Isolates ofC Research Articles

Genetic analysis of Clavibacter toxicus, the agent of annual ryegrass toxicity.

Multilocus enzyme electrophoresis was used to examine the relatedness of 52 isolates of Clavibacter toxicus, the agent of annual ryegrass toxicity. These included 37 Western Australian (WA) field isolates sampled in 3 distinct locations over a 2-year period, and 15 isolates sampled from 6 different host plant species in 3 states in Australia over approximately 8 years. Seventeen reference strains for the related genera Curtobacterium, Rhodococcus and Arthrobacter were examined for comparison. The 69 isolates were divided into 29 electrophoretic types (ETs), separated by genetic distances of 0.06 to 0.81. The C. toxicus isolates fell into 12 ETs, 11 of which formed a tightly clustered group separated by a genetic distance of 0.23 or less. Thirty-one of the WA field isolates of C. toxicus fell into a single ET, and four into another ET. Clavibacter toxicus therefore formed a closely related group which was genetically distinct from the other plant pathogenic species, and a dominant widely disseminated strain of the species was identified in WA.

Allozyme variation and racial differentiation in SwedishCarex lepidocarpa s.l.(Cyperaceae)

Two morphological races have previously been recognized within the sedge,Carex lepidocarpa, in Sweden. These largely allopatric races are accorded specific status, asC. lepidocarpa s.s.andC. jemtlandica, in Scandinavian floras. A study of allozyme variation in populations from 80 Swedish sites supports the morphological evidence for racial differentiation withinC. lepidocarpa. The two races differ from each other in terms of allele frequencies at polymorphic loci and also show different levels of within-population genetic diversity. Material that is morphologically referable toC. lepidocarpa s.s.is characterized by relatively high levels of allozyme variation, both within and between populations.Carex lepidocarpa s.s.is widespread in southern Sweden. In contrast, material that is morphologically assignable toC. jemtlandicashows low levels of within-population genetic diversity, and there is little differentiation between the geographically separated isolates ofC. jemtlandicain northern Sweden and on the Baltic island of Gotland. The high degree of morphological similarity and moderate levels of genetic differentiation between the two races withinC. lepidocarpaindicate that it is more appropriate to recognize the races as subspecies than as species. The low levels of genetic variation inC. jemtlandica, both within and between populations, suggest thatC. jemtlandicamay have arisen fromC. lepidocarpa(or a near ancestor ofC. lepidocarpa) as a result of population fragmentation and isolation in glacial refugia, or during the process of post-glacial colonization of Scandinavia. Lack of allozyme evidence for extensive hybridization between the two races ofC. lepidocarpa, despite their ability to hybridize freely where their ranges overlap at present, supports the suggestion that the two races have had separate post-glacial histories.

Vegetative compatibility among isolates ofColletotrichum gloeosporioides from almond in Israel

Anthracnose, caused byColletotrichum gloeosporioides, is the major disease of almond in Israel. Pathogen attack of young fruit results in fruit rot and leaf wilting. Seventy isolates ofC. gloeosporioides were obtained from affected almond fruits collected at 11 sites during 1991–2 and 1994. Chlorate-resistant nitrate-nonutilizing (nit) mutants were generated from each isolate and used in complementation (heterokaryon) tests. The formation of complementary stable heterokaryons between mutants from different isolates showed that all the isolates belonged to a single vegetative compatibility group. Representative isolates ofC. gloeosporioides from almond did not form heterokaryons with local isolates ofColletotrichum from anemone and avocado, indicating that the almond isolates constitute a distinct subspecific group withinC. gloeosporioides.

A biochemical test based on pyrazinamidase activity for rapid differentiation of corynebacteria

A simple, rapid and inexpensive test is described based on the activity of the enzyme pyrazinamidase, which hydrolyses pyrazinamide to pyrazinoic acid. This test differentiates most of the corynebacteria possessing this enzyme from the exotoxin producing human pathogenic members of this group, i.e.,C. diphtheriae, C. ulcerans andC. pseudotuberculosis, which do not produce this enzyme. In our laboratory, 16 isolates ofC. diphtheriae and 75 isolates of non-diphtheric corynebacteria were rapidly identified, over the past three years.

Flagellin gene polymorphism analysis ofCampylobacter jejuniinfecting man and other hosts and comparison with biotyping and somatic antigen serotyping

Flagellin gene sequence polymorphisms were used to discriminate amongst 77 strains of Campylobacter jejuni from sporadic and outbreak-associated human enteric infections, and from chickens, sheep and calves. The results were assessed in relation to Lior biotyping and serotyping (Penner somatic antigens). Eight DNA PCR-RFLP patterns (genotypes) were identified by analysis of HinfI fragment length polymorphisms in flagellin gene (flaA) polymerase chain reaction (PCR) products. One genotype (F-1) was a feature of 55% of strains. Strains within the genotypes were heterogeneous with respect to somatic antigens with 12 serogroups represented amongst the C. jejuni isolates of flaA type F-1. Serogroups Pen 1, 2 and 23 were the commonest (45%) amongst the 20 different serogroups represented. Several unique clusters of isolates with diverse biotypes were defined, and one cluster (F-7/Pen 23) contained epidemiologically implicated outbreak strains as well as sheep and calf isolates. We conclude that HinfI flaA typing is reproducible and offers high typability, and its combination with serogrouping provides a novel approach to characterizing isolates of C. jejuni with improved discrimination.

A molecular characterization ofClostridium difficileisolates from humans, animals and their environments

It is generally accepted that most patients with Clostridium difficile-associated diarrhoea acquire the organism from the environment. Recently we demonstrated that household pets may constitute a significant reservoir of C. difficile through gastrointestinal carriage in up to 39% of cats and dogs. These findings suggested that direct transmission from household pets, or contamination of the environment by them, may be a factor in the pathogenesis of C. difficile-associated diarrhoea. To investigate this possibility, we examined isolates of C. difficile from humans, pets and the environment by restriction enzyme analysis (REA) and restriction fragment length polymorphism (RFLP) typing using enhanced chemiluminescence. Both REA and RFLP typing methods used Hind III digests of chromosomal DNA. A total of 116 isolates of C. difficile from pets (26), veterinary clinic environmental sites (33), humans (37) and hospital environmental sites (20) was examined. REA was far more discriminatory than RFLP typing and for all isolates there were 34 REA types versus 6 RFLP types. There was good correlation between the REA types found in isolates from pets and from the veterinary clinic environment, and between isolates from humans and from those found in the hospital environment. There was, however, no correlation between REA type of C. difficile found in pets and isolates of human origin. We conclude that there may still be a risk of humans acquiring C. difficile from domestic pets as these findings may be the result of geographical variation.

Gastrointestinal carriage ofClostridium difficilein cats and dogs attending veterinary clinics

Cats and dogs being treated at two veterinary clinics were investigated for gastrointestinal carriage of Clostridium difficile using selective solid and enrichment media. Thirty-two (39.5%) of 81 stool samples yielded C. difficile. There were significant differences in isolation rates between clinics, 61.0% of animals being positive at one clinic compared to 17.5% at the other (Chi-square, P less than 0.005). Of 29 animals receiving antibiotics, 15 (52.0%) harboured C. difficile while 11 (23.9%) of 46 animals not receiving antibiotics were positive (Chi-square, P less than 0.01). There was no difference in carriage rate between cats (38.1%) and dogs (40.0%). The environment at both veterinary clinics was surveyed for the presence of C. difficile. Fifteen of 20 sites at one clinic were positive compared to 6 of 14 sites at the other clinic. Both cytotoxigenic and noncytotoxigenic isolates of C. difficile were recovered from animals and environmental sites. These findings suggest that household pets may be a potentially significant reservoir of infection with C. difficile.

Interaction Between a Velvetleaf Isolate ofColletotrichum coccodesand Thidiazuron for Velvetleaf (Abutilon theophrasti) Control in the Field

Spraying with tank mix combinations of the fungusColletotrichum coccodesand the plant growth regulator thidiazuron increased mortality, decreased biomass and height of velvetleaf, and increased soybean yield in field experiments. The velvetleaf isolate ofC. coccodeswas compatible with thidiazuron, and tank mix combinations consistently improved control over either component alone. In the moist 1986 season the fungus and the growth regulator interacted synergistically to increase velvetleaf mortality. In the drier 1987 season no significant interaction was detected between the two components.

A microbiological etiology for gastritis and peptic ulceration.

AbstractThe relationship betweenCampylobacter pyloriinfection and gastroduodenal disease was investigated using antral biopsies from 102 patients undergoing endoscopy.C. pyloriwas isolated from the majority of patients with histologically defined gastritis (89%), gastric ulcer (100%), and duodenal ulcer (94%), but only in 6% of patients with normal gastric mucosa. No obvious epidemiological association betweenC. pyloriinfection and recent travel, domestic pets, diet, dental hygiene, drug treatment or symptoms could be established. The susceptibility of 38 of these isolates ofC. pylorito 12 antibiotics was determined.The serum anti‐C.pyloriantibody response was determined by an ELISA technique using an acid extractable antigen on the solid phase, in order to improve the sensitivity and specificity. There was a significant increase in antibody level (p=0.001) of patients with histologically defined gastritis over patients with a normal mucosa. The majority of patients (96%) with a culturableC. pyloriinfection had raised anti‐C.pyloriantibodies. However, some patients with neither C.pyloriinfection nor gastritis had serum antibodies. Possible reasons for this are discussed.The results suggest that a serodiagnostic assay forC. pyloriinfection may be of value in the assessment of gastroduodenal disease.

Anthracnose of Florida Beggarweed (Desmodium tortuosum) Caused byColletotrichum truncatum

Greenhouse and growth chamber studies were conducted to determine conditions for infection of the fungal pathogenColletotrichum truncatum(Schw.) Andrus and Moore on Florida beggarweed (Desmodium tortuosum(Sw.) DC. # DEDTO and to determine the host specificity of this fungus. Optimum conditions for disease development were 14 to 16 h incubation in 100% relative humidity (RH) at 24 to 29 C. Control of Florida beggarweed with 105to 107C. truncatumspores/ml was greatest in the cotyledon stage and decreased with plant age. Ten of 18Desmodiumspecies tested were susceptible to the Florida beggarweed isolate ofC. truncatumbut 13 other plant species and varieties were resistant and 61 were immune.

Two enzymes involved in biosynthesis of the host-selective phytotoxin HC-toxin

Cochliobolus carbonum race 1 produces a cyclic tetrapeptide HC-toxin, which is necessary for its exceptional virulence on certain varieties of maize. Previous genetic analysis of HC-toxin production by the fungus has indicated that a single genetic locus controls HC-toxin production. Enzymes involved in the biosynthesis of HC-toxin have been sought by following the precedents established for the biosynthetic enzymes of cyclic peptide antibiotics. Two enzymatic activities from C. carbonum race 1 were found, a D-alanine- and an L-proline-dependent ATP/PP(i) exchange, which by biochemical and genetic criteria were shown to be involved in the biosynthesis of HC-toxin. These two activities were present in all tested race 1 isolates of C. carbonum, which produce HC-toxin, and in none of the tested race 2 and race 3 isolates, which do not produce the toxin. In a genetic cross between two isolates of C. carbonum differing at the tox locus, all tox(+) progeny had both activities, and all tox(-) progeny lacked both activities.

Temperature responses of tropical to warm temperateCladophora species in relation to their distribution in the North Atlantic Ocean

The relationship between distribution boundaries and temperature responses of some North AtlanticCladophora species (Chlorophyta) was experimentally examined under various regimes of temperature, light and daylength. Experimentally determined critical temperature intervals, in which survival, growth or reproduction was limited, were compared with annual temperature regimes (monthly means and extremes) at sites inside and outside distribution boundaries. The species tested belonged to two phytogeographic groups: (1) the tropical West Atlantic group (C. submarina: isolate from Curacao) and (2) the amphiatlantic tropical to warm temperate group (C. prolifera: isolate from Corsica;C. coelothrix: isolates from Brittany and Curacao; andC. laetevirens: isolates from deep and shallow water in Corsica and from Brittany). In accordance with distribution from tropical to warm temperate regions, each of the species grew well between 20–30°C and reproduction and growth were limited at and below 15°C. The upper survival limit in long days was <35°C in all species but high or maximum growth rates occurred at 30°C.C. prolifera, restricted to the tropical margins, had the most limited survival at 35°C. Experimental evidence suggests thatC. submarina is restricted to the Caribbean and excluded from the more northerly American mainland and Gulf of Mexico coasts by sporadic low winter temperatures in the nearshore waters, when cold northerly weather penetrates far south every few years. Experimental evidence suggests thatC. prolifera, C. coelothrix andC. laetevirens are restricted to their northern European boundaries by summer temperatures too low for sufficient growth and/or reproduction. Their progressively more northerly located boundaries were accounted for by differences in growth rates over the critical 10–15°C interval.C. prolifera andC. coelothrix are excluded or restricted in distribution on North Sea coasts by lethal winter temperatures, again differences in cold tolerance accounting for differences in their distribution patterns. On the American coast, species were probably restricted by lethal winter temperatures in the nearshore and, in some cases, by the absence of suitable hard substrates in the more equable offshore waters. Isolates from two points along the European coast (Brittany, Corsica) ofC. laetevirens showed no marked differences in their temperature tolerance but the Caribbean and European isolates ofC. coelothrix differed markedly in their tolerance to low temperatures, the lethal limit of the Caribbean isolate lying more than 5°C higher (at ca 5°C).

Influence of Temperature Adaptation on Glucose Metabolism in a Psychrotrophic Strain of Cytophaga johnsonae

Selective enrichment of yellow-orange-pigmented, gram-negative bacteria related to Cytophaga johnsonae from lake sediment was dependent on low temperatures (ca. 5 degrees C). However, this temperature effect was abolished when excessive amounts of dissolved organic carbon (10 mM N-acetylglucosamine) were added. A psychrotrophic freshwater isolate of C. johnsonae was used to study the physiological versatility of this group. Exponential growth rates were found to be dependent on the temperature to which the cells used as the inocula were acclimated. Glucose incorporation and respiration were also dependent upon the acclimation temperature of the inocula. Patterns of CO(2) evolution obtained from position-labeled [C]glucose indicated that glucose was predominantly metabolized via the Embden-Meyerhof-Parnas pathway, which, however, was greatly reduced at 25 degrees C when the concentration of glucose was as low as 5 muM/liter. Transport, respiration, and incorporation of glucose (0.2- to 20,000-muM/liter concentrations) into macromolecular cellular compounds were characterized by multiple K(m) values which were a function of substrate concentration and temperature. It appeared possible that these multiple K(m) values reflected the changing participation of the Embden-Meyerhof-Parnas pathway in glucose metabolism. These results may provide a physiological explanation for the selective enrichment of psychrotrophic freshwater cytophagas. Moreover, they exhibit the limits of interpreting kinetic data based on conventional heterotrophic potential measurements, especially when some complications may arise from temperature and substrate adaptations of the more versatile members of the chemoorganotrophic microflora such as C. johnsonae.

Waterborne transmission ofCampylobacter enteritis

Campylobacter jejuni is an important cause of human diarrheal disease throughout the world and likeSalmonella enteritidis, has a large animal reservoir which includes most of man's domestic animals. Until recently, it has been difficult to trace the chain of transmission from animals to man because of inadequate environmental sampling techniques and means to distinguish strains. Recent improvements in these techniques have made environmental studies more feasible in 2 water-related out-breaks.In 1 study,C. jejuni was found to be an important cause of sporadic, summertime diarrheal disease among hikers in national wilderness areas of Wyoming. In this setting, illness was significantly associated with drinking untreated surface water. SubsequentlyC. jejuni was isolated from surface water, including mountian streams, and from animals in the area. Some of the environmental isolates were serotypically identical to strains isolated from humans.A second study occurred as a result of an outbreak of Campylobacter enteritis in a community in northern Illinois which was epidemiologically associated with the community water system.Campylobacter jejuni was isolated from several surface water sources and from the implicated water system. These studies demonstrate that environmental isolation ofC. jejuni is now possible and may add to our understanding of disease transmission.

Comparison ofin Situ andin Vitro survival ofCandida albicans in seawater

The survival in seawater of several laboratory and field isolates ofCandida albicans was investigated. Initial studies were madein vitro (flasks) to confirm previous reports. Frequent sampling of viable cells showed that flask experiments, even repeated, produced varied patterns of survival in this closed system. As an alternative, multiple experiments were run in untreated seawater in dialysis bags and plexiglas chambers at ambient temperature (17‡ to 22‡C) in flowing seawater. Die-off rates of all cultures tested in dialysis bags were very rapid in the first day and may have been related to high levels of dissolved organic carbon in the tubing. Distilled water-or acid-washed bags did not yield significantly higher survival rates in all cases. When plexiglas chambers closed with Nuclepore membranes were used, survival rates decreased to 5% to 15% of the original population after 6 days. Chamber data were more uniform and represented approximately a twofold increase in survival over that shown previously inin vitro (flask) studies. Some evidence was obtained in all three test systems for the greater survival rate of a field isolate ofC. albicans compared with that noted for a laboratory (ATCC) strain. The results are considered to more accurately depict the survival ofC. albicans in summer temperate recreational waters.

An agglutination test for the serodiagnosis ofCorynebacterium sepedonicum

Antisera specific forCorynebacterium sepedonicum were produced in three rabbits and in a goat. Bacterial cell agglutination titers of 640, 1280, and 1280 were recorded for the rabbit sera and 320 for the goat antiserum. All antisera reacted equally well with twelve isolates ofC. sepedonicum obtained from Canada, California, Montana, Oregon, Washington, and Wisconsin. No cross-reactivity was observed when the anti-C. sepedonicum sera were tested against five otherCorynebacterium spp.and seven other species of plant pathogenic bacteria.

Antibiotic Resistance of Bacteria in Water Containing Ornamental Fishes

Water containing ornamental fishes was found to frequently contain countable numbers of bacteria that were resistant to one or more antibiotic or chemotherapeutic agents. The multidrug-resistant strains most commonly isolated were lactose-fermenting Citrobacter freundii. The overall resistance of these aquaria strains was greater than the previously described resistance of clinical isolates of C. freundii. Although the strains examined appeared to lack R-factors, this pool of resistant bacteria may have public health implications.

Variables influencing susceptibility testing of Cryptococcus neoformans to 5-fluorocytosine.

The minimum inhibitory concentration (MIC) of 5-fluorocytosine (5-FC) was determined for 65 isolates of Cryptococcus neoformans by using a twofold serial tube dilution method. The MIC was profoundly influenced by incubation temperature, inoculum size, and duration of incubation. By using a standard set of test conditions, 100% of 49 pretreatment isolates of C. neoformans were susceptible to 10 mug of 5-FC per ml or less, and 9 (56%) of 16 isolates recovered during or after 5-FC therapy were massively drug resistant (MIC > 320 mug/ml). With the standard test conditions recommended here, the tube dilution method was found to be both accurate and reproducible, and the results correlated with the treatment status of patients.

5-fluorocytosine resistance in Cryptococcus neoformans.

Isolates of Cryptococcus neoformans from six patients were obtained before and after unsuccessful therapy with 5-fluorocytosine (5-FC). Post-therapy isolates exhibited massive and stable 5-FC resistance. The frequency of drug-resistant mutants in susceptible isolates of C. neoformans was <0.001% (70.4 +/- 17.9 per 10(7) cryptococci), whereas mutant frequencies in resistant isolates approached 100%. Non-drug-induced, spontaneously appearing 5-FC resistant mutants were documented in four susceptible isolates of C. neoformans by use of the statistical method of fluctuation analysis. Mutation rates on these same four isolates ranged from 1.2 x 10(-7) to 4.8 x 10(-7). Total intracellular uptake and incorporation of cytosine-5-(3)H (CyH(3)) and 5-fluorocytosine-2-(14)C (5-FC(14)) into a trichloroacetic acid-insoluble fraction were markedly reduced in six isolates with in vivo-acquired resistance when compared with susceptible pretreatment strains from the same patients. Five of these six isolates also had acquired massive resistance to 5-fluorouracil (5-FU), suggesting that a mutation in the uridine-5'-monophosphate pyrophosphorylase was responsible for drug resistance. The sixth isolate, which remained susceptible to 5-FU, appeared to have a defect in a cytosine-specific permease accounting for 5-FC resistance. A single isolate with in vitro-acquired 5-FC and 5-FU resistance had no reduction in uptake or incorporation of CyH(3) or 5-FC(14). The mechanism of resistance in this isolate is discussed.

In vitro activity of 5-fluorocytosine against Candida and Torulopsis species.

One hundred yeasts were studied. Tests included detailed identification and determination of 24- and 48-hr minimal inhibitory concentrations and minimal fungicidal concentrations of 5-fluorocytosine (5-FC). Final identifications included 57 isolates of Candida albicans, 15 isolates of C. tropicalis, 13 isolates of C. parapsilosis, 7 isolates of Torulopsis glabrata, 3 isolates of C. guilliermondii, 2 isolates each of C. stellatoidea and Cryptococcus neoformans, and 1 isolate of Candida krusei. Twenty-three original identifications were in error; these involved mostly C. albicans, C. tropicalis, C. parapsilosis, and T. glabrata. Inhibitory end point readings based on 24 hr of incubation were misleading. Whereas 79 of 91 isolates of Candida appeared to be inhibited at 24 hr by 12.5 mug or less of 5-FC/ml, only 52 were inhibited at 48 hr; whereas only 12 isolates appeared to be resistant to 100 mug/ml after 24 hr, 37 were resistant after 48 hr. Susceptibility varied amount the different species. C. tropicalis was the most susceptible, with 10 of 15 isolates (66.7%) being inhibited by 12.5 mug or less/ml and 7 (46.7%) being killed by 100 mug or less/ml. C. albicans was similarily susceptible; 33 of 57 isolates (57.9%) were inhibited by 12.5 mug or less/ml and 25 (43.9%) were killed by 100 mug or less/ml. C. parapsilosis was quite resistant, as only 4 of 13 isolates (30.8%) were inhibited by 12.5 mug or less/ml and 3 (23.1%) were killed by 100 mug or less/ml.