Isolation and morphological characterization of a mycelial mutant of Candida albicans.

In this paper we describe the isolation of a novel strain of Candida albicans which is a mycelium at ambient temperatures. Mutagenesis of C. albicans ATCC 10261 with N-methyl-N-nitro-N-nitrosoguanidine followed by plating on solid media at 28 degrees C yielded colony morphology variants which were characterized by a raised, rough-surfaced colony of irregular outline in marked contrast to the flat, shiny circular colonies of the parental 10261 strain. One mutant colony, hOG301, was studied in detail. Strain hOG301 was stable and exhibited mycelial morphology over a wide temperature range (5 to 40 degrees C) in several media. The hyphae comprising hOG301 mycelium were examined by light microscopy, scanning electron microscopy, and transmission electron microscopy and showed morphological features described in the literature as being typical of both true hyphae and pseudohyphae. In contrast to 10261, hOG301 was not pathogenic after intraperitoneal injection in mice. This is the first report of a mycelial C. albicans that is stable at ambient temperatures.     

"White-opaque transition": a second high-frequency switching system in Candida albicans

A second high-frequency switching system was identified in selected pathogenic strains in the dimorphic yeast Candida albicans. In the characterized strain WO-1, cells switched heritably, reversibly, and at a high frequency (approximately 10(-2] between two phenotypes readily distinguishable by the size, shape, and color of colonies formed on agar at 25 degrees C. In this system, referred to as the "white-opaque transition," cells formed either "white" hemispherical colonies, which were similar to the ones formed by standard laboratory strains of C. albicans, or "opaque" colonies, which were larger, flatter, and grey. At least three other heritable colony phenotypes were generated by WO-1 and included one irregular-wrinkle and two fuzzy colony phenotypes. The basis of the white-opaque transition appears to be a fundamental difference in cellular morphology. White cells were similar in shape, size, and budding pattern to cells of common laboratory strains. In dramatic contrast, opaque cells were bean shaped and exhibited three times the volume and twice the mass of white cells, even though these alternative phenotypes contained the same amount of DNA and a single nucleus in the log phase. In addition to differences in morphology, white and opaque cells differed in their generation time, in their sensitivity to low and high temperatures, and in their capacity to form hypae. The possible molecular mechanisms involved in high-frequency switching in the white-opaque transition are considered.     

Characterization of form variants of Xenorhabdus luminescens.

From Xenorhabdus luminescens XE-87.3 four variants were isolated. One, which produced a red pigment and antibiotics, was luminescent, and could take up dye from culture media, was considered the primary form (XE-red). A pink-pigmented variant (XE-pink) differed from the primary form only in pigmentation and uptake of dye. Of the two other variants, one produced a yellow pigment and fewer antibiotics (XE-yellow), while the other did not produce a pigment or antibiotics (XE-white). Both were less luminescent, did not take up dye, and had small cell and colony sizes. These two variants were very unstable and shifted to the primary form after 3 to 5 days. It was not possible to separate the primary form and the white variant completely; subcultures of one colony always contained a few colonies of the other variant. The white variant was also found in several other X. luminescens strains. DNA fingerprints showed that all four variants are genetically identical and are therefore derivatives of the same parent. Protein patterns revealed a few differences among the four variants. None of the variants could be considered the secondary form. The pathogenicity of the variants decreased in the following order: XE-red, XE-pink, XE-yellow, and XE-white. The mechanism and function of this variability are discussed.     

Characterization of form variants of Xenorhabdus luminescens.

From Xenorhabdus luminescens XE-87.3 four variants were isolated. One, which produced a red pigment and antibiotics, was luminescent, and could take up dye from culture media, was considered the primary form (XE-red). A pink-pigmented variant (XE-pink) differed from the primary form only in pigmentation and uptake of dye. Of the two other variants, one produced a yellow pigment and fewer antibiotics (XE-yellow), while the other did not produce a pigment or antibiotics (XE-white). Both were less luminescent, did not take up dye, and had small cell and colony sizes. These two variants were very unstable and shifted to the primary form after 3 to 5 days. It was not possible to separate the primary form and the white variant completely; subcultures of one colony always contained a few colonies of the other variant. The white variant was also found in several other X. luminescens strains. DNA fingerprints showed that all four variants are genetically identical and are therefore derivatives of the same parent. Protein patterns revealed a few differences among the four variants. None of the variants could be considered the secondary form. The pathogenicity of the variants decreased in the following order: XE-red, XE-pink, XE-yellow, and XE-white. The mechanism and function of this variability are discussed.     

Selection from gonococci grown in vitro of a colony type with some virulence properties of organisms adapted in vivo.

Gonococci from subcutaneously implanted chambers in guinea pigs produced, on agar, more than 95% small colonies showing a "double highlight" (DH) effect in oblique reflected light combined with transmitted light. Laboratory strains of gonococci produced some DH colonies, but other showed a single highlight (SH) or no highlight (NH). Selection of DH colonies and comparison of their organisms with gonococci grown in vivo and with those from SH colonies, showed that the DH character was associated with high infectivity for guinea-pig chambers, resistance to killing by human phagocytes and heavy pilation. Furthermore, DH colonies were found in the first culture of three fresh samples of urethral pus. Thus, the DH colony characteristic may be a more reliable criterion of pathogenicity of gonococcal isolates than systems used previously. There were, however, some differences between the gonococci grown in vivo and the DH colony types. The gonococci grown in vivo and cultured once on solid medium possessed one or two antigens which differed from those of DH (or SH) colonies. They also formed smooth suspensions (which separated slowly) in saline, compared with the rough suspensions (which separated quickly) formed by gonococci from DH (or SH) colonies. Finally, the organisms grown in vivo were resistant to killing by human serum whereas the DH (and SH) colony types were susceptible; the resistance of the organisms grown in vivo was lost during one subculture on agar suggesting that the property is a phenotypic characteristic. Hence, in addition to selecting DH colony types the conditions in vivo produce organisms which differ, probably phenotypically, from cultured organisms.     

Reduced azole susceptibility of oral isolates of Candida albicans from HIV-positive patients and a derivative exhibiting colony morphology variation.

Approximately 50% (15/28) of a selection of oral isolates of Candida albicans from separate individuals infected with the human immunodeficiency virus (HIV) exhibited low susceptibility to ketoconazole as determined by hyphal elongation assessment. Nine of these isolates exhibited colony morphology variation or switching at 37 degrees C, of which six expressed low ketoconazole susceptibility. To determine whether colony morphology variation could give rise to derivatives with reduced azole susceptibility, several high-frequency switching variants of three HIV-patient isolates were recovered and assessed. All but one of the variants expressed similar azole susceptibility profiles to their respective parental strains. However, the C. albicans derivative 132ACR expressed significantly reduced susceptibility to ketoconazole in comparison to its parental strain 132A. In whole cells, on the basis of total growth the switched derivative 132ACR was markedly less susceptible than its parental isolate 132A to ketoconazole at 10 microM. A much smaller difference was observed with fluconazole at 10 microM, with the switched derivative 132ACR exhibiting a threefold lower susceptibility compared with the parental isolate 132A. The incorporation of [14C]acetate in control and azole-treated cells of both organisms was higher for the parental strain. When cell lysates of strain 132A and its derivative 132ACR were incubated with [14C]mevalonic acid and ketoconazole, the IC50 for 14C-label incorporation into C-4 demethyl sterols was fivefold higher for lysates of the switched derivative 132ACR compared with those of the parental strain 132A. With fluconazole the IC50 value for the derivative 132ACR was 25-fold higher than for strain 132A. The 14-sterol demethylase of the switched derivative 132ACR was possibly less sensitive to azole inhibition than that of the enzyme of strain 132A. These studies indicated that colony morphology variation in vitro can generate derivatives with stable, reduced azole susceptibility without prior exposure to azoles.     

Wall mannoproteins in cells from colonial phenotypic variants of Candida albicans

Candida albicans ATCC 26555 switched at high frequency (10(-1) to 10(-3)) between several phenotypes identified by colony morphology on a defined mineral amino-acid-containing agar medium supplemented with arginine and zinc (LAZ medium). When cells taken from colonies exhibiting distinct morphologies were plated directly onto LAZ agar, spontaneous conversion to all the variant phenotypes occurred at combined frequencies of 2.1 x 10(-1) to 9.5 x 10(-3). However, when cells taken from the different colonial phenotypes were plated directly onto an undefined medium (yeast extract/peptone/dextrose; YPD medium), or first incubated in liquid YPD medium and then cloned on YPD agar, all colonies observed exhibited the same phenotype (smooth-white). When cells from the smooth-white colonies were plated as clones on LAZ agar, the original switch phenotype reappeared. These results suggest that environmental conditions such as the growth medium (and possibly the temperature) influence switching by suppressing phenotype expression, but have no effect on genotype. The variant colony morphologies also appeared to be associated with differences in the relative proportions of yeast and mycelial cells. Zymolyase digests of wall preparations obtained from cells belonging to different colonial phenotypes were analysed by SDS-PAGE. After blotting to nitrocellulose paper, the mannoproteins were stained with Concanavalin A, with a polyclonal antiserum enriched in antibodies against mycelium-specific wall components, and with a monoclonal antibody raised against a high-molecular-mass mannoprotein band (260 kDa) specific to the walls of mycelial cells. The results suggest that phenotypic switching might be associated with changes in the degree of glycosylation in high-molecular-mass mannoproteins, or in the way these mannoproteins are bound to other cell wall components.     

Effects of hyperbaric oxygen on the growth and properties of Pseudomonas aeruginosa

Growth of Pseudomonas aeruginosa in 2 atmospheres absolute of 100% oxygen at 37 degrees C produced two types of abnormal colonies--stunted, rough colonies, termed dwarfs, and large, domed, mucoid colonies, termed giants. The occurrence of these variants depended upon the partial pressure of oxygen and the inoculum size. Subculture of dwarf or giant colonies produced a mixture of both colony types after incubation in hyperbaric oxygen, and colonies of normal appearance after incubation in air. Electron micrographs of ultrathin sections showed that cells from dwarf colonies had a more clearly defined envelope region than cells from normal colonies. Giant colony and normal colony-derived cells were of similar appearance. Whole cells from giant colonies contained more carbohydrate, readily extractable lipid, neutral lipid and free fatty acid than cells from normal colonies; the two cell types showed similar contents of 2-keto,3-deoxyoctonic acid and total phospholipid, but different proportions of individual phospholipids. Cells from dwarf, giant and normal (air-grown) colonies were incubated in air on nutrient agar containing either polymyxin, tetracycline or phenoxyethanol. Relative to cells from normal colonies, cells from dwarf colonies showed enhanced resistance to all three agents and cells from giant colonies showed enhanced resistance to polymyxin and tetracycline only. The resistance of cells from variant colonies was lost following a single subculture in air in the absence of antibacterial agents. It was concluded that the envelopes of cells from dwarf and giant colonies differed both from each other and from those of normal cells. These differences, and the formation of variant colonies, appeared to result from bacterial adaptation to hyperbaric oxygen rather than from mutation.     

Comparative characteristics of spore-forming and asporogenic strains of Bacillus thuringiensis

Comparative characteristics of sporogenous and asporogenous Bacillus thuringiensis strains is carried out. Asporogenous strains are found to differ from wild type strains in a number of criteria, including colony morphology, character of growth on rich and poor media and UV-sensitivity. Sporogenous strains form R colonies, they are more stable and more rare produce variants forming S colonies. S colonies are typical for asporogenous mutants, and under the cultivation in unfavourable conditions (elevated temperature, a shift of pH, a change of an incubation regime) asporogenous strains dissociate with a high frequency into R form. Initial strains, which are multiple auxotrophs, under certain conditions can form "prototrophic" revertants which are unstable when incubated on rich media. Suppressor mutation is supposed to be a possible mechanism of the origination of "prototrophs".     

A Simplified Method for the Isolation of Bacteroides nodusus from Ovine Foot-rot and Studies on its Colony Morphology and Serology

A simple method for the isolation of Bacteroides nodosus from foot-rot lesions in sheep is described together with observations on the colony morphology of freshly isolated strains and variant colonies arising on subculture in the laboratory. Serological results from a study of English isolates demonstrate a multiplicity of types based on the K antigens as well as additional antigens common to all strains.     

Phenotypic switching and beta-N-acetylhexosaminidase activity of the pathogenic yeast Trichosporon asahii

The pathogenic yeast Trichosporon asahii is the major causative agent of deep-seated trichosporonosis in immunocompromised patients. Although infection by this microorganism is becoming increasingly frequent, information related to its pathogenicity and virulence factors is still limited. Therefore, we investigated phenotypic switching in colony morphology, and the production of extracellular enzymes as a virulence factor. Sixty-one clinical isolates of T. asahii produced four different morphological types on Sabouraud dextrose agar (SDA): 69% WF (white farinose), 18% WP (white pustular), 10% Y (yellowish white), and 3% WC (white cerebriform). Strains of the three major types (WF, WP, and Y) produced two to five colony types when cultured on SDA at 37 C. The frequency of switching between colony types was 10(-2) to 10(-4), as in Candida albicans and Cryptococcus neoformans. Notably, most of the colonies switched to the smooth (S) type irreversibly, at frequencies of 10(-2) to 10(-3). No secreted aspartic proteinase or phospholipase activity was detected in T. asahii, while beta-N-acetylhexosaminidase activity, which catalyzes the hydrolysis of terminal nonreducing N-acetyl-D-hexosamine residues in N-acetyl-beta-D-hexosaminides, was found. Furthermore, enzymatic activity of the S type was significantly greater than that of the parent type in all strains. No other clinically relevant Trichosporon species (T. mucoides, T. inkin, and T. ovoides ) produced this enzyme. These results provide basal information for understanding the pathogenic potential of T. asahii.     

Bacterial differentiation within Moraxella bovis colonies growing at the interface of the agar medium with the Petri dish.

Moraxella bovis was found to colonize the interface between agar and the polystyrene Petri dish, producing circular colonies when the inoculum was stabbed at a single point. The bacteria occurred in a thin layer of nearly uniform thickness, and colonial expansion occurred in at least two temporal phases. In the first phase, the radial colonial expansion was slow and non-linear. In the second phase, the radial expansion was linear. The interfacial colonies possessed three characteristic concentric growth zones. At the periphery was a narrow ring zone that enclosed another wider ring zone, which, in turn, surrounded a central circular zone. Different bacterial phase variants were recovered from these zones. The two outer ring zones yielded bacteria that formed agar surface colonies of spreading-corroding morphology, while cells from the innermost zone always yielded colonies with a different morphology. The uniform thickness of the colonies implied that replication was restricted to the outermost ring, and that the bacteria within the inner ring and inner circle had entered a quiescent state. The inner ring appeared to represent the lag in time needed for the replicative form to differentiate into the quiescent form. A different kind of variant was associated with wedge-shaped sectors within the colonies. The greatest number of these clonal variants appeared shortly after inoculation and their frequency decreased after the onset of linear growth. The period of slowest colonization coincided with highest frequency of clonal variant expression. It is proposed that the proliferative rate of the parental bacterial population exerted selective pressure on the expression of new clonal variants.     

Induction and selection of the minute-rough (MR) colonial variant of Candida albicans

Summary An adenine requiring strain ofCandida albicans, WC-7, forms large smooth colonies. When grown at 37° C under conditions of severe adenine deprivation, WC-7 cultures accumulate variant cells (MR variants) which produce minute, rough colonies. The variants are stable in that they persist upon repeated selective subculturing. However, they do exhibit high rates of reversion to their large, smooth colony progenitor form. It is shown that cultural conditions which favor the appearance of variants in WC-7 populations create a metabolic stress within the WC-7 cells which leads to their direct transformation into variants. These same conditions also impart a selective growth advantage to variant cells over WC-7 cells. Considerations of the genetic properties of the variants and the factors involved in their induction argue strongly that variant cells originate through alteration of a non-genic, hereditary determinant.     

Changes in internal and external pH accompanying growth of Candida albicans: studies of non-dimorphic variants

Non-dimorphic variants of Candida albicans, which were unable to form germ tubes or mature hyphae in media containing amino acids, glucose and salts or N-acetylglucosamine or serum, were prepared from two hyphal positive laboratory strains using a physical separation method. The hyphal-minus phenotype was stable and may be due to mutations or phenotypic variation. The variant strains maintained their internal pH within narrower bounds as compared to their parental wild-types. When exposed to conditions that normally supported the induction of germ tubes the cytoplasmic pH of the wild type strains increased from 6.8 to over pH 8.0 within 5 min while in the variants the rise in internal pH was only about 0.3 pH units. The wild type strains acidified the growth medium more rapidly than the variants. The results suggest that the control of internal pH is directly or indirectly associated with the regulation of dimorphism. The variants had unaltered cell volumes and specific growth rates. The hyphal-minus phenotype was however fully reversible since revertants occurred spontaneously on serum containing agar.     

Phenotypic switching of Cryptococcus neoformans can influence the outcome of the human immune response

The human pathogenic fungus Cryptococcus neoformans exhibits the phenomenon of phenotypic switching, a process that generates variant colonies that can differ in morphology, virulence and other characteristics such as capsular glucuronoxylomannan (GXM) size and structure. A previous study established that mucoid colony (MC) variants of C. neoformans were more virulent and elicited a different inflammatory response than smooth colony (SM) variants. In this study, we investigated the interaction of cells from MC and SM variants and their respective GXMs with human T cells and monocytes. Specifically, we measured CD40, CD80 and CD86 expression, lymphoproliferation and interleukin (IL)-4, IL-10, interferon (IFN)-gamma and IL-12Rbeta2 expression in the presence and absence of variant cells and their GXMs. For some immune parameters, both MC and SM strains produced similar results, in particular no differences were observed in IL-4 induction. However, for other critical parameters, including CD86 expression, lymphoproliferation and IL-10 production, the MC variant had effects that can be expected to impair the immune response. Hence, a single C. neoformans strain can elicit several different immune responses depending on the colony type expressed, and this is unlikely to be accounted for by differences in phagocytosis only. The results provide a potential explanation for the higher virulence of the MC variant based on the concept that these cells inhibit the development of a vigorous immune response. Furthermore, the results suggest a mechanism by which phenotypic switching can generate variants able to evade the immune response.     

Colonial and Cellular Polymorphism in Xenorhabdus luminescens

A highly polymorphic Xenorhabdus luminescens strain was isolated. The primary form of X. luminescens was luminescent and nonswarming and produced a yellow pigment and antimicrobial substances. The primary form generated a secondary form that had a distinct orange pigmentation, was weakly luminescent, and did not produce antimicrobial substances. Both the primary and secondary forms generated a set of colony variants at frequencies that exceeded normal rates for spontaneous mutation. The variant forms include nonswarming and swarming forms that formed large colonies and a small-colony (SC) form. The primary and secondary forms generated their SC forms at frequencies of between 1 and 14% and 1 and 2%, respectively. The SC forms were distinct from their parental primary and secondary forms in colony and cellular morphology and in protein composition. The cellular morphology and protein patterns of the nonswarming and swarming colony variants were all very similar. The DNA fingerprints of all forms were similar. Each SC-form colony reverted at high frequency to the form from which it was derived. The proportion of parental-type cells in the SC-form colonies varied with age, with young colonies containing as few as 0.0002% parental-type cells. The primary-to-secondary switch was stable, but all the other colony forms were able to switch at high frequencies to the alternative colony phenotypes.     

Hereditary variation and genetic recombination in Koji-molds (Aspergillus oryzae and Asp. sojae). I. Natural variation

Natural variation in monospore lines of Koji-molds (Asp. oryzae and Asp. sojae), isolated from commercial Koji material or soil and from laboratory stock cultures, has been observed. We can divide the 58 strains of Koji-molds investigated into two groups; one group consists of inconstant strains which are very liable to produce natural variants, and the other consists of strains which remain constant through successive single spore culture. The inconstant strains develop colonies bearing various proportions of conidia and aerial mycelium (X-type). They generally form large conidia (Asp. oryzae var. magnasporus) but sometimes medium sized conidia (Asp. oryzae s. str.), which produce large conidia occasionally. The colonies of the constant strains show abundant conidial formation and smooth surfaces (C-type). The conidia are mostly small (Asp. oryzae var. microsporus) but sometimes medium in size (Asp. oryzae s. str.). The colony types of the variants are as follows: C (Conidial type, whole colony covered with conidia), M (mycelial type), R (restricted in growth rate), St (sterile type, little sporulation on all media tested), Nit (requiring reduced nitrate, very faint growth on Czapek's agar), and LS (semi lethal, growth cease immediately after germination). Pedigree cultures of the 8 inconstant strains have been made, but no definite segregation ratios for each variant type have been recognized through successive generations. The LS and N types commonly occur spontaneously from the M-type.     

Direct Cord Reading Medium for Isolation of Mycobacteria

A study of growth and colony morphology of mycobacteria was performed on 7H10 medium and compared with the same medium to which WR 1339 was added. Collection strains of human, bovine, and atypical mycobacteria, in addition to 1,199 sputa, were planted on both media. The results showed that WR 1339 modifies the growth and colony morphology of human, bovine, atypical group I, and, to a lesser degree, atypical groups II, III, and IV mycobacteria. The great majority of human and bovine strains exhibit definite cords when examined at a magnification of 100 times. The colonies are larger when WR 1339 is added, especially if there is a small number of colonies. The addition of WR 1339 spreads the growth on the surface of the agar, producing a thin but larger colony as compared with 7H10 medium alone on which the colonies grow more vertically, thus producing thick but smaller colonies. WR 1339 spreads the growth, producing thin and transparent colonies where the cords are oriented in uniplane and easily visible directly on the isolation media. Half of the positive sputum cultures were easily identified at 12 days. The presence of typical cords permits a quick screening diagnosis of species directly on the isolation media and, in most instances, elimination of the possibility of atypical or contaminating colonies.     

Culture media for differential isolation of Lactobacillus casei Shirota from oral samples

This study aimed to develop a solid culture medium for differential isolation of the probiotic strain Lactobacillus casei Shirota (LcS) and for selective cultivation of lactobacilli present in oral samples. Type strains of lactobacilli and isolates from commercial probiotic products were inoculated onto modified de Man Rogosa Sharpe agar (termed 'LcS Select'), containing bromophenol blue pH indicator, vancomycin and reducing agent L-cysteine hydrochloride for differential colony morphology development. L. casei Shirota cultured on the novel medium produced distinctive colony morphologies, different from other lactobacilli tested. LcS-characteristic colonies were recovered on LcS Select medium from samples of saliva and tongue plaque following a four-week probiotic intervention study. The viable count of presumptive LcS colonies correlated with those isolated on a non-commercial lactitol-LBS-vancomycin agar (LLV) developed for a selective isolation of LcS from faeces. The novel LcS Select medium proved suitable for differential isolation of the probiotic strain L. casei Shirota from oral samples containing mixed microbial populations. It can also be used for selective growth of vancomycin-resistant lactobacilli. There are few available culture media that are sufficiently selective to enable isolation of probiotic strains from mixed populations. LcS Select medium provides a cheaper, yet effective tool in this context.     

Agar underlay method for recovery of sublethally heat-injured bacteria

A method of recovering sublethally heat-injured bacteria was developed. The procedure (termed the agar underlay method) uses a nonselective agar underlaid with a selective medium. In a two-chambered petri dish, the Lutri plate (LP), a nonselective agar is inoculated with a population of sublethally heat-injured bacteria. After a 2-h repair incubation period, selective agar is added to the bottom chamber of the LP and incubated. By diffusing through the nonselective top agar, selective agents from the underlay medium impart selectivity to the system. By the agar underlay method, recovery rates of the heat-injured food-borne pathogens Escherichia coli O157:H7 and Salmonella typhimurium were not different (P > 0. 05) from recovery rates determined with nonselective media. Sublethally heat-injured cells (60 degrees C for 1.5 min in buffer or 80 degrees C for 30 s on meat surfaces) grew and produced a typical colony morphology and color reaction when the agar underlay procedure was used with the appropriate respective selective agars. Unlike agar overlay methods for injury repair, the agar underlay procedure allows the typical selective-medium colony morphology to develop and allows colonies to be more easily picked for further characterization. Higher recovery rates of heat-injured fecal enterococci from bovine fecal samples and total coliforms from animal waste lagoons were obtained by the agar underlay method with selective agars than by direct plating on the respective selective media.