Cell Wall Deficient Forms Research Articles

Atypical behaviour and survival ofStreptococcus pyogenesL forms during intraperitoneal infection in rats

Groups of rats were injected intraperitoneally with cell wall-deficient (L) forms of Streptococcus pyogenes, with their parental (S) forms, as well as with a combined inoculum of both forms (S+L). Peritoneal exudate samples were harvested on days 1, 3, 7, 15 and 30 after challenge and were investigated by microbiological, electron microscopic, cytometric and biochemical methods. Parental S forms were isolated from peritoneal exudate samples up to day 15 post infection, while L form cultures were isolated until the end of the examined interval. Electron microscopic examination revealed continuous adhesion of L forms on the macrophage surface as well as intracellular persistence inside them. It was demonstrated that the intraperitoneal inflammatory response to L form infection was higher than to the other infections and the monocyte-macrophage populations were predominant. The established atypical behaviour and long survival of S. pyogenes L forms in the rat's peritoneum could explain some of the mechanisms of the pathogens' persistence as well as the reasons for chronic streptococcal infections.

Atypical behaviour and survival of Streptococcus pyogenes L forms during intraperitoneal infection in rats

Groups of rats were injected intraperitoneally with cell wall-deficient (L) forms of Streptococcus pyogenes, with their parental (S) forms, as well as with a combined inoculum of both forms (S+L). Peritoneal exudate samples were harvested on days 1, 3, 7, 15 and 30 after challenge and were investigated by microbiological, electron microscopic, cytometric and biochemical methods. Parental S forms were isolated from peritoneal exudate samples up to day 15 post infection, while L form cultures were isolated until the end of the examined interval. Electron microscopic examination revealed continuous adhesion of L forms on the macrophage surface as well as intracellular persistence inside them. It was demonstrated that the intraperitoneal inflammatory response to L form infection was higher than to the other infections and the monocyte-macrophage populations were predominant. The established atypical behaviour and long survival of S. pyogenes L forms in the rat’s peritoneum could explain some of the mechanisms of the pathogens’ persistence as well as the reasons for chronic streptococcal infections.

Update on sarcoidosis.

The local inflammatory response in sarcoidosis appears to be a Th1-mediated process. Evidence exists that the systemic immune response, however, shows a Th2 predominance. Mycobacteria species, particularly cell wall-deficient forms, continue to be candidates for an infectious cause of the disease. Evidence for a possible role for human herpesvirus 8 has also been submitted. The relationship between sarcoidosis and infectious pathogens remains open to investigation. Both Sjögren's syndrome and common variable immunodeficiency display clinical and immunopathologic overlap with sarcoidosis. Blau's syndrome, a genetic disorder of autosomal dominant inheritance, may be confused with infantile sarcoidosis. Vasculitis in sarcoidosis may be underappreciated. Virtually any size vessel may be involved, thus adding to the multiplicity of clinical settings in which sarcoidosis can occur.

Growth of acid fast L forms from the blood of patients with sarcoidosis.

Acid fast cell wall deficient forms (CWDF) of bacteria have been grown from blood, bronchial washings, and ocular anterior chamber fluid from patients with sarcoidosis. A monoclonal antibody raised against Mycobacterium tuberculosis whole cell antigen (H37RV) was used to characterise further CWDF grown from the blood of patients with sarcoidosis. Blood from 20 patients with active sarcoidosis and from 20 controls was cultured using methods favourable for the growth of CWDF. Isolates were further characterised by indirect fluorescent antibody analysis using a monoclonal antibody highly reactive with M tuberculosis. CWDF were grown from the blood of 19 of 20 subjects with sarcoidosis. All isolates stained positively with the monoclonal antibody and with a modified Kinyoun stain. No organisms were grown from the blood of controls. These data demonstrate that CWDF can be grown from the blood of nearly all patients with active sarcoidosis. The results confirm that the organisms are mycobacterial in origin and are similar, if not identical, to M tuberculosis. Their role in the pathogenesis of sarcoidosis is unknown.

Bacillus thermosphaericus sp. nov. a New Thermophilic Ureolytic : Bacillus Isolated from Air

Bacillus -strains representing the dominant thermophilic aerobes in municipal landfill and urban air are described. 16s rDNA sequencing studies showed that these strains represented a distinct lineage with the radiation of the genus Bacillus . The strains were strictly aerobic, growing between 32°C and 64°C, unable to use sugars as source of carbon and energy and formed sphaerical spores in terminally swollen sporangia. The cell morphology varied between cocci, short rods sometimes S-layered, extremely long rods and sphaerical cell wall deficient forms. Liberated spores exhibited hairy appendages. The guanine + cytosine content in DNA was 36 to 37%. and the peptidoglycan type was A4α-lysine-asparagine. The strains shared the peptidoglycan type and GC% with B. sphaericus but were distinguished from this species by 16S rDNA sequencing, the amino acid in the interpeptide bridge of the peptidoglycan, true thermophily and whole cell fatty acid composition. Since the strains exhibited no high similarity to any previously described Bacillus species, the traits are proposed as a new species B. thermosphaericus sp. nov., with P-11 as the type strain.

Protoplast formation, L-colony growth, and regeneration ofClostridium beijerinckii NRRL B-592 and B-593 andClostridium acetobutylicum ATCC 10132

Protocols for protoplast formation, L-colony cultivation, and regeneration ofClostridium beijerinckii NRRL B-592, B-593 andC. acetobutylicum ATCC 10132 were developed. Two osmotically reinforced media were formulated. Protoplasts of B-592, B-593, and ATCC 10132 grew as cell wall-deficient forms (L-colonies) when plated on the first medium (BLM) and continued to do so through at least 3 passages on this medium. The second (BRM) permitted the L-colonies to regenerate cell walls after transfer to this medium. TransferredC. beijerinckii B-592 L-colonies reverted to bacillary colonies at a frequency of 25%. Likewise, L-colonies of B-593 andC. acetobutylicum ATCC 10132 could be regenerated at frequencies of 7.0 and 8.6%, respectively. Thus, these procedures are suitable for genetic engineering of these industrial microorganisms using protoplast manipulation techniques.

Investigation of mycobacteria in Crohn's disease tissue by Southern blotting and DNA hybridisation with cloned mycobacterial genomic DNA probes from a Crohn's disease isolated mycobacteria.

A mycobacterial aetiology for Crohn's disease (CD) has been suggested. Slow growing mycobacteria indistinguishable from M paratuberculosis, the causative agent of enteritis in ruminants (Johne's disease) have been isolated from CD tissues. We have used cloned genomic DNA probes derived from a CD isolated mycobacteria strain Ben, to investigate the presence of mycobacterial DNA sequences in CD tissues. DNA was extracted from total tissue from 17 CD and four control gut specimens. DNA was digested with restriction endonucleases, electrophoresed and transferred to nylon membranes by Southern blotting and hybridised to radiolabelled DNA probes. No mycobacterial DNA was detected in any tissue sample studied. Reconstitution experiments with known numbers of in vitro cultured mycobacteria showed sensitive detection of mycobacterial DNA. DNA extracted from mouse liver, infected with M lepraemurium revealed a strong hybridisation signal and showed the applicability of the experimental approach to the detection of mycobacterial DNA in naturally infected tissues. The results do not provide evidence for the involvement of mycobacteria in the pathogenesis of CD but do not exclude the possibility of low levels of infection in subsets of intestinal cells with spheroplast or cell wall deficient forms of mycobacteria.

Cell-wall deficient bacteria in inflammatory bowel disease.

Cell wall deficient forms of bacteria were isolated from 40-60% of patients with active inflammatory bowel disease, 15-25% of patients with inactive disease and only 5-7% of controls. The recovery rate from Crohn's disease and ulcerative colitis tissue filtrates did not differ significantly, but was raised in both groups in comparison with controls (p less than 0.01). It is concluded that a range of such bacteria have a possible aetiological role in inflammatory bowel disease.

Spheroplastic phase of mycobacteria isolated from patients with Crohn's disease.

Two strains of an unclassified Mycobacterium species were isolated after 18 and 30 months of incubation of media inoculated with resected intestinal tissues from patients with Crohn's disease. These strains represented the third and fourth isolates of this organism from Crohn's disease patients. Ultrastructural examination of this strain and two previously isolated strains revealed the presence of spheroplasts which eventually transformed into the bacillary form of a previously unrecognized Mycobacterium species. These cell wall-deficient forms did not stain with conventional dyes and failed to grow on hypertonic media. Restriction polymorphism of the ribosomal DNA genes was used to determine the relationship between the cell wall-deficient and bacillary forms. Identical restriction patterns of the ribosomal DNA genes were found between the spheroplasts and Mycobacterium sp. isolates with EcoRI, BamHI, and XhoI restriction endonucleases, thus providing definitive evidence of their origin. Unidentified spheroplasts were isolated from an additional 12 patients with Crohn's disease, of which 7 of 10 seroagglutinated with antiserum prepared against the Mycobacterium sp. Spheroplasts were isolated from 16 of 26 (61%) patients with Crohn's disease but not from tissues of 13 patients with ulcerative colitis or 13 patients with other diseases of the bowel. These findings support the role of mycobacteria as etiologic agents in some cases of Crohn's disease.

Histologic observations of pleomorphic, variably acid-fast bacteria in scleroderma, morphea, and lichen sclerosus et atrophicus.

Variably acid-fast coccoid forms, suggestive of cell wall deficient forms of mycobacteria, were observed in the dermis in microscopic sections of skin from six patients with generalized scleroderma, 10 patients with localized scleroderma (morphea), and four patients with lichen sclerosus et atrophicus (LSA). These coccoid forms were found within the collagen bundles, around the adnexae (hair shafts, pilosebaceous units, eccrine glands), and less commonly around the blood vessels and nerves. These coccoid forms may be related to cocci and also to granular coccoid elements of corynebacteria-like coccobacilli, which, on occasion, can be cultured from the skin of these three diseases. The findings in this study support the three-decade old hypothesis concerning the constant association of pleomorphic acid-fast bacteria with scleroderma. The study also suggests that closely related diseases, such as morphea and LSA, are also associated with the presence of similar appearing microbes.

Spheroplast formation of Mycobacterium smegmatis and morphological aspects of their reversion to the bacillary form.

Cell wall-deficient forms (spheroplasts) of Mycobacterium smegmatis strain P53 were prepared by combined treatment with glycine, lysozyme, and lytic enzyme no. 2 as the spheroplasting agents. Quantitative mass conversion to spherical forms was effected by pretreatment of the intact cells with 1.2% glycine in nutrient broth, followed by transfer to spheroplasting medium containing the above agents. Two apparent modes of reversion to the bacillary form were observed under electron microscopy. The first one was initiated by budding from the spheroplasts. The buds gradually elongated to become the mycelial form, which showed branching, septation, and fragmentation. The second resulted from the intracellular formation of tiny cells, possibly the elementary bodies, and their release from the spheroplasts.

Variably Acid‐fast Bacteria in a Rare Case of Coexistent Malignant Lymphoma and Cutaneous Sarcoid‐like Granulomas

A 74-year-old woman presented clinical, laboratory, and histologic evidence of coexistent cutaneous sarcoid-like granulomas, malignant lymphocytic lymphoma, and multiple basal cell carcinomas, all occurring within slightly more than a one-year period. Variably acid-fast coccoid forms were observed in the histologic sections of the cutaneous, noncaseating granulomas; and similar, variably acid-fast, extra and intracellular coccoid forms and granular bodies were seen in the lymph nodes showing lymphoma. The possible relationship between sarcoid-like granulomas, sarcoidosis, and malignancy is discussed, as well as the possible role of cell wall deficient forms (L-forms) of mycobacteria in the pathogenesis of these diseases.

Preparation of protoplasts and whole cell ghosts from Mycobacterium smegmatis.

Cell wall-deficient forms of Mycobacterium smegmatis were produced in growth medium containing D-cycloserine and horse serum. These cells were transformed into protoplasts with EDTA and lysozyme. Subsequent lysis by nucleases followed by osmotic shock produced membrane vesicles (whole cell ghosts).

Antibiotic Treatment of Penicillin-Sensitive Streptococcal Endocarditis

Controversy regarding optimal antimicrobial therapy for penicillin-sensitive streptococcal endocarditis persists. Is penicillin alone adequate? Is an aminoglycoside antibiotic such as streptomycin also required? This controversy is readdressed in two reports in this issue ofThe Journal(pp 1801 and 1807). Their common conclusion is that single-drug therapy with penicillin may be sufficient. In evaluating this conclusion, let us consider (1) the mechanisms of action of the pertinent antibiotics, (2) the results of experimental studies of penicillin-aminoglycoside synergism, (3) similar-clinical reports during the past 35 years, and (4) certain aspects of the natural history of this disease. Penicillin does not enter the bacterial cell. It inhibits the enzymatic formation of pentapeptide bridges cross-linking the peptide side chains of polysaccharide polymers, which constitute the cell wall. Thus, the final stage in bacterial cell-wall synthesis is impaired, making the bacterium more susceptible to lysis.3Penicillin does not, however, act on cell-wall—deficient forms

Penetration and interaction with haemoglobin of Corynebacteria-like microorganisms into erythrocytes in vitro

Following 24 h incubation of normal blood in the presence of the microorganism, the evolution of cell wall deficient forms within the erythrocytes and a process of oxidation of the haemoglobin may be observed.

Infective endocarditis associated with cell wall-deficient bacteria: Electron microscopic findings in four cases

Although cell wall-deficient bacteria have been isolated in vitro from cases of endocarditis, no pathogenic role has been established for these forms in human disease. One criterion difficult to satisfy is the demonstration of these variants in human tissue, and electron microscopic documentation has not been reported. Cardiac valvular vegetations from four cases of endocarditis were examined by electron microscopy because of unusual histologic features of minimal inflammation and organization and small organisms that stained poorly by Gram stain. Although cell wall-complete bacteria were identified in the specimens, each showed the presence of cell wall-deficient forms within the vegetations; these variants predominated in three cases. Since manifestations of infective endocarditis were present in three cases and conventional cultures were negative, the evidence indirectly suggests a pathogenic role for these aberrant bacteria in human disease.

The Whipple Riddle

Whipple disease (intestinal lipodystrophy) is a systemic disorder that involves not only the small bowel and lymph nodes but also the liver, spleen, heart, brain, and other organs. Histopathologically, the disease is characterized by the ubiquitous macrophages that give a positive diastase-resistant PAS staining reaction. Although Whipple disease can be treated with antibiotics, its etiology remains one of the unsolved riddles of medicine, that is, perhaps, until now. Clancy and associates 1 recently reported in the British Medical Journal that a cell wall-deficient form (CWD) of an α-hemolytic Streptococcus was grown from a prolonged monolayer cell culture of a lymph node taken from a patient with Whipple disease. Serological cross reactivity was shown between the organisms and the material within Whipple disease macrophages. In vitro studies characterized the organisms as a facultative intracellular parasite that caused the accumulation within cells of PASpositive material. Moreover, parenteral injection of this CWD organism into

Isolation and characterization of an aetiological agent in Whipple's disease.

A cell wall deficient form of an alpha-haemolytic streptococcus was grown from a prolonged monolayer cell culture of a lymph node taken from a patient with Whipple's disease. Serological cross reactivity was shown between the organism and the material within Whipple's disease macrophages positive for diastase-resistant periodic acid-Schiff (D./P.A.S.). In vitro studies characterized the organism as a facultative intracellular parasite which caused the accumulation within cells of D./P.A.S.-positive material. These results suggest that a pathogenic bacterium is the essential aetiological agent and that the culture of Whipple's disease tissues in hypertonic media may have practical value.

A Cell-Wall Deficient Form of the Pneumococcus in a Case of Pneumonia

The role of cell-wall deficient forms (CWDF) of bacteria in initiating and prolonging or both an infection is controversial. CWDF reverting to Diplococcus pneumoniae were cultured from two blood specimens through use of special laboratory techniques. Though the effect on the CWDF on this patient's unfavorable response to treatment cannot be determined, evidence of the etiology of his pneumonia which eluded physicians was provided. The role of cell-wall deficient forms (CWDF) of bacteria in initiating and prolonging or both an infection is controversial. CWDF reverting to Diplococcus pneumoniae were cultured from two blood specimens through use of special laboratory techniques. Though the effect on the CWDF on this patient's unfavorable response to treatment cannot be determined, evidence of the etiology of his pneumonia which eluded physicians was provided.

Structures suggesting cell-wall-deficient forms detected in circulating erythrocytes by fluorochrome staining.

Cell-wall-deficient (CWD) forms of bacteria are associated with certain cases of idiopathic septicemia. In this preliminary study of blood examined immediately after venipuncture, structures with a morphology characteristic of CWD forms were seen parasitizing the erythrocytes. These inclusions were usually circumferential, but in some cases they protruded from the red cells. The CWD forms were detected by staining with Gould's rhodamine-labeled muramidase, which reacted similarly to acridine orange but with greater specificity. A blocking test, employing unlabeled muramidase, indicated the specificity of the reaction between muramidase and the microbial substrate. Reaction of the forms with muramidase indicates their bacterial, rather than mycoplasmal, nature. Thus in vivo CWD forms have a detectable component of muramic acid, at least in certain cases. Sixty-eight individuals with a diagnosis of fever of unknown origin were tested, with 51 nondebilitated individuals serving as controls. More intraerythrocytic forms reacting with muramidase were found in the patients than in the controls. Nearly 40% of the cases had a relatively high incidence of erythrocyte parasitism. In some instances when freshly drawn blood was examined, the structures, which appear to be microbial, extended in rhizoid filaments from the erythrocytes.