Charles H. Zierdt

Blastocystis hominis: Pathogenic potential in human patients and in gnotobiotes

Abstract Germfree guinea pigs were inoculated orally, in some experiments, and intracecally, in others, with Blastocystis hominis and the enteric flora from symptomatic patients. Other germfree guinea pigs received the parasite from axenic culture and still others from monoxenic culture with Proteus vulgaris . Fourteen of 43 animals inoculated orally with B. hominis and patients enteric flora developed B. hominis infections and those with particularly heavy infections developed watery diarrhea of more than 1 weeks duration immediately prior to sacrifice. Similar results were obtained from intracecal inoculations in that 13 of 28 animals developed infections and those with the greatest numbers of B. hominis had watery diarrhea for more than 1 week prior to sacrifice. Gross pathologic changes in these animals were mostly unremarkable, with only a slight hyperemia observed in several of the symptomatic animals. Microscopic examination, however, revealed frequent penetration of intestinal epithelium by B. hominis and the parasites in significant numbers were observed within the epithelium. There was a slight increase in cellularity in the lamina propria but parasites were not observed therein and their presence in the epithelium did not provoke an inflammatory response. Only one of eight animals inoculated from monoxenic cultures developed B. hominis infection (asymptomatic), and infections were not produced in animals inoculated from axenic culture. As a result of our observations of diarrhea in patients with particularly heavy infections with B. hominis together with the demonstration of similar symptoms in animals heavily infected with this parasite, we believe B. hominis may occasionally be related causally to the production of such symptoms by a mechanism not completely understandable.

Ultrastructure and light microscope appearance of Blastocystis hominis in a patient with enteric disease.

SummaryThe patient reported had a fulminant, refractory diarrhea of unknown etiology producing about 81 of diarrheal fluid daily which required continously large volumes of intravenous fluid therapy. No protozoon other thanBlastocystic hominis was present. Bacterial counts were very low in the fecal material because of continuous antibiotic therapy.Blastocystic hominis was present in large numbers, averaging for a 5-day period, 8.3×106/ml of diarrheal fluid. Treatment with metronidazole for 12 days had no effect on either the diarrhea or the numbers ofB. hominis present. The patient died from aspiration pneumonia without a firm diagnosis of his underlying disease. Identification of an unusual form ofB. hominis seen only in diarrheal fluid was confirmed through cultivation, specific fluoresecent antibody staining, as well as by transmission electron microscopy and light microscopy using hematoxylin staining and Nomarski interference contrast.The most significant result of this study is the description of ultrastructure of the in vivo trophozoite form ofB. hominis. The persistence of certain morphologic features of the in vivo and in vitro forms ofB. hominis is described. In vivo,B. hominis has a larger nucleus than in vitro, more prominent, well structured nculeoli and hundreds of mitochondria with numerous delicate saccular cristae in a clear electron-lucent matrix and complex internal structure. Many cytoplasmic vesicles lined with ribosomes are present. The in vivo structure differs from the granular culture form ofB. hominis, which has a smaller nucleus (nuclei), no distinct nucleoli, mitochondria characterized by a uniform structureless electron dense matrix, and few cytoplasmic, ribosome-fined vesicles. A feature retained in both in vivo and in vitroB. hominis is a distinct crescentic band of nuclear chromatin.

Blastocystis hominis: Axenic cultivation

Abstract Blastocystis hominis, an intestinal parasite of humans, had been previously grown only with benefit of a bacterial flora. Bacteria were eliminated in the presence of 4000 μg/ml of ampicillin and 1000 μg/ml of streptomycin. Amphotericin B (50 μg/ml) was added only to eliminate yeasts or filamentous fungi. Blastocystis hominis was found to be a strict anaerobe. It was essential to use a prereduced modified biphasic egg medium, and cultures were incubated in anaerobic jars. The bacterial flora of the conventional cultures was eliminated gradually, over a months time, and 6–10 transfers. Two lines of each of 8 strains of axenized B. hominis have been transferred weekly for 2 yr, one with antibiotics and one without. Cultures for bacteria, mycoplasma, and L forms have remained negative. It is now possible to study pure cultures of B. hominis without the previously essential bacterial flora.

Ultrastructure of blastocystis hominis

SummaryUltrastructural studies of modified Boeck-Drbohlav egg slant cultured Blastocystis hominis reveal ameba, granular, and vacuolated forms of this organism. The ameba form shows a thick filamentous surface layer, a less conspicuous solid central body than the granular and vacuolated forms and very large mitochondria. The granular form differs from the ameba form by a granular transformation of the enlarged central body (vacuole). Multiple prominent nuclei and nucleoli and a well developed Golgi apparatus and endoplasmic reticulum are present in the cytoplasmic layer surrounding the central body. The granules within the central body are of three types judged by size, shape, and electron density. One of these is believed to be a reproductive granule. The vacuolated form has a large, membrane-bounded central body displacing all other organelles against the cell membrane. Small granules of many morphological types are present within the developing central body (vacuole). The usual division by binary fission may be concurrent with central body maturation.

STUDY OF THE ANAEROBIC CORYNEBACTERIA

ABSTRACT Reduction in number of anaerobic Corynebacterium species is proposed. C. liquefaciens, C. pyogenes, C. granulosum, C. parvum, C. diphteroides (sic) and C. anaerobium are synonyms of C. acnes (Gilchrist) Eberson 1918 and should be so treated. Evidence is presented separating C. acnes from the genus Propionibacterium where recent proposals have placed it. C. acnes NCTC 737, ATCC 6919 is proposed as neotype strain.

Endosymbiosis in Blastocystis hominis

Abstract Examination of eight strains of axenically grown Blastocystis hominis by Nomarski interference optics revealed the presence in all strains of intracellular bacterialike spheres and rods, which were named alpha. These structures were confirmed by transmission (TEM) and freeze fracture (FEM) electron microscopy. The endosymbiont was spherical to rod-shaped. A limiting membrane was present, but never a cell wall. Alpha could not be grown outside of the B. hominis cell. There was a direct relationship of increasing endosymbiont numbers and B. hominis cell size. Cells containing hundreds of alpha were from 80 to 200 μm in diameter. Conventional B. hominis cells (4–7 μm) contained few or no endosymbiont. B. hominis in fecal specimens contained the endosymbiont.

Freeze-etch studies of the granular and vacuolated forms of Blastocystis hominis

SummaryFreeze-etch studies of modified Blastocystis hominis granular and vacuolated forms cultivated in Boeck-Drbohlav egg slants reveal marked structural differences between the inner (covering central body) and outer cytoplasmic membranes. The outer cytoplasmic membrane has pores, approximately 500 Å (50 nm) in diameter, which are evenly distributed throughout the membrane surface. The inner cytoplasmic membrane possesses intramembranous particles and indentations. The indentations are distributed in a pattern similar to outer membrane pores. Outer and inner membranes may communicate directly by means of the pore-indentation system. The nucleus is delimited by two membranes. The outer nuclear membrane contains intramembranous particles which are twice as numerous as those of the inner nuclear membrane. The individual features of the central body, cytoplasmic organelles, and general shape of B. hominis observed by thin section electron microscopy have been confirmed by freeze-etch electron microscopy.

Comparative analysis of lipid composition in axenic strains of Blastocystis hominis

1. Six axenic strains of Blastocystis hominis varied in content of lipids from 12 to 43 pg total lipid/cell. With all strains, phospholipid content was about 39% of total lipids. 2. Neutral lipid fractions of B. hominis were resolved into nine constituents, of which seven were identified tentatively. Sterol esters, principally esters of cholesterol, were the major neutral lipid constituent, accounting for 49-63% of the neutral lipids, and at least 30% of the total lipids. 3. Polar lipids were resolved into eleven constituents, of which nine were identified tentatively. Phosphatidylcholine was the major polar lipid constituent of all strains, accounting for 53-63% of the polar lipids, and about 22% of the total lipids.

Cyniclomyces guttulatus (Saccharomycopsis guttulata) — culture, ultrastructure and physiology

Organisms that form an essential extra inner lining of selected areas of the stomach mucosa occur in mice, rats and some other animals. The yeast Cyniclomyces guttulatus (Saccharomycopsis guttulata) was shown in this study to line the stomach of domestic and feral rabbits, guinea pigs, and chinchillas. The layer of yeast cells formed a loose barrier between lumen contents and mucosal surface. A rapid rate of multiplication in the stomach provided yeast cells that blended in with stomach lumen contents, passed throught the gut, and were finally excreted in large numbers in fecal pellets. Ascospore formation occurred during passage through the large intestine. The layer of yeast cells lining the stomach had no evident salubrious nor deleterious effect on the animal. C. guttulatus grew rapidly from stomach contents or single fecal pellets in a new enriched semisolid medium. Growth was good at pH 1 through 8 on the solidified enriched medium. A very unusual characteristic of C. guttulatus is optimal growht at 38° C, and growth at 42° C, with failure to grow below 30° C. TEM demonstrated a very thick, laminated cell wall which had a thick, filamentous external coating. There were mitochondria, polyribosomes, lipid droplets, and an unusually large central nucleus. The developing spore nucleus became extremely electron dense and encapsulated, along with condensed mitochondria, ribosomes, short membrane sections and other organelles, in a dense lamellar covering.

Autolytic nature of iridescent lysis in Pseudomonas aeruginosa.

Attempts to demonstrate a filterable agent to be the cause of iridescent lysis inPseudomonas aeruginosa were uniformly negative. It was not possible to transmit the principle by needle transfer from iridescent plaques to non-iridescent cultures, and plaques produced byP. aeruginosa bacteriophages were never iridescent. Iridescent lysis and bacteriophage lysis were subjected to antibiotics, anti-metabolites, agar at different pH values, antisera to bacteriophage-lysed and to iridescent-lysed bacteria, different oxygen concentrations, and to different nutritional sources. Certain antibiotics, notably tetracycline, streptomycin and polymyxin inducedde novo or enhanced formation of metallic lysis in nutrient agar surface cultures ofP. aeruginosa. Bacteriophage was not induced. Antimetabolites of amino acids, carbohydrates and vitamins inhibited iridescence, or inhibited it at high concentrations and enhanced it at low concentrations. Bacteriophage action was unaffected. Metallic lysis was completely inhibited at pH 6.0; it was inhibited on media containing dye or bile salt and at lowered oxygen concentrations. Bacteriophage action was not affected under these conditions. Antisera to iridescent lysates and to bacteriophage lysates ofP. aeruginosa were tested. Phage antiserum strongly neutralized phage lysis but had no effect on iridescent lysis; antisera to iridescent lysates had no effect on either. No evidence for phage mediation of iridescent lysis was seen in any of the experiments. Iridescent lysis ofP. aeruginosa was demonstrated to be based on metabolic autolysis.