Sarabelle Madoff

The role of pleuropneumonia-like organisms in genitourinary and joint diseases.

THE high incidence of acute joint disease in male patients with positive prostatic cultures, in addition to the knowledge that animals infected with L organisms frequently have arthritis, suggests ...

ISOLATION AND IDENTIFICATION OF PPLO

The first strains of the pleuropneumonia group of organisms were discovered in 1898 by the cultivation of filtered material from bovine pleuropneumonia. While sharing the property of filterability with the viruses, the ability of these organisms to grow on cell-free media did not prevent early workers from classifying them with viral agents. It was not until the late 1930s, after Klienebergerl had noted the association with Streptobacillus moniliformis of organisms resembling those of bovine pleuropneumonia, that interest in this unique group of organisms was stimulated. This 14 form, as she designated it. was isolated and propagated on agar medium. Indeed, in the following years, both saprophytic and pathogenic strains resembling those of bovine pleuropneumonia were isolated on agar media from a variety of sources including man, animals, sewage, soil, and vegetable matter. It soon became apparent that all these strains, in addition to their small size, had other important properties in common. The organisms are very soft and friable, they are pleomorphic, and their size varies from the border line of resolution by light microscopy to several fi in diameter. The conclusion that the L, of S. moniliformis was a variant of the bacillus was based on the demonstration of its reversion to the parent bacillus.2 With the discovery that L forms could be isolated from many other bacteria, the distinction was made between those forms isolated from nature as pleuropneumonialike organisms (PPLO) and those derived from bacteria in the laboratory: the L forms of b a ~ t e r i a . ~ More recently, increasing interest has attracted many new workers to the study of the pleuropneumonialike organisms. However, to the uninitiated observer, not only is the identification of PPLO difficult, but their distinction from L forms may be confusing. In our opinion, the difficulty that the newcomer may encounter in the isolation of these interesting forms lies, principally, in their initial recognition on agar media. It is for this reason that, in presenting the methods used in our laboratory for the study of PPLO, we emphasize their recognition and identification in agar cultures.

Specific, high-affinity binding sites for angiotensin II on Mycoplasma hyorhinis

Mycoplasmataceae are known to express various proteins that are similar to those present in mammals. We report a strain of Mycoplasma hyorhinis isolated from opossum kidney cells with specific, high-affinity binding sites for human angiotensin II (Kd = 5.1 +/- 1.9 nM). In contrast, two strains of M. hominis revealed no specific binding. These binding sites resembled mammalian angiotensin II receptors by their high affinity and by their sensitivity to dithiothreitol. However, they are different from mammalian angiotensin II receptors in that they bind angiotensin I with high affinity (Kd = 1.6 +/- 0.29 nM) but not angiotensin III (Kd approximately 330,000 nM). [125I]-angiotensin II binding was not inhibited by angiotensin receptor subtype antagonists DuP 753 and CGP 42112A but it was sensitive to bacitracin and aprotinin. Positions Asp1, Ile5, His6 and Pro7 were essential for binding to M. hyorhinis as deletion of these residues led to a more than 10,000-fold decrease in affinity.

In vitro sensitivity of some bacteria, their L forms and pleuropneumonia-like organisms to antibiotics.

Summary The sensitivity of PPLO strains of various origin and of several bacteria and their L forms were examined in the presence of 9 antibiotics. The sensitivity of L forms to the various antibiotics with the exception of penicillin was comparable to that of the parent bacterium. However, the L forms of streptococci and of one Vibrio strain were consistently less sensitive to bacitracin than their bacterial forms. All PPLO strains, like the L forms, were highly resistant to penicillin. The sensitivities of the parasitic strains to the various antibiotics were uniform; they were markedly resistant to bacitracin and erythromycin, like some bacteria. The 3 saprophytic strains were noticeably less resistant than the parasitic strains to several antibiotics, especially to erythromycin. The sensitivities of all strains of PPLO, of bacteria and of L forms of these bacteria were on the whole comparable.

Differences between oral and genital strains of human pleuropneumonia-like organisms.

Conclusion and Summary Pleuropneumonia-like strains isolated from the human genitourinary tract and from the buccal cavity, respectively, differ in the appearance of the colonies, in serological properties and, to a certain extent, in nutritional requirements. It is likely that they represent different species. In addition to the easily cultivatable strains, colonies of much smaller size develop occasionally on the plates inoculated from either source. These small colonies retain their identity in transfer. It is uncertain whether they are variants of the usually cultivated strains.

Infection of a traumatic pelvic hematoma with Mycoplasma hominis.

Fever developed in a previously healthy young man who had sustained extensive pelvic trauma. Mycoplasma hominis was isolated in pure culture from six of seven specimens taken from a retroperitoneal hematoma over a one-week period, and mycoplasmacidal antibodies were present in high titer in the convalescent-phase serum. The fever abated after thorough surgical drainage of the infected hematoma.

In vitro detection of Mycoplasma fermentans binding to B-lymphocytes in fresh peripheral blood using flow cytometry

Previous reports have shown, using fluorescent probes conjugated to the organism, that Mycoplasma fermentans fuses with about 12% of peripheral blood lymphocytes. However, no lymphocyte subset was specified. To elucidate the specific subset of lymphocytes involved, we developed a three-color flow cytometric assay to detect M. fermentans binding to fresh peripheral blood cells. In our assay, two strains of M. fermentans were grown in SP4 glucose broth, mixed with fresh whole blood samples (n > 20), and incubated at 37 degrees C. The blood samples were then stained with a polyclonal antibody to M. fermentans, a monoclonal antibody to B-lymphocytes (CD19), and a monoclonal antibody to T-lymphocytes (CD3). Using three-color flow cytometry, we obtained data confirming binding of M. fermentans to 10%-15% of peripheral blood lymphocytes with minimal granulocyte or monocyte staining detected. Flow cytometric analysis showed that early binding appears predominantly directed towards B-lymphocytes (86.7 +/- 9.0%), and that this binding could not be blocked by antibodies directed towards common B lymphocyte cell surface antigens. M. fermentans binding to B-lymphocytes occurred within 5 min of in vitro inoculation, reached a maximum within 30-60 min (94-97%), and thereafter plateaued. The binding was concentration dependent over a three log dilution using 10(3) color changing units as standard. Binding to T-lymphocytes was minimal (<5% positive). B lineage tumor cells or peripheral blood B cells obtained from HIV infected individuals demonstrated reduced binding of M. fermentans. This assay provides a good method to study the cellular interactions of mycoplasma and may help to elucidate pathogenic mechanisms of mycoplasma infections.

Characterization of cytopathogenicity of aquarium seal mycoplasmas and seal finger mycoplasmas by light and scanning electron microscopy

Two recent mycoplasma isolates (one from an aquarium seal and one from a patient with the clinical entity known as seal finger) have been evaluated for cytopathogenicity in tracheal explant cultures. Examinations were performed in direct comparison to Mycoplasma phocidae, an isolate from an epidemic of seal pneumonia along the New England coast (USA) in 1979-1980. The study revealed similar inhibitory effects on ciliated tracheal epithelial cells and clumping of cilia by attached mycoplasmas; no exfoliation of respiratory epithelial cells was observed. In addition, cytopathic effects caused by the mycoplasmas were distinguished from effects of aging in non-infected explants under long-term in-vitro cultivation conditions. The general meaning of mycoplasmas in seals is discussed in detail in this paper.

INDUCTION AND IDENTIFICATION OF L-FORM OF BACTERIA*

We present observations in this paper on the development of L-forms in several groups of bacteria using various inducing agents, and comment on the definition of L-forms and on the evidence necessary for their identification. A study was undertaken to determine the relative tendency of several strains of Group A and Group D streptococcus and of Bacillus species to produce L-forms on exposure to penicillin, glycine and lysozyme. Particular attention was given to the manner in which the L-forms were produced, their morphology and reversion to the parent bacterium. The development of L-forms is similar in all cases: After exposure to the inducing agent, growth of some of the bacteria continues without division. I t consists in increase in the bulk of the cell and extension in all directions. Round and spherical bodies are produced in this way which may be very large compared to the bacteria. Growth of the granules which produce the L-forms is observed only on agar media at a certain stage of the development of the large bodies. The properties of the L-forms and the criteria for their definition are based on the growth of these small granules. L-forms induced by exposure of bacteria to lysozyme have been referred to as “protoplast cultures.”’ Our studies indicate that protoplasts are formed in exceptional strains on exposure to lysozyme. Transferred to agar, some of the protoplasts develop into large bodies, and the subsequent development of these large bodies to L-forms follows the same pattern as with the other inducing agents, In the same strains, exposure to penicillin and glycine induces the formation of large bodies and L-forms without the intermediary stage of protoplasts. Direct transformation of bacteria to large bodies and thence to L-forms is the commonly observed phenomenon. L-cultures obtained with either inducing agent are morphologically similar and the growing elements in the L-colonies d o not correspond to protoplasts. One apparent difference between penicillinand lysozyme-induced L-forms i? that the latter show a greater tendency to revert to the bacterial form. Photographs a to g illustrate the development of L-forms from a strain of Streptococcus faecalis under the influence of lysozyme. This is ATCC strain 9790 received from Dr. G . D. S h o c k ~ n a n . ~ , ~ It produced L-forms in the presence of penicillin and glycine also. Photograph a indicates the protoplasts produced in the cultures exposed to lysozyme in broth containing sucrose. Transferred t o agar, the protoplasts develop into large bodies (photographs b and c) and small granules start to grow out of the large bodies (photograph d ) . The large bodies grow further to irregular forms on the surface of the agar, and the small granules of the L-forms grow into the agar (photographs e and f). The fully developed colonies on the surface of agar are surrounded by large bodies (photograph g). A similar sequence was observed with a strain of B . subtilis exposed to lysozyme. (photographs h-l) . While the streptococcus required an osmotically protective media, this bacillus developed protoplasts and then L-forms on media

Serological Reactions of L Type Cultures isolated from Proteus.

Summary The serological properties of 3 Proteus strains and the L forms isolated from them have been studied. The L antigens were agglutinated both by the bacillary and L immune rabbit sera in a relatively low titre and agglutination occurred in large loose flocci. Clear filtrates of L emulsions gave a good precipitation. The reactions of 2 L type cultures were strain specific. A marked common reaction was present between the bacillary and L forms of 2 strains both in the production of antibodies and in the in vitro tests. There was only a slight overlapping in the reactions of the L forms and of the bacilli in the third strain. The similarity of the serological reactions of the bacillary and L forms of 2 Proteus strains is further evidence for the genetical identity of the L forms and the bacilli. The nature of the serological dissimilarity between the bacillary and L forms of one strain, apparent both in in vitro tests and in antibody production, can not be interpreted at present.