Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

Extracellular polysaccharide-protein complexes produced by selected strains ofCandida albicans (Robin) Berkhout

Water-soluble extracellular polysaccharide-protein complexes of a molecular weight of about 200,000 were isolated from glucose-containing nutrient media after cultivation of slightly, moderately and highly virulent strains ofCandida albicans (Robin) Berkhout. Chemical, physico-chemical and immunological properties of these compounds were studied. The complexes contain 74–86% of mannose, 21–31% of glucose, 1–1.5% of glucosamine and 11–14% of proteins built from 17 aminoacids. The polysaccharideprotein complexes are immunologically active and differ from each other by their precipitation ability if added to specific antisera prepared against moderately highly virulentCandida albicans strains.     

Identification of Candida isolated from the cutaneous candidiasis by the combined use of confirmatory medium and slide agglutination with monofactorial antibodies

Forty strains ofCandida and one ofTorulopsis were isolated from patients with cutaneous candidiasis. The isolates comprised 29 strains ofC. albicans, 7 strains ofC. tropicalis, 2 strains ofC. guilliermondii, and one each ofC. parakrusei, C. lipolytica, andT. famata were identified by the ordinary method. Besides the common pathogenC. albicans, a few other species ofCandida may be etiologic organisms of cutaneous candidiasis. These strains were re-examined by combined use of sucrose agar slants and slide agglutination tests with IgG monofactorial antibodies as a rapid identification method, especially for determining serotypes ofC. albicans. The new method was useful and reliable for rapid identification ofC. albicans and related species. All strains ofC. albicans isolated from skin lesions proved to be standard serotypes ofC. albicans.

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Zusammenfassung Vierzig Stämme vonCandida und eins vonTorulopsis wurden aus Kranken mit kutanen Candidamykosen isoliert. Neunundzwanzig Stämme vonC. albicans, 7 vonC. tropicalis, 2 vonC. guilliermondii, und je einer vonC. parakrusei, C. lipolytica undT. famata wurden mit dem ordinären Methode identifiziert. Außer dem gewohnlichen Erreger,C. albicans, konnten auch ein Paar andere Spezies vonCandida als den Erreger betrachtet werden. Sechsunddreißig Stämme vonC. albicans undC. tropicalis wurden mit der von uns verbesserten kombinierten serologischen und biologischen Methode untersucht, besonders um den Serotypus vonC. albicans festzusetzen. Die neue Methode war gut und zuverlässig als die rapide Identification vonC. albicans und verwandten Spezies. Alle aus der Hautläsion isoliertenC. albicans waren der in Japan allgemeine Serotypus vonC. albicans.

     

Occurrence of atypical lymphocytes following intravenous injection of Candida albicans in mice

The occurrence of atypical lymphocytes has been observed in the course of experimental acute infection withCandida albicans in mice. In animals injected intravenously with 2.5 × 10⁶ ofCandida albicans cells, an increased number of monocytes was seen in 24 hours. Monocytes showed toxic vacuolisation in most instances in protoplasm and sometimes in the nuclei. Only a few atypical lymphocytes could be seen at that time. In the following days the number of monocytes diminished and the number of atypical lymphocytes increased. After four days atypical lymphocytes constituted frequently over 20 % of white cells. The autopsy of sacrificed or dead animals with the presence of such elevated percentages of atypical lymphocytes showed enlargement of cervical lymphnodes in all animals. In mice infected with 1.4 × 10³ ofCandida albicans cells, no level higher than 12% of atypical lymphocytes was seen. Pictures were returning to normal with only a few atypical lymphocytes present among the animals which survived for two months after infection withCandida albicans.This work was supported partially byDora Kaplan, Joan Sloan, Cathy Cooper. Memorial Funds and the Roon Foundation.     

Adhesive Properties and Hydrolytic Enzymes of Oral <Emphasis Type="Italic">Candida albicans</Emphasis> Strains

Several virulence factors in Candida albicans strains such as production of hydrolytic enzymes and biofilm formation on surfaces and cells can contribute to their pathogenicity. For this, control of this opportunistic yeast is one of the factors reducing the nosocomial infection. The aim of this study was to investigate biofilm formation on polystyrene and polymethylmethacrylate and the production of hydrolytic enzymes in Candida albicans strains isolated from the oral cavity of patients suffering from denture stomatitis. All strains were identified by macroscopic, microscopic analysis and the ID 32 C system. Our results showed that 50% of the total strains produced phospholipase. Furthermore, protease activity was detected in seven (35%) strains. All Candida albicans strains were beta haemolytic. All C. albicans strains adhered to polystyrene 96-well microtiter plate at different degrees, and the metabolic activity of C. albicans biofilm formed on polymethylmethacrylate did not differ between tested strains. The atomic force micrographs demonstrated that biofilm of Candida albicans strains was organized in small colonies with budding cells.     

„Room temperature“ incubation for chlamydospore production by Candida albicans

Summary Occasional failure ofCandida albicans to produce chlamydospores on potato-carrot chlamydospore agar could not be attributed to variations in the preparation of the medium including autoclaving and lyophilization. Chlamydospore production was, however, very sensitive to temperature. 104 strains ofC. albicans were grown for 3 days on potato-carrot agar at 16, 20, 25, 30, and 37° C. While at 25° C (the optimal temperature) 93 % of the strains sporulated, a variation of only 5° C either way caused a serious reduction in the performance and only 43 % of the strains sporulated. Sporulation at both extremes of temperature was negligible. A check of temperature variations in the laboratory over a 24 week period during winter months showed that for almost half that period, as expressed in total hours, the temperature remained below 21.1° C (70° F.). Thus room temperature incubation for chlamydospore production inC. albicans may not be sufficient in many cases. Production of chlamydospores on potato-carrot agar was also found to be much superior to that on corn meal agar.     

A combination rapid and standard method for identification of Candida albicans

A medium consisting of an aqueous extract of zein, lactose, and Tween 80 is used together with an overlay of 1 % Tween 80 and coverslipping to provide a combined rapid (germ tube) and standard (chlamydospore) method for diagnosis ofCandida albicans. The method is exquisitely sensitive for diagnosis ofC. albicans but lumps chlamydospore-producing strains ofC. tropicalis withC. albicans.     

The distribution of transglucosyl-amylase amongCandida yeasts

Candida transglucosyl-amylase (Sawai, 1958, 1960; Sawai and Hehre, 1962) was found to be produced intracellularly by three of 28 species within theCandida genus, i.e. by 29 of 35C. tropicalis strains, all of 15C. albicans strains and oneC. claussenii. The novel capacity of this enzyme to catalyze transglucosylation from starch was substantiated by the observation that preparations from all the positive strains were highly effective in causing such transfer to glycerol, and by the fact that isomaltose was synthesized from dextrin by the action of all preparations checked for this capacity (21 strains). Two strains ofC. pelliculosa produced an amylase of a different kind, while representatives of the remaining 24Candida species tested (one or two strains of each species) gave no evidence of amylase production. In view of the narrow and apparently specific distribution of transglucosyl-amylase within the genus, it seems possible that production of this enzyme might be useful as an aid in the taxonomic differentiation ofCandida yeasts.A preliminary account of this work was presented at the 60th National Meeting of the Society of American Bacteriologists, held at Philadelphia, Pa., U.S.A. (Sawai and Hehre, 1960).     

Immunoelectrophoresis to detect differences between strains of Candida albicans

Summary Aqueous extracts of two parental strains ofCandida albicans and two mutants obtained from these strains were studied through immunoelectrophoresis and differential staining for protein and polysaccharide fractions after electrophoresis. Differences in antibody-antigen reactions, and protein and polysaccharide fractions could be detected between these strains. These differences reflected changes in synthesizing ability and genetic information inherent in these yeast cells.     

Comparative media studies in the isolation ofCandida albicans from pregnant women

Summary Rice agar and corn meal agar, with and without Tween 80, were evaluated clinically as directly inoculable selective and differential media for the isolation ofC. albicans from vulvovaginal specimens taken from pregnant women. Chlamydospore formation on these media was investigated as a criterion for the identification ofC. albicans.Of 301 patients cultured, 118 (39.2 %) gave positive cultures for yeast-like fungi of the genusCandida. Of 118 strains for which fermentation patterns were determined, 69 (58.5 %) gave the pattern forC. albicans. Of these, 56 (81.1 %) formed chlamydospores.Tween 80 was found to exert a very stimulating influence on chlamydospore production. Rice agar with Tween 80 appeared to be the most efficient medium for eliciting chlamydospores. However, since strains of 4 out of 6 species ofCandida isolated were found to sporulate it was concluded that chlamydospore formation is not a reliable criterion for the speciation ofC. albicans.Each of the 4 media served satisfactorily as a directly inoculable selective medium for the isolation of yeast-like fungi of the genusCandida from vulvovaginal specimens. None of the media appeared to preferentially stimulate chlamydospore production inC. albicans.Dr.Smith is Associate Professor in the Department of Microbiology; Dr.Taubert is a Fellow in the Department of Obstetrics and Gynecology; Mr.Towns is Laboratory Assistant in the Department of Microbiology.Supported in part by a grant from the Lederle Medical Faculty Awards Committee and in part by United States Public Health Service Grant E-3068.     

The serological grouping of Candida albicans strains isolated in the Kraków province in the years 1963–1965 by means of agar gel diffusion test

Summary One hundred forty sevenCandida albicans strains isolated from different clinical specimens were serologically differentiated. Agar gel diffusion test inBjörklund modification was applied inCandida albicans grouping. Autolysates prepared from the strains under study and from the group A and B standard strains ofHasenclever were used as antigens against anti-Candida albicans group A rabbit immune sera. Among 147 examined strains isolated from 107 patients, 137 strains were classified as belonging to group A and only 10 (9.3 %) strains as belonging to group B.     

An experimental model of ascending pyelonephritis due toCandida albicans in rats

We developed a new experimental model of ascendingCandida pyelonephritis in female rats with leukopenia and vesicoureteral reflux. Rats were treated transperitoneally with cyclophosphamide (200 mg/kg) to induce leukopenia 3 days before and transurethrally with diluted acetic acid solution to induce vesicoureteral reflux 1 day before inoculation ofCandida albicans strain, ATCC 10259 (containing 10⁷ cells). Microscopy revealed acute pyelonephritis in whichCandida cells invaded from the fornix and/or papilla into the medulla within 3 days after inoculation. Between 7 and 28 days after inoculation, chronic pyelonephritis reached the cortex. The incidence of pyelonephritis increased gradually and was approximately 80% after 7 days.Candida colony counts of bladder urine specimens obtained by direct puncture were significantly greater in rats with pyelonephritis extending into the parenchyma than in those with pyelonephritis located along the pelvis (p<0.01). These results suggest that this rat model shows the characteristic feature of ascending pyelonephritis due toC. albicans and that the severity ofCandida pyelonephritis can be estimated fromCandida counts of bladder urine.     

Immunochemical studies on mannans of the genusSaccharomyces

Antigenic mannans isolated from the cells ofSaccharomyces fermentati, Saccharomyces rosei,Saccharomyces delbrueckii, Torulopsis colliculosa, Candida albicans andSaccharomyces cerevisiae were examined for their reactivity withSaccharomyces fermentati andCandida albicans antisera. Mannans ofTorulaspora as well asCandida albicans showed high cross-reactivity with the investigated antisera, which could be due to the presence of long side chains established by the partial acetolysis method. The low specific rotations ofSaccharomyces fermentati, Saccharomyces rosei andTorulopsis colliculosa mannans indicate a predominance of β-glycosidio linkages, whereasSaccharomyces delbrueckii andCandida albicans mannans possess predominantly α-linkages.Saccharomyces cerevisiae mannan showed different structural and immunological properties.     

Epidemiological investigations of oral Candida Albicans

Using a gargle-rinse technique, the oral cavities of 103 volunteers were sampled and cultured for the presence ofCandida albicans. Thirty-six (33.95 %) were positive forC. albicans, including 14 females and 22 males. Sixty-four subjects, including negative controls, were placed on treatment regimes of a pre-sleep gargle-rinse with either sterile distilled water (W) or Cepacol® Mouthwash/Gargle (C). The possible effects of ambient temperature, diet, age, sex, and mouthwash use on oralC. albicans levels are illustrated and discussed, including some evidence for familial endemicity. On simulated sporadic or continuous mouthwash use, some individuals showed statistically significant reductions in oralC. albicans flora, whereas others had biologically significant reductions that were not confirmed statistically. A few originally negative individuals developed non-persistent lowC. albicans counts on one or two days. Total bacterial counts were made for 32 subjects, for most of whom biologically significant reductions were obtained, although the counts were highly variable and erratic. The data support the concept that a reduction in oralC. albicans does not lead to an increase in total bacterial flora, and vice versa.with the technical assistance ofAlyce R. Schmitt Paper 741, Department of Botany, The Ohio State University. This investigation was supported by a research grant form the Wm. S. Merrell Co., Cinninnati, O.     

A proteomic approach to understanding the development of multidrug-resistant<Emphasis Type="Italic"> Candida albicans</Emphasis> strains

Resistance of the pathogenic yeast Candida albicans to the antifungal agent fluconazole is often caused by the overexpression of genes that encode multidrug efflux pumps (CDR1, CDR2, or MDR1). We have undertaken a proteomic approach to gain further insight into the regulatory network controlling efflux pump expression and drug resistance in C. albicans. Three pairs of matched fluconazole-susceptible and resistant clinical C. albicans isolates, in which drug resistance correlated with stable activation of MDR1 or CDR1/2, were analyzed for differences in their protein expression profiles. In two independent, MDR1-overexpressing, strains, additional up-regulated proteins were identified, which are encoded by the YPR127 gene and several members of the IFD (YPL088) gene family. All are putative aldo-keto reductases of unknown function. These proteins were not up-regulated in a fluconazole-resistant strain that overexpressed CDR1 and CDR2 but not MDR1, indicating that expression of the various efflux pumps of C. albicans is controlled by different regulatory networks. To investigate the possible role of YPR127 in the resistance phenotype of the clinical isolates, we constitutively overexpressed the gene in a C. albicans laboratory strain. In addition, the gene was deleted in a C. albicans laboratory strain and in one of the drug-resistant clinical isolates in which it was overexpressed. Neither forced overexpression nor deletion of YPR127 affected the susceptibility of the strains to drugs and other toxic substances, suggesting that the regulatory networks which control the expression of efflux pumps in C. albicans also control genes involved in cellular functions not related to drug resistance.Communicated by D. Y. Thomas     

Investigations into the role of fungi in pulmonary diseases

Summary 32 cases of non tuberculous chronic pulmonary diseases and 10 cases of chronic pulmonary tuberculosis not responding to the routine treatment, were investigated to find out if fungi played any role in their etiology. Fungi were isolated from 9 cases of non-tuberculous and 2 cases of tuberculous pulmonary infections.Fungi as such seem to play some role in the etiology of Pulmonary Diseases and probably also help in keeping up the disease in tuberculous cases. Mycostatin is the drug of choice in the treatment of pulmonary moniliasis.Sputa and bronchial aspirates from 32 cases of non-tuberculous chronic pulmonary diseases and 10 cases of chronic pulmonary tuberculosis, not responding to the routine treatment, were repeatedly examined to find out if fungi played any role in their etiology. Fungi were isolated from 9 cases of non-tuberculous (C. albicans from 8 andActinomyces bovis from 1) and two cases (C. albicans from both) of tuberculous pulmonary infections. All the strains ofC. albicansandA. bovis isolated were tested for their sensitivity against Nystatin. Only strains ofC. albicans were found to be sensitive to Nystatin. Two cases treated with Mycostatin improved rapidly.

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Zusammenfassung Sputum and Bronchialsekret von 32 Fällen von nicht-tuberkulösen chronischen Lungenkrankheiten und von 10 Fällen von chronischer Lungentuberkulose, die auf Routinebehandlung nicht ansprachen, sind wiederholt daraufhin untersucht worden, ob Pilze eine aetiologische Rolle spielten. Pilze sind in 9 nicht-tuberkulösen Fällen (C. albicans in 8,Actinomyces bovis in einem Falle) und in 2 tuberkulösen Fällen (C. albicans in beiden) isoliert worden.Alle isolierten Stämme vonC. albicans andA. bovis sind auf ihre Sensitivität gegen Nystatin untersucht worden. NurC. albicans war sensitiv gegen Nystatin. Zwei Fälle, die mit Mycostatin behandelt wurden, zeigten rasche Besserung.

     

Differentiation of Coryne-bacterium diphtheriae of the mitis type found in diphtheria and ozaena. II. The rate of rapidity of glucose fermentation by C. diphtheriae type mitis and C. belfanti

Summary Eighty strains ofC. diphtheriae typemitis and 80C. belfanti strains were tested for the rate of rapidity of glucose fermentation according to the method ofParsons andFrobisher. 90% ofC. diphtheriae mitis strains, in contrast to only 13.7% ofC. belfanti strains, fermented glucose in 1 to 2 days. 76% ofC. belfanti strains fermented glucose in 3 to 4 days, whereas some strains needed 8 to 9 days to complete the fermentation. So the results of this test revealed next to that of nitrate reduction, a further difference between the strains ofC. diphtheriae typemitis found in diphtheria and ozaena.     

Influence of lactobacilli on the adhesion ofStaphylococcus aureus andCandida albicans to fibers and epithelial cells

The ability of organisms to adhere to and form biofilms on fibrous materials is believed to be an important initiating step in the induction of several diseases, such as toxic shock syndrome. Using anin vitro assay, a moderately hydrophobic strain ofStaphylococcus aureus (water contact angle 35°) and a hydrophilicCandida albicans (shown by a hexadecane test) were highly adherent to commercial diaper fibers. The lumen side of the diaper was porous and the fibers were very hydrophobic (>140°), but the internal section was very hydrophilic (0°), presumably for adsorption purposes. There was evidence that adhesion of the pathogens was inhibited when one of fiveLactobacillus strains was present. Surfaces precoated with lactobacilli inhibited staphylococcal adhesion by 26–97%, and candida by 0–67%. When the lactobacilli were used to challenge adherent pathogens, there was 99% displacement of theS. aureus and up to 91% displacement ofC. albicans. HydrophobicL. acidophilus 76 (54°) and T-13 (80°) were the most effective of fiveLactobacillus isolates tested at interference by precoating. The moderately hydrophilicL. casei varrhamnosus GR-1 (33°) was the most effective at displacing the yeast. Experiments with uroepithelial cells also showed that the lactobacilli could significantly interfere with the adhesion of both pathogens to the cells. The results demonstrate the rapidity with which two pathogens adhered to fibers and epithelial cells, and raised the possibility that members of the normal female urogenital flora might interfere with infections caused by these organisms.     

Proteolysis and pathogenicity of Candida albicans strains

Summary Proteolysing strains ofCandida can be recognized in serum-protein-agar pH 5.0 (SPA pH 5.0) and can be isolated from clinical specimens on the basis of their proteolytic activity after addition of Penicillin, Streptomycin and Chloromycetin to the medium.When injected into white mice, only the proteolysing strains ofC. albicans cause extensive peritonitis and infection of all viscera. This possible association of proteolytic properties and pathogenic action ofC. albicans strains is discussed.

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Zusammenfassung Proteolysierende Stämme der GattungCandida können auf Serum-Protein-Agar pH 5,0 (SPA pH 5,0) mit Antibiotica-Zusatz aus klinischen Untersuchungsmaterialien anhand ihrer Proteolyse-Aktivität isoliert werden. Diese stammspezifische Enzym-Aktivität kann mit Hilfe der Folin-Technik bestimmt und in Beziehungen zu pathologisch-anatomischen Veränderungen im Tierexperiment gebracht werden, nachdem in der weißen Maus nur proteolysierendeC. albicans-Stämme eine ausgedehnte Peritonitis und Infektion der parenchymatösen Organe verursacht haben. Inwieweit eine Beziehung zwischen diesen Eigenschaften und der noch ungeklärten Menschen-Pathogenität vonC. albicans-Stämmen besteht, muß weiteren Untersuchungen vorbehalten bleiben.

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This investigation was supported by grants from the Deutsche Forschungsgemeinschaft.

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Presented at the 4th meeting of the International Society for Human and Animal Mycology, New Orleans, July 31st–August 2nd, 1967.     

A comparative study on cell wall antigens and cell surface hydrophobicity in clinical isolates ofCandida albicans

Characterization of common cell surface-bound antigens inCandida albicans strains, particularly those expressed in the walls of mycelial cells might be useful in the diagnosis of systemic candidiasis. Hence, antigenic similarities among wall proteins and mannoproteins fromC. albicans clinical serotype A and B isolates, were studied using polyclonal (mPAbs) and monoclonal (MAb 4C12) antibodies raised against wall antigens from the mycelial form of a commonC. albicans serotype A laboratory strain (ATCC 26555). Zymolyase digestion of walls isolated from cells of the different strains studied grown at 37°C (germination conditions), released, in all cases, numerous protein and mannoprotein components larger than 100 kDa, along with a 33–34 kDa species. The pattern of major antigens exhibiting reactivity towards the mPAbs preparation was basically similar for all the serotype A and B isolates, though minor strain-specific bands were also observed. The immunodeterminant recognized by MAb 4C12 was found to be absent or present in very low amounts inC. albicans isolates other than the ATCC 26555 strain, yet high molecular weight species similar in size (e.g., 260 kDa) to the wall antigen against which MAb 4C12 was raised, were observed, particularly in wall digests from serotype A strains. Cell surface hydrophobicity, an apparently important virulence factor inC. albicans, of the cell population of each serotype B strain was lower than that of the corresponding serotype A counterparts, which is possibly due to the fact that the former strains exhibited a reduced ability to form mycelial filaments under the experimental conditions used.Abbreviations CSH cell surface hydrophobicity - IIF indirect immunofluorescence