Morphology and Reproductive Processes of the L Forms of Bacteria II. Comparative Study of L Forms and Mycoplasma with the Electron Microscope

Representative electron micrographs, from the study of eight strains of L forms and one strain of Mycoplasma, are presented. A- and B-type L forms were derived from two strains of Proteus, two other L forms were derived from a diphtheroid and from a staphylococcus strain, and two strains (designated as LX) were isolated from L forms derived from a group A beta-hemolytic streptococcus and from a staphylococcus. The Mycoplasma strain was isolated from goats. Sections were made of young colonies grown within agar and from parts of surface colonies embedded in the agar. B-type L colonies of Proteus were produced by inoculation of bacteria into media containing penicillin. The large bodies developing from the bacteria and the organisms in B-type L colonies of Proteus, like the parent bacteria, had a cell wall consisting of a plasma membrane and an outer cell wall. The loss of rigidity in the cell wall indicated an alteration in its structure. The A-type L cultures of Proteus consisted of irregular branching masses extending in several directions, of small dense organisms corresponding to the elementary corpuscles present in cultures of Mycoplasma, and of intermediary forms. In contrast to the B-type, all organisms in the A-type colonies were surrounded by a single unit membrane corresponding to the plasma membrane of bacteria. The structures inside the cell membrane, both in the A- and B-type, seemed to correspond to the structure of the parent bacteria, which contained ribosomes and threads of DNA. The elementary corpuscles formed chains and filaments, and, apparently, these corpuscles took part in the multiplication by gradual enlargement. The organisms seen in the cultures of all L forms and Mycoplasma studied, except in the B-type L forms of Proteus, corresponded in size, shape, and structure, as well as in the development of elementary corpuscles, to the organisms in the A-type L form of Proteus. In contrast to the spherical organisms usually seen in broth cultures, the organisms in young cultures of Mycoplasma, which were grown within the agar, were similar in morphology, as well as in the discernible structure of the organisms, to L forms. Significant morphological and structural differences were not apparent between the L forms and Mycoplasma (in cultures grown within agar media) under the conditions of this investigation.     

An Unclassified Gram-Negative Rod Isolated From the Pharynx on Thayer-Martin Medium (Selective Agar)

An oxidase-positive, small gram-negative rod was isolated on Thayer-Martin medium (TM) inoculated with pharyngeal swabs obtained during surveys to detect Neisseria carriers. In one survey, this organism was isolated from 48% of the subjects, and 50 or more colonies were present on the majority of the primary isolation plates. Other characteristics of the organism, which has been given the provisional designation "TM-1," include: delayed production (2 to 10 days) of acid from glucose, formation of gas during nitrate reduction, and the frequent formation of "pits" in the agar surface. On TM, nonpitting colonies of TM-1 are morphologically similar to colonies of Neisseria gonorrhoeae and N. lactamica. Comparison of the characteristics of TM-1 strains with other similar fastidious gram-negative organisms encountered in clinical laboratories indicates that TM-1 is a distinct species. Further studies are required before proper taxonomic placement can be made.     

紫穗槐根瘤菌的分离与回接鉴定

采用YMA培养基对新鲜的紫穗槐(Amorpha fruticosa L.)根瘤提取物进行根瘤菌的分离培养,获得的菌落为圆形乳白色,表面凸起,直径0.4~0.5 cm,革兰反应阴性,显微观察显示为短杆状,分离菌株回接原宿主植物,同时接种于经60℃15 min水浴加热处理的紫穗槐种子,蛭石盆栽实验显示,接种此菌的紫穗槐植株都有结瘤,并与对照在结瘤数、瘤重、植株鲜重与干重间都有显著差异,实验证明所分离的菌株均为紫穗槐根瘤菌。     

Isolation and characterization of fecal bacteria capable of 16 alpha-dehydroxylating corticoids.

For more than a decade it has been known that the fecal flora of humans and rats includes organisms capable of 16 alpha-dehydroxylating corticoids, but their identity has remained unknown. To isolate these organisms, Mueller-Hinton agar plates were seeded with fresh feces from Proteus-free rats and incubated anaerobically. On an average, 1 of every 35 colonies consisted of organisms synthesizing 16 alpha-dehydroxylase. Isolation of the individual colonies yielded two obligate anerobes, strains 144 and 146, which elaborated the enzyme. The steroid transformation could be attained by the microbial culture alone in prereduced media or in aerobic media in the presence of Escherichia coli. Although both strains were phenotypically similar to Eubacterium lentum, they differed between themselves in their enzymatic equipment.     

Mesophilic aerobic Gram negative cellulose degrading bacteria from aquatic habitats and soils

New procedures have been developed for the isolation and purification of aerobic and facultatively anaerobic bacteria able to utilize cellulose as sole source of carbon and energy. Wood pulp medium was used for enrichment, and bacterial cellulose, obtained from cultures of Acetobacter aceti subsp. xylinus , was employed as carbon substrate during purification and for the rapid screening of colonies for cellulolytic activity. The methods have revealed several new groups of Gram negative cellulose-degrading bacteria, including organisms that form differentiated colonies superficially similar to myxobacterial sori. The organisms formed several phenetic clusters, three of which contained reference strains of Cellvibrio fulvus, Pseudomonas fluorescens var. cellulosa and Cytophaga hutchinsonii . No cellulose degrading cluster included non-cellulose degrading strains. Most of the cellulose degraders studied were flagellated and, of these, the majority had polar or lophotrichous flagella, although one cluster included peritrichously flagellated organisms. The cellulose degraders in this study included five organisms that grew on nitrate-free medium; these appeared in two different clusters. A few Gram positive isolates appeared to belong to the genera Streptomyces and Thermoactinomyces .     

Microbes from apiarian sources: Bacillus spp. in frass of the greater wax moth

Frass from the greater wax moth, Galleria mellonella, obtained from feral colonies of honey bees, Apis mellifera; from managed honey bee colonies; and from a laboratory culture of the wax moth was sampled for aerobic Gram-positive spore-forming rods. One hundred eighty-five strains belonging to the genus Bacillus were isolated, and most were identified. One hundred and three of the isolates were from frass from the wax moth culture, 61 were from frass from managed honey bee colonies, and 21 were from frass from feral honey bee colonies. The species most frequently isolated varied with the source. Fifty-eight isolates from frass from managed honey bee colonies were B. cereus which was isolated from this source only, but B. sphaericus was the most frequent isolate from frass from the wax moth culture. Bacillus megaterium and organisms belonging to the B. alvei-B. thiaminolyticus spectrum were the most frequent isolates from frass from feral honey bee colonies. Most strains isolated produced caprylate esterase-lipase, leucine aminopeptidase, and phosphoamidase. The numbers of isolates, the species, and the enzymatic activity of the strains varied with the source of the frass. In fact, the complete microbial complement varied with the source. These results are discussed in relation to possible roles of Bacillus spp. in the nutrition of the wax moth as well as the microbial ecology of the honey bee colony.     

Immunochemical study of nutritionally variant streptococci

Nutritionally variant streptococci (NVS) have been characterized by their growth as satellite colonies around colonies of staphylococci or several other gram-positive or gram-negative bacterial strains. The majority of the NVS strains were isolated from patients with subacute bacterial endocarditis. Organisms identified as NVS were subdivided into three serotypes by rocket-line electrophoresis and hemagglutination inhibition assays. Ninety-nine of 103 strains expressed one or more of the three serotype antigens; however, a group antigen was not demonstrated in the various extracts of these streptococci. Surface protein studies confirmed the NVS differentiation into serotypes. Serotype I organisms expressed surface protein(s) specific for the serotype, whereas the serotype II and III NVS demonstrated common protein(s) on their surface. Furthermore, SDS extraction released a greater amount of radioiodinatable surface protein from serotypes I and III bacteria than serotype II. Finally, there was no correlation between the serotype or the disease of the patients from which the NVS strains were isolated.     

Pollution indicator bacteria associated with municipal raw and drinking water supplies.

A total of 3819 bacterial cultures isolated from municipal water samples were identified using a combination of Enterotubules and confirmatory media. Frequency distributions for the different genera or groups of bacteria were similar for raw water and drinking water isolations, except for Escherichia organisms which doubled their frequency in raw water. Differences between the membrane filter (MF) and presence-absence (P-A) test with regard to types of organisms isolated were limited to Klebsiella organisms which were preferentially cultured from MF plates. Members of the genus Enterobacter were isolated more than twice as frequently as any of the other coliform genera dealt with in this study. Aeromonas organisms were detected almost as often as such individual genera as Escherichia, Citrobacter, or Klebsiella. Although non-lactose fermenting colonies (false-negatives) of the coliform genera would not be detected by the MF technique, their lack of detection would likely be offset by the Aeromonas colonies (false-positives). At least 25% of the coliform isolates were either anaerogenic or non-lactose fermenters and would therefore go undetected by the most probably number (MPN) technique.     

Diseases affect cold-water corals too: Eunicella verrucosa (Cnidaria: Gorgonacea) necrosis in SW England

The first recorded incidence of cold-water coral disease was noted in Eunicella verrucosa, a coral on the international 'red list' of threatened species, at a marine protected area in SW England in 2002. Video surveys of 634 separate colonies at 13 sites revealed that disease outbreaks were widespread in SW England from 2003 to 2006. Coenchyme became necrotic in diseased specimens, leading to tissue sloughing and exposing skeletal gorgonin to settlement by fouling organisms. Sites where necrosis was found had significantly higher incidences of fouling. No fungi were isolated from diseased or healthy tissue, but significantly higher concentrations of bacteria occurred in diseased specimens. Of 21 distinct bacteria isolated from diseased tissues, 19 were Vibrionaceae, 15 were strains of Vibrio splendidus and 2 others closely matched Vibrio tasmaniensis. Vibrios isolated from E. verrucosa did not induce disease at 15 degrees C, but, at 20 degrees C, controls remained healthy and test gorgonians became diseased, regardless of whether vibrios were isolated from diseased or healthy colonies. Bacteria associated with diseased tissue produced proteolytic and cytolytic enzymes that damaged E. verrucosa tissue and may be responsible for the necrosis observed. Monitoring at the site where the disease was first noted showed new gorgonian recruitment from 2003 to 2006; some individuals had died and become completely overgrown, whereas others had continued to grow around a dead central area.     

Small Colonies of Clostridium sticklandii Resulting from Nitrosoguanidine Treatment and Exhibiting Defects in Catabolic Enzymes

Several strains of Clostridium sticklandii, isolated from small colonies arising after treatment with 1-methyl-3-nitro-1-nitrosoguanidine, exhibited markedly depressed activities of certain catabolic enzyme systems known to provide energy to the organism in the form of adenosine triphosphate. In some of these strains the levels of glycine reductase, the ability to ferment lysine to fatty acids and ammonia, and formate-dependent 2,3-5-triphenyltetrazolium chloride reduction were only 0 to 10% of that of the wild type. Another subgroup of mutants exhibited activities of some of these enzymes from 1.3 to 3 times higher than those of the wild type. Small-colony mutants of an obligate anaerobe, like those of oxygen-utilizing organisms, can therefore be due to defects in one or more of their energy-providing systems. The merits of small-colony formation as an auxiliary marker for the isolation of catabolic mutants are discussed.     

GROWTH, PRIMARY PRODUCTIVITY, AND NITROGEN FIXATION POTENTIAL OF NODULARIA SPP. (CYANOPHYCEAE) IN WATER FROM A SUBTROPICAL ESTUARY IN THE UNITED STATES

Nodularia is a halotolerant, filamentous, dinitrogen-fixing cyanobacterium that forms massive blooms in some coastal oceans, estuaries, and saline lakes worldwide. Although the genus is globally distributed, its blooms are sporadic and appear to be confined to certain water bodies. Blooms are frequently associated with phosphorus enrichment; therefore Nodularia may benefit from increased anthropogenic nutrient loading to coastal waters. We studied the potential for Nodularia to grow in the nitrogen-limited Neuse River Estuary (North Carolina, U.S.A.) with laboratory growth experiments in Neuse River Estuary water and by examining physico-chemical data from the estuary. Analysis of nutrients (nitrogen and phosphorus), salinity, and temperature data from the Neuse River Estuary between 1994 and 1998 revealed that suitable conditions for Nodularia prevailed during the summer of each of these years for time spans ranging from 1.5 to 5 months. Growth of two laboratory strains in Neuse River Estuary water was as fast or slightly slower than in artificial growth medium, as long as the culture inoculum had phosphorus reserves. Phosphorus addition did not stimulate growth of already phosphorus-sufficient inocula. Phosphorus starvation of the inoculum before the experiment decreased growth rates in the estuarine water unless additional phosphorus was supplied. Although phosphorus addition had a stimulatory effect on dinitrogen fixation and productivity, the effect differed for the two Nodularia strains. Results suggest that growth of Nodularia in North Carolinian estuaries is possible, and that such growth would be phosphorus-limited at times. Phosphorus availability may determine the times and locations for potential establishment of Nodularia in this and similar estuarine ecosystems.     

Characterization of Bacteroides Species Isolated From Soft Tissue Infections in Cats

A total of 77 strains of Gram negative anaerobes belonging to the genus Bacteroides and isolated from 60 subcutaneous abscesses and 10 cases of pyothorax in cats, have been examined morphologically and biochemically. Colony pigmentation, gas chromatography and biochemical analysis placed them into two major categories-those which produced pigmented colonies (Group 1) and those which failed to produce pigmented colonies after 14 d on laked blood agar (Group 2). All 29 strains in Group 1 produced acetic, propionic, isobutyric, butyric, isovaleric and succinic acids but failed to ferment carbohydrates, and were classified as B. asaccharolyticus. All organisms in Group 2 produced acetic, propionic, isobutyric, isovaleric and succinic acids and were divided into four categories based on indole production and bile tolerance. Designation to species was then decided on the basis of phenylacetic acid production and sugar fermentation tests. This sequence of analysis of results enabled confident speciation of some groups of these organisms despite some biochemical variation of the cat strains when compared to human type strains.     

Growth characteristics of the slow-growing actinobacterium Frankia sp. strain CcI3 on solid media

Frankia sp. strains are slow-growing Actinobacteria that form N₂-fixing root nodules on certain angiosperm trees and shrubs. These organisms are generally cultured in liquid media as routine growth on solid media is impractical because of lengthy incubation periods. Liquid media is problematic for filamentous organisms because unicellular spores are difficult to isolate, and mutants are difficult to separate. The goal of this study was to optimize Frankia CcI3 growth on solid media. Starting with an appropriate basal medium containing pyruvate as a carbon source, various peptones were individually tested to determine if the time needed for colonies to appear could be reduced. Bacto™ Proteose Peptone #3 at 1.6 mg ml⁻¹ final concentration promoted the best growth with colonies visible after 3 days, compared with the 7- to 10-day incubation required for control unamended media. Gellan gum gave a more rapid growth response as compared with agar. Increased calcium concentrations were required to promote solidification of gellan gum. Sporulation occurred within 2 weeks of plating CcI3 with colonies changing color from cream to reddish-brown. Scanning and transmission electron micrographs revealed the architecture of colonial development. Colonies developed in a lens-shaped manner mainly by penetration into the gel. Older colonies had numerous empty hyphae with sporangia developing near or at the surface and with crystalline material, presumably pigment, interspersed among the hyphae. Occasional vesicles were seen mixed within the colonies. Colonial development of Frankia CcI3 is reminiscent of that described for other members of the filamentous Suborder Frankineae.     

Cell Wall Composition and Associated Properties of Methicillin-Resistant Staphylococcus aureus Strains

Methicillin-resistant (MR) Staphylococcus aureus strains have previously been reported to be deficient in surface negative charge; this has been correlated with methicillin resistance and ascribed to a deficiency of teichoic acid at the cell surface (A. W. Hill and A. M. James, Microbios 6:157-167, 1972). Teichoic acid was present in walls of MR organisms as revealed by appreciable phosphate levels and detection of ribitol residues. Phosphate levels in walls from five MR strains (0.54 to 0.77 mumol/mg of wall) were lower than in three unrelated methicillin-sensitive (MS) strains (0.86 to 1.0 mumol/mg of wall). However, two MS strains derived from two of the MR strains had wall phosphate levels very similar to those of the MR strains. No evidence for unusual wall polymers was found. Simple deficiency of wall teichoic acid does not result in methicillin resistance since an independently isolated teichoic acid-deficient strain (0.1 mumol of phosphate per mg of wall) was not methicillin resistant. In studies of biological properties possibly related to wall teichoic acid, it was discovered that walls isolated from MR organisms grown in the presence of methicillin autolyzed more rapidly than those isolated from organisms grown in the absence of the drug. Since methicillin resistance is enhanced by NaCl and suppressed by ethylenediaminetetraacetate, the effects of these compounds on autolysis of isolated walls were studied. NaCl (1.0 M) and ethylenediaminetetraacetate (1.0 mM) inhibited the autolysis of walls isolated from MR and MS strains. An MR strain bound phage 47, 52A, and 3A only slightly less well than their respective propagating strains.     

Isolation and identification of yeasts associated with vineyard and winery by RFLP analysis of ribosomal genes and mitochondrial DNA

Yeast colonies isolated from vineyard and cellar substrates were analysed in the present study. Yeast species assessment was carried out by amplification and digestion of a region of the ribosomal RNA gene repeat unit. Saccharomyces strains were also characterised using mitochondrial DNA restriction analysis. Oxidative basidiomycetous yeasts without enological potential were predominant in the vineyard environment. Yeasts associated with grape skin depend on grape variety, vintage and degree of grape maturation. These species from grape surface constituted the predominant microbiota in must and they developed during the first stages of the process. Yeasts colonies were also isolated and identified from the walls of a fermentation vat some days before the harvest. Contrary to what was expected, Saccharomyces cerevisiae was not the major species isolated as Candida sorbosa represented 76% of the species isolated. Saccharomyces strains isolated from the fermentation vat had been previously isolated in wine fermentations in this cellar. Therefore, these strains should be considered as constant residents of this winery.     

6-cyanopurine, a color indicator useful for isolating mutations in the nif (nitrogen fixation) genes of Klebsiella pneumoniae.

6-Cyanopurine (6-CP) can be used as a color indicator for certain classes of nif (N2 fixation) mutations in Klebsiella pneumoniae. Under N2-fixing conditions, Nif+ colonies and most Nif- colonies are purple on media containing 6-CP. Twenty-two Nif- mutants with altered color on medium containing 6-CP were isolated. All white mutants contained mutations in the regulatory genes, nifAA-nifL. Mutants which were more darkly colored than the wild type had mutations distributed among six nif genes. Medium with 6-CP was used to isolate Nif- mutants with deletions internal to the nif genes, and 6-CP was used to identify strains depressed for nitrogenase synthesis in the presence of NH4+.     

Characterization of "Pasteurella" piscicida isolated from white perch and cultivated yellowtail.

The morphological, physiological, and biochemical characteristics of twelve "Pasteurella" piscicida strains isolated from white perch and yellowtail are described and the present uncertain taxonomic status of the organisms is discussed. The organisms isolated were gram-negative rods showing bipolar staining and pleomorphism. No spores or flagella were observed. They were non motile and viscid colonies were formed. Growth was observed in a temperature range of 20 to 30 C and the salinity range of growth was between 0.5% and 4.0%. They were aerobic and facultative anaerobic. Oxidase and catalase were produced. Nitrates were not reduced to nitrites. Lysine and ornithine decarboxylases were not produced but arginine dihydrolase was produced. The egg yolk reaction was positive. Tween 80 and tributyrin were decomposed. Phosphatase was produced. Beta hemolysis was revealed on a medium containing defibrinated blood from chickens or carp but not from mammals. Methyl red and Voges-Proskauer tests were positive, and acetoin was produced from 2,3-butanediol. Glucose was fermented without gas production. Acid was produced from fructose, mannose, galactose, sucrose, maltose, dextrin and glycerol. These organisms differed from all other members of the genus Pasteurella with respect to nitrate reduction, arginine dihydrolase, Voges-Proskauer and methyl red tests. The formation of viscid colonies and inability to grow in a medium without sodium chloride or at 37 C were additional characteristics of these organisms.     

Common Antigenic Determinant in a Rumen Organism and in Salmonellae Containing the Antigen O4

Nineteen of 28 strains of rumen organisms isolated from a cow on a high roughage diet and identified morphologically as butyrivibrios, reacted to a low agglutinin titer with salmonella antisera, forming five groups. However only one strain reacted with polyvalent O salmonella antiserum. This strain reacted with O4 factor serum and with antisera to Salmonella strains containing the antigen O4, and agglutinin absorption tests showed the presence of an antigen identical to O4. When 16 further strains of butyrivibrio-like rumen organisms isolated from three cows and one steer were examined, one possessed an antigen similar to but not identical with the antigen O9, and two strains reacted with specific O6,7 factor serum but were not examined further. These four strains were presumptively identified by physiological tests as butyrivibrios. The possible site of antigenic stimulation by such organisms is discussed.     

Phenotypic heterogeneity of Pseudomonas corrugata strains from southern Italy

Fifty-five strains of Pseudomonas corrugata isolated in southern Italy were characterized phenotypically and compared with 23 strains of different origins. At least two main cultural types with rough or smooth colonies were observed. Strains with rough colonies produced a diffusible pigment in culture. On the basis of their nutritional profiles, Ps. corrugata strains formed a distinct phenon most closely related to fluorescent Pseudomonas spp. isolated from tomato pith necrosis-diseased plants. Three major groups of strains were differentiated within the Ps. corrugata phenon on the basis of utilization of 2-ketogluconate, meso-tartrate, hystamine, DL- glycerate and induction of a hypersensitive reaction on tobacco. Some Ps. corrugata strains belonging to group 1 and 3 which did not produce pigment in culture produced IAA in a colorimetric test. Variability in the serological reaction of the Italian strain was observed. None of the three antisera utilized reacted with all strains. Some strains isolated from diseased plants from the same greenhouse showed different nutritional profiles and reacted with different antisera. Fifteen lipopolysaccharide (LPS) patterns were observed. Strains were divided into two groups on the basis of their protein profiles. The heterogeneity which had already been observed in a world-wide study on Ps. corrugata was confirmed in strains from this restricted area.     

Characterization of “Pasteurella” piscicida Isolated from White Perch and Cultivated Yellowtail

The morphological, physiological, and biochemical characteristics of twelve “Pasteurella” piscicida strains isolated from white perch and yellowtail are described and the present uncertain taxonomic status of the organisms is discussed. The organisms isolated were gram-negative rods showing bipolar staining and pleomorphism. No spores or flagella were observed. They were non motile and viscid colonies were formed. Growth was observed in a temperature range of 20 to 30 C and the salinity range of growth was between 0.5% and 4.0%. They were aerobic and facultative anaerobic. Oxidase and catalase were produced. Nitrates were not reduced to nitrites. Lysine and ornithine decarboxylases were not produced but arginine dihydrolase was produced. The egg yolk reaction was positive. Tween 80 and tributyrin were decomposed. Phosphatase was produced. Beta hemolysis was revealed on a medium containing defibrinated blood from chickens or carp but not from mammals. Methyl red and Voges–Proskauer tests were positive, and acetoin was produced from 2, 3-butanediol. Glucose was fermented without gas production. Acid was produced from fructose, mannose, galactose, sucrose, maltose, dextrin and glycerol. These organisms differed from all other members of the genus Pasteurella with respect to nitrate reduction, arginine dihydrolase, Voges–Proskauer and methyl red tests. The formation of viscid colonies and inability to grow in a medium without sodium chloride or at 37 C were additional characteristics of these organisms.