Strains ofC Research Articles

Nutritional requirements ofCytophaga johnsonae and some of its auxotrophic mutants

A defined medium for growing cells ofCytophaga johnsonae was developed for use in studies of the genetics of gliding motility of this organism. The defined medium, containing only mineral salts and any of six sugars, supports good growth of actively motile cells of several strains ofC. johnsonae. Characteristics of growth in the defined medium and ability of cells to use alternative sources of carbon and nitrogen are reported. Certain compounds, when added to solid media, inhibit colony spread ofC. johnsonae without affecting motility of individual cells. Amino acid requirements were determined for 41 of 43 auxotrophic mutants isolated following penicillin selection of unmutagenized cells growing in the minimal medium.

Identification of catalase-producingCampylobacter species based on biochemical characteristics and on cellular fatty acid composition

A total of 50 catalase-positive campylobacters from human and animal sources were studied. The nomenclatural type strains ofCampylobacter coli, C. jejuni, C. fetus, andC. fetus subsp.venerealis, a typical strain of the nalidixic acid-resistant thermophilic group, and various clinical isolates were characterized by bacteriological tests and by gas-liquid chromatographic analysis of their cellular fatty acids. The tests most useful in the differentiation of the various catalase-positive species were growth at 25 and 42°C, H2S production, tolerance to nalidixic acid and to 2,3,5-triphenyltetrazolium chloride, and hippurate hydrolysis. The latter test was the only reliable means to differentiate betweenC. coli andC. jejuni. Differences between.C. fetus andC. jejuni/coli were confirmed by cellular fatty acid compositions. The bacteriological results indicated thatC. fetus andC. jejuni were distinct species, although within the thermophilic campylobacters there were several related taxa that included bothC. coli andC. jejuni strains with typicalC. coli and some thermophilic strains ofC. fetus subsp.fetus at the extremes.

'1001' Campylobacters: cultural characteristics of intestinal campylobacters from man and animals.

The cultural characteristics of 1220 Campylobacter strains from a variety of sources are described. Forty-two were identified as Campylobacter fetus ssp. fetus (Véron & Chatelain, 1973), 1120 as members of the C. jejuni/C. coli group, and 58 did not conform to any known description. Sixteen of the latter strains had the basic characteristics of C. fetus but were atypical in certain other respects. The other 42 strains had the thermophilic characteristics of the jejuni/coli group, but were resistant to nalidixic acid and had other features in common; it is possible that they represent a new species. They were isolated from 19% of locally caught wild seagulls but only occasionally from other animals and man.Growth at 25 degrees C clearly distinguished strains of C. fetus from those of the jejuni/coli and the nalidixic acid-resistant thermophilic (NARTC) groups. Maximum growth temperature was less reliable for this purpose, and 43 degrees C was found to be better than the traditional 42 degrees C. By arranging the results of three tests (tolerance to 2,3,5-triphenyltetrazolium chloride, growth at 30.5 and 45.5 degrees C) serially in the form of a schema comprising nine categories, the jejuni/coli strains fell into two main groups resembling the Institute Pasteur C. jejuni and C. coli type strains, but these groups could not be clearly defined owing to the existence of strains with intermediate characteristics.Most of the strains from cattle resembled C. jejuni, whereas those from pigs resembled C. coli; poultry strains occupied a more intermediate position. Strains from man and other animals were of mixed types, but most human strains resembled C. jejuni rather than C. coli. The type distribution pattern that most nearly matched that of human indigenous strains was given by a half-and-half mixture of strains from cattle and poultry.

Application of serological typing to the investigation of outbreaks of Clostridium perfringens food poisoning, 1970-1978.

Serological typing was used as an epidemiological tool in the investigation of 524 outbreaks of Clostridium perfringens food poisoning in the United Kingdom and 37 outbreaks in other countries. Five thousand five hundred and fifty-four (77%) of 7245 strains of C. perfringens associated with the 561 outbreaks were typable with the 75 Food Hygiene Laboratory antisera; in 354 (63%) of these outbreaks a specific serotype was established as being responsible for the outbreak. An assessment is made of the ability of two additional sets of antisera, prepared against 34 American and 34 Japanese strains of C. perfringens, to increase the number of strains which can be typed. The extent of cross-reaction between the three sets of antisera was determined and the results are discussed in relation to the source and history of the type strains.

Appetitive response of the nematodeCaenorhabditis elegans to oxygen

Using a technique of recording the behavior of individual nematodes during exposure to various solutions, it was demonstrated thatC. elegans made more reversal behaviors after transfer to solutions of lower oxygen tension than higher. The response was stronger after the first hour in the apparatus than initially. This change was not dependent on reduced oxygen availability during the initial period. Starvation is the most likely cause of this change. A variety of mutant strains ofC. elegans that are defective in response to most known chemotactic stimuli, including two strains that have been shown to be severely abnormal in the ciliated endings of all sensory neurons of the worm's snout, all responded to changes in oxygen tension. This observation suggests that oxygen is sensed internally rather than by specialized peripheral receptor cells.

Combined activity of minocycline and amphotericin B in vitro against medically important yeasts.

The capacity of minocycline to enhance the activity of amphotericin B against Candida albicans, Torulopsis glabrata, Cryptococcus neoformans, and non-albicans Candida was examined in vitro utilizing a time-killing curve technique. Synergism was apparent at 4 h with 5 of 5 strains of C. albicans, 8 of 8 strains of C. neoformans, and 1 of 12 strains of non-albicans Candida. Synergism was apparent at 24 h with the remaining 11 strains of non-albicans Candida and all 5 strains of T. glabrata. C. neoformans was the most susceptible of the yeasts to the minocycline-amphotericin B combination; seven strains showed a 3-log or greater reduction in colony count in 4 h and all strains showed this reduction in 24 h at amphotericin B concentrations of 0.4 mug/ml or less in the presence of minocycline.

Synergistic Action of Amphotericin B and Antioxidants Against Certain Opportunistic Yeast Pathogens

Results of earlier turbidimetric growth experiments showed that certain antioxidants prolonged the antifungal activity of amphotericin B (AB) against Candida albicans, presumably by retarding autoxidative destruction of the drug. Viability studies were designed to examine this in more detail. Subinhibitory concentrations of either butylated hydroxyanisole, n-propyl gallate, or nordihydroguaiaretic acid in combination with fungistatic levels of AB exerted synergistic fungicidal activity against two strains of C. albicans and one of C. parapsilosis. Although synergism was not seen in tests with a strain of Torulopsis glabrata, antioxidants prolonged the inhibitory action of AB against this organism. On the basis of these findings and other considerations that are discussed, it is suggested that stabilization of AB and the ability to act synergistically with AB represent two distinctly different effects of the antioxidants.

Ferredoxin and Formyltetrahydrofolate Synthetase: Comparative Studies withClostridium acidiurici, Clostridium cylindrosporum, and Newly Isolated Anaerobic Uric Acid-Fermenting Strains

Six strains of Clostridium acidiurici and three strains of C. cylindrosporum were isolated from soil samples by enrichment culture with uric acid as the source of carbon, nitrogen, and energy. The newly isolated strains were characterized by their spore morphology and the amounts of glycine and formate formed by the fermentation of uric acid. The strains were easily identified as belonging to one species or the other on the basis of spore morphology and formate production. The crystal properties and spectra of the native ferredoxins of all the strains isolated and the amino acid composition and partial carboxy-terminal sequence of all their apoferredoxins were determined. All the ferredoxins were tested for cross-reactivity with antiserum to C. acidiurici ferredoxin by microcomplement fixation. Five of the six C. acidiurici strains, which had ferredoxins with amino acid compositions identical to that from C. acidiurici, also showed immunological identity (immunological distance = 0.0). These results suggest sequence identity. The one strain with a different amino acid composition failed to show complete cross-reactivity. Two of the three C. cylindrosporum strains have ferredoxin amino acid compositions identical to that from C. cylindrosporum. The third strain had a minimum of five differences in sequence. All C. cylindrosporum strains had ferredoxins that differed considerably from C. acidiurici strains (minimum of eight to nine differences), and none of these ferredoxins cross-reacted with antisera to C. acidiurici ferredoxin. Antisera were prepared to formyltetrahydrofolate synthetase from C. acidiurici and C. cylindrosporum, and all possible comparisons were made by using immunodiffusion and microcomplement fixation. There is more intraspecies variation in the synthetases than in the ferredoxins; however, the results suggest considerable interspecies differences in both proteins. These results suggest a low degree of genomic relatedness between the two species, which contrasts sharply with their apparent high degree of phenotypic similarity.

Susceptibility of Clostridium perfringens Isolated from Human Infections to Twenty Antibiotics

The proper choice of antibiotic for Clostridium perfringens infections in patients allergic to penicillin is not clear; the usual recommendations and recent in vitro studies disagree. We tested the susceptibility of 57 strains of C. perfringens to eight penicillins, seven cephalosporins, two tetracyclines, clindamycin, chloramphenicol, and rifampin by the agar dilution method. All strains were inhibited by (per milliliter) 4 mug or less of any of the penicillins, chloramphenicol, or clindamycin and 8 mug or less of any of the cephalosporins tested. Penicillin G and amoxicillin inhibited all strains at 0.12 mug or less per ml. Only 54% of the strains were inhibited by 1 mug of tetracycline per ml. Penicillin G remains the drug of first choice for infections with C. perfringens; it need not be added to a regimen containing a penicillinase-resistant penicillin given parenterally in high doses. The cephalosporins should be considered as alternative drugs for penicillin-allergic patients. Clindamycin and chloramphenicol are also effective. Tetracyclines cannot be depended upon in clostridial infections without in vitro testing, which is impracticable for initial empirical therapy.

In vitro susceptibility patterns of beta-hemolytic streptococci.

The antibacterial drug nalidixic acid (Nal) can suppress the growth of Candida albicans at levels of the drug normally found in urine. Growth suppression increases as drug levels are increased, and Nal also causes a similar proportional inhibition of the synthesis of all cellular macromolecules. However, growth temperature (25 versus 37 C) and the divalent cations Mg(2+) and Mn(2+) can increase C. albicans resistance to Nal. Also, nitrogen depletion of Candida shows that Nal-treated and untreated cells exhibit no difference in leucine uptake during readaptation to nitrogen. In Nal-treated, nitrogen-starved cells, ribonucleic acid and deoxyribonucleic acid (DNA) biosynthesis are less affected than in unstarved Nal-treated cells, but of the two nucleic acids DNA synthesis is the most affected. Nal-resistant strains of C. albicans exhibit a slight toxicity for macromolecular synthesis. Nal treatment of a synchronized population of Saccharomyces cerevisiae results in an increase in the culture mean doubling time of, at most, 20%, but Nal causes the loss of synchronous cell division. With a synchronized population of Kluyveromyces lactis, Nal causes an increase in the mean doubling time of upwards of 300%, with synchrony of cell division being maintained. It is known that S. cerevisiae asynchronously synthesizes mitochondrial DNA during the cell cycle, whereas with K. lactis it is synchronous. Thus, with C. albicans Nal toxicity is dependent both on the dose and the physiological state of the cell. Furthermore, Nal inhibits growth of yeast with synchronous mitochondrial DNA synthesis more adversely than yeast with asynchronous mitochondrial DNA synthesis.

Inhibition of Clostridium botulinum by Strains of Clostridium perfringens Isolated from Soil

Thirty-one soil samples were examined for the presence of organisms capable of inhibiting growth and toxin production of strains of Clostridium botulinum type A. Such organisms were found in eight samples of soil. Inhibiting strains of C. perfringens were found in five samples, of C. sporogenes in three and of Bacillus cereus in three. Three of the C. perfringens strains produced an inhibitor effective on all 11 strains of C. botulinum type A against which they were tested, seven of eight proteolytic type B strains, one nonproteolytic type B strain, five of nine type E strains and all seven type F strains, whether proteolytic or nonproteolytic. They did not inhibit any of 26 type C strains, 6 type D strains, 4 type E strains, or 24 C. sporogenes strains. In mixed culture, an inhibitor strain of C. perfringens repressed growth and toxin production by a C. botulinum type A strain even though it was outnumbered by the latter about 40 times. It also repressed growth and toxin production of C. botulinum in mixed culture of soils in which this latter organism naturally occurred when cooked meat medium but not when trypticase medium was used.

Direct enzymatic repair of deoxyribonucleic acid single-strand breaks in dormant spores.

With the alkaline sucrose gradient centrifugation method, it was found that dormant spores of Clostridium botulinum subjected to 300 krads of gamma radiation showed a distinct decrease in deoxyribonucleic acid (DNA) fragment size, indicating induction of single-strand breaks (SSB). A two- to threefold difference in radiation resistance of spores of two strains of C. botulinum, 33A (37% survival dose [D(37)] = 110 krads) and 51B (D(37) = 47 krads), was accompanied by relatively larger DNA fragments (molecular weight 7.9 x 10(7)) obtained during extraction from the radiation-resistant strain 33A and smaller DNA fragments (molecular weight 1.8 x 10(7)) obtained under identical conditions from radiation-sensitive strain 51B. The apparent number of DNA SSB produced by 300 krads in strains 33A and 51B was 0.37 and 3.50, respectively, per 10(8) daltons of DNA. Addition of 0.02 M ethylenediaminetetraacetic acid (EDTA) to spore suspensions during irradiation doubled the apparent number of SSB in strain 33A but had no effect on strain 51B. In vivo, 0.02 M EDTA present during irradiation to 100 to 300 krads decreased survival of spores of 33A by about 30% but had little or no effect on 51B. Survival of 33A was also reduced by about 45% when the spores were irradiated while frozen in dry ice (-75 C) and, after irradiation, immediately exposed to 0.03 M EDTA for 1 h to inhibit repair in the dormant spores. These results suggest that the highly radiation-resistant strain 33A may be able to accomplish repair of SSB during irradiation or after irradiation under nonphysiological conditions, i.e., in the dormant state. This repair can be inhibited by EDTA. Sedimentation patterns show that DNA from spores of both strains 33A and 51B did not show any postirradiation repair during the first 6 h of germination, as opposed to Bacillus subtilis spores, which exhibit repair immediately after germination. These observations suggest the existence of direct repair in physiological dormant spores of strain 33A in the cryptobiotic resting state in the absence of germination. The repair seems to be similar to that of polynucleotide ligase activity shown to be operative in some vegetative cells. Apparently radiation-sensitive strains such as 51B and B. subtilis are generally poor in DNA repair enzyme activity under conditions of spore dormancy, which may account for the approximately threefold difference in radiation sensitivity or DNA fragility of different strains, or both.

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.

Interconversion of type C and D strains of Clostridium botulinum by specific bacteriophages.

These studies show that Clostridium botulinum types C and D cultures can be cured of their prophages and converted to either type C or D depending on the specific phage used. Strains of types C and D were cured of their prophages and simultaneously ceased to produce their dominant toxins designated as C(1) and D, respectively. Cured nontoxigenic cultures derived from type C strain 162 were sensitive to the phages from the toxigenic type C strain 162 and type D strain South African. When cured nontoxigenic cultures derived from strain 162 were infected with the tox(+) phages from the 162 strain of type C and the South African strain of type D, they then produced toxin neutralized by types C and D antisera, respectively. Cured nontoxigenic cultures isolated from the type D South African strain were only sensitive to the parent phage, and, when reinfected with the tox(+) phage, they produced toxin neutralized by type D antiserum. Type C strain 153 and type D strain 1873, when cured of their respective prophages, also ceased to produce toxins C(1) and D, but, unlike strain 162 and the South African strain, they continued to produce a toxin designated as C(2). When the cured cultures from strains 153 and 1873 were infected with the tox(+) phage from type D strain 1873, the cultures simultaneously produced toxin that was neutralized by type D antiserum. When these cured cultures were infected with the tox(+) phage from type C strain 153, the cultures produced toxin that was neutralized by type C antiserum. These studies with the four strains of C. botulinum confirm that the toxigenicity of types C and D strains requires the continued participation of tox(+) phages. Evidence is presented that types C and D cultures may arise from a common nontoxigenic strain.

Isolation of Clostridium absonum and Its Cultural and Biochemical Properties

A new procedure for isolation of Clostridium absonum was devised. Sixtyseven strains of C. absonum were isolated from 135 soil samples, but no strain of C. absonum could be found from human fecal samples. The lecithinase, hemolysin, and lethal toxin in the culture filtrates of this species exhibited low avidity for C. perfringens type A antitoxin. The three activities were inseparable by the present method of purification. A reinvestigation of biochemical properties revealed that incomplete suppression of lecithinase reaction by C. perfringens type A antitoxin and no fermentation of raffinose, melibiose, and starch are useful criteria to differentiate C. absonum from C. perfringens, and that positive, although weak, gelatin liquefaction and fermentation of trehalose are useful to differentiate it from C. paraperfringens.

Diphthericin types, bacteriophage types and serotypes of Corynebacterium diphtheriae strains isolated in Australia.

A dipthericin typing scheme has been constructed using 441 strains of Corynebacterium diptheriae isolated in eastern Australia from 1962 to 1971. Ten types have been distinguished using seven strains of C. diphtheriae and two strains of C. belfanti as indicators of the diphthericins produced by the newly isolated strains. Strains grouped into types L2, L3 and L3a were found only in Melbourne and types L1 and L4 were predominant in Sydney. Type L5 strains were isolated intermittently throughout the period of study and were found in all eastern states. Numerical analysis of the characteristics of the strains suggests that associations exist between, on the one hand, diphthericin type and, on the other hand, bacteriophage type, serotype and biochemical activity.

Quantitative immunoelectrophoretic methods as tools for a polyvalent approach to standardization in the immunochemistry of Candida albicans.

Quantitative immunoelectrophoresis allows a polyvalent approach to immuno-chemical problems, i.e., identification, quantitation, and thereby standardization of antigens and antibodies in complex mixtures, for example as in crude extracts of microorganisms and their corresponding antisera. This approach is a short cut to conventional standardization of single purified substances, since the polyvalent approach gives a precise quantitative impression as described in this investigation and therefore enables the immunochemist to precisely select the right substance(s) to be purified for further standardization and characterization. To secure the precision of the results, in complex systems it is a sine qua non to select a complex antibody standard, a complex antigen standard, and to describe the complex standard precipitate pattern which again allows a quantitative study of the reproducibility of methods and procedures. The present article describes how such a standardization was approached in this laboratory within the field of Candida albicans immunochemistry. By means of crossed immunoelectrophoresis, 78 water-soluble antigens were demonstrated and enumerated in an antigen standard prepared from one strain of C. albicans A (B 311 Hasenclever). The antibody standard consisted of purified and concentrated rabbit antibodies. The migration velocity of each antigen was indicated in relation to purified human albumin. Not all precipitates could be seen in one immunoplate; therefore a standardized procedure was worked out showing reproducibly 54 precipitates. The reproducibility of quantitation by the crossed-immunoelectrophoresis procedure was determined for each of 30 antigens by repeated measurements; the relative standard deviations ranged from 2.4 to 15.4% and were below 10% for 24 antigens. A simple standardized antigen production procedure was described in great detail, and by quantitative determinations on 30 antigens the procedure was found to be satisfactorily reproducible. By means of crossed-line immunoelectrophoresis in the modification called absorption of antibodies in situ, it was found that strain B 311 contained no specific antigens in comparison to eight other strains of C. albicans. Thirty antigens were quantitated in antigen preparations made by the standardized procedure from the eight strains, and each antigen concentration was expressed as a percentage of the antigen standard. For each antigen a significant inter-strain variation was observed. Of 30 antigens, fifteen were satisfactorily stable after storage at -20 C for 1(1/2) years. The laboratory methods and procedures described in this article thus work with high precision and allow a rapid collection of quantitative data concerning many individual antigens and their corresponding antibodies without purification of antigens. A new complex antigen standard can be made with satisfactory precision from strain B 311. The production of a similar complex antibody standard is a major problem; therefore the main problem in the intra-laboratory standardization seems to be the change from one complex standard to another. In inter-laboratory standardization on the complex level, there seems to be a minimum demand that the first laboratory distributes the antibody standard and that other laboratories use the methods and procedures of the first laboratory.

A taxonomic study of the genusCalamagrostis on Mount Yakeishidake

Populations of seven species ofCalamagrostis growing on Mt. Yakeishidake were studied from a viewpoint of taxonomy. Four of them (C. sesquifolia, C. fauriei, C. matsumurae, C. gigas) were composed of tetraploid plants with 2n=28 and high pollen fertility.C. matsumurae andC. gigas had sympatric contacts, but no sign of their hybridization could be detected. The population ofC. sachalinensis on this mountain included hexaploids (2n=42) and octoploids (2n=56), and no obvious morphological or ecological differences could be found between them. The population ofC. sachalinensis was estimated to include various ecological races which may be continuously graded. InC. langsdorffi, only octoploid plants were found. Plants with 2n=56+0−1B were found in the population ofC. hakonensis. The pollen features of the collections ofC. langsdorffii, C. sachalinesis andC. hakonensis showed that apomictic seed formation may be more or less developed in them. Populations of these species on Mt. Yakeishidake were clearly separated morphologically. It was concluded that the populations of the seven species examined each have a unity and that strains ofC. langsdorffii, C. sachalinensis andC. hakonensis found on Mt. Yakeishidake are confined to those of a derived type.

Rapid, direct tissue culture test for toxigenicity of Corynebacterium diphtheriae.

A method for testing toxigenicity of Corynebacterium diphtheriae in tissue culture is described. The technique, called the colony overlay test (COT), involves inoculating material from an isolated colony of C. diphtheriae to a small area on the surface of an agar medium which overlays a monolayer of toxin-susceptible HeLa cells. If toxin is produced during incubation at 37 C, it diffuses to the tissue monolayer and destroys the cells below the inoculation site. Twenty-four hours after inoculation, organisms are killed and tissue cells are fixed with formaldehyde. The agar overlay is then removed, and the monolayer is stained with crystal violet. Toxin-affected areas fail to stain or stain poorly. A second plate with antitoxin incorporated in the overlay serves as a control for specificity. Forty-eight strains of C. diphtheriae were tested by the COT, guinea pig, and in vitro, gel diffusion tests. The COT is as specific as the other two tests, is easy to read, and can be used to test large numbers of isolates for toxin action more conveniently than by animal inoculation.

Fatty acid composition of paracolons: Arizona, Citrobacter, and Providencia.

The fatty acid compositions of stationary-phase cultures of Arizona arizonae, Citrobacter freundii, Providencia alcalifaciens, Providencia stuartii, and Providencia sp. were studied. The major fatty acids of A. arizonae, C. freundii, and Providencia were 16:0, 16:1, 17:cyclopropane, and 19:cyclopropane. The fatty acid compositions of the two strains of A. arizonae examined were similar to each other, but the three strains of C. freundii differed from one another in their fatty acid compositions. In both A. arizonae and C. freundii, the relative quantities of saturated, unsaturated, and cyclopropane fatty acids were similar to those which have been found in stationary-phase cultures of other members of the Enterobacteriaceae. The three strains of Providencia also differed from one another in their fatty acid compositions. In all three strains, the total quantity of unsaturated fatty acids was larger and that of the cyclopropane fatty acids was smaller than those found in stationary-phase cultures of other enteric bacteria.