 reversed into bacterial forms, 8 failed to reverse and were referred to the stable L-forms; the rest 25 L-cultures perished during the 8th--10th passage. This led to a supposition that the relapses and exacerbations of the infectious process in pyelonephritis were associated with the change of the L-forms into bacterial ones, and that the persistence of the L-forms in the kidney tissue promoted the maintenance of the chronic process.     

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.     

Isolation of L-Forms in a Clinical Microbiology Laboratory

Previous studies have demonstrated that L-forms of bacteria may play a role in persistent, chronic, or recurrent urinary-tract infections. A 2-year program was initiated to determine the feasibility of culturing for L-forms on a routine basis, and to determine the effectiveness of such a program. In relation to the total number of specimens, few L-forms were actually isolated. In comparison with the amount of equipment and technician time required, the return was negligible; only 0.5% of all urine specimens were positive for L-forms. An increase to only 1.2% was noted when culturing for L-forms was limited to patients with a diagnosis of bacteriuria or pyelonephritis. It is recommended that this technique be reserved for those patients with a long history of recurrent urinary-tract infections, after other attempts to cure the patient have met with failure.     

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。     

Role of anaerobic streptococci in human pathology. Report I. Etiological significance of anaerobic streptococci in chronic tonsillitis]

The authors describe a method of isolation of anaerobic streptococci from the blood. A high seeding efficiency of anaerobic streptococci from the depth of the tonsils (75.1%) and from their surface (59.7%) and also from the blood (36%) pointed to the leading role of these microbes in the etiopathogenesis of chronic tonsillitis. In healthy individuals anaerobic streptococci are revealed (by culture method) on the surface of the tonsils but rarely (6%). Str. micros are the most incident in chronic tonsillitis (in 85.3% of cases-from the depth of the tonsils and in 77.5%-from the surface), whereas Str. anaerobius and Str. intermedius occur in individual cases only (8.4-7.5% and 6.3-15%, respectively). Pure culture of Str. micros was found in 36% of cases. Pathogenetic scheme of development of bacteriemia in chronic tonsillitis and rheumatism is presented. The isolated cultures were found to be resistant to the widely-used antibiotics.     

慢性肾盂肾炎L型细菌感染及耐药分析

目的了解L型细菌在慢性肾盂肾炎的感染及耐药状况。方法对71例患者清洁中段尿做普通细菌培养(B型)、L型细菌培养(L型)及耐药分析。结果细菌阳性率为77.5%,其中单独L型阳性率为49.3%、B型与L型混合感染为15.5%,而B型阳性率仅为12.7%。主要是大肠埃希菌,其次是葡萄球菌;青霉素及头孢噻肟均有较高的耐药率(88.9%及73.6%)。结论L型细菌在慢性肾盂肾炎感染中占主导,β-内酰胺类药物有较高的耐药性,临床治疗应据药敏结果合理选择及时调整抗生素。     

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。     

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.