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PubMed1990

Nucleotide sequences of an Australian and a Canadian isolate of potato leafroll luteovirus and their relationships with two European isolates.

The genomes of an Australian and a Canadian isolate of potato leafroll virus have been cloned and sequenced. The sequences of both isolates are similar (about 93%), but the Canadian isolate (PLRV-C) is more closely related (about 98% identity) to a Scottish (PLRV-S) and a Dutch isolate (PLRV-N) than to the Australian isolate (PLRV-A). The 5'-terminal 18 nucleotide residues of PLRV-C, PLRV-A, PLRV-N and beet western yellows virus have 17 residues in common. In contrast, PLRV-S shows no obvious similarity in this region. PLRV-A and PLRV-C genomic sequences have localized regions of marked diversity, in particular a 600 nucleotide residue sequence in the polymerase gene. These data provide a world-wide perspective on the molecular biology of PLRV strains and their comparison with other luteoviruses and related RNA plant viruses suggests that there are two major subgroups in the plant luteoviruses.

Australia↗
PubMed2006

Mycobacterium kumamotonense Sp. Nov. recovered from clinical specimen and the first isolation report of Mycobacterium arupense in Japan: Novel slowly growing, nonchromogenic clinical isolates related to Mycobacterium terrae complex.

Three mycobacterium strains isolated from clinical specimens in Japan were provisionally assigned to the genus Mycobacterium based on their phenotypical characteristics. These isolates were further investigated to determine their specific taxonomic statuses. Mycolic acid analysis and 16S rRNA gene, rpoB, and hsp65 sequence data for the isolates showed that they are most similar to M. terrae complex. DNA-DNA hybridization studies indicated that the three strains were of two species and were distinguishable from M. terrae, M. nonchromogenicum, and M. hiberniae. Therefore, these strains represent two novel species within the genus Mycobacterium. However, one potential new species should have been considered as M. arupense with the 16S rRNA gene and hsp65 sequences similarities of 99.8% and 100% respectively; it was isolated from human specimens in the United States and was proposed in June 2006 as a new species. This report describes the first isolation of M. arupense in Japan, suggesting that the organism is clinically relevant. In addition, we propose the novel species designation Mycobacterium kumamotonense sp. nov. The type strain is CST 7247(T) (=GTC 2729(T), =JCM 13453(T), =CCUG 51961(T)).

Bacterial Proteins↗
PubMed1977

Selective isolation of allergens. I. Reaction of house dust mite extracts with tridacnin and concanavalin A and examination of the allergenicity of the isolated components.

Tridacnin, a lectin from the clam Tridacna maxima was found to precipitate with crude extracts from the house dust mites Dermatophagoides farinae and Dermatophagoides pteronyssinus. Using tridacnin in direct precipitation and affinity chromatography studies, a carbohydrate-rich preparation of high molecular weight was isolated from D. farinae extracts. The isolated preparation gave one precipitin line when used with tridacnin in gel diffusion and immunoelectrophoresis experiments but two bands were seen on polyacrylamide disc gels after electrophoresis at pH 8.9. Radioallergosorbent test (RAST) studies demonstrated that the isolated material reacted strongly with sera from human subjects allergic to house dust mites and accounted for a significant proportion of the IgE binding capacity of the crude D. farinae extracts. Only one out of fifteen mite-allergic subjects gave a positive response when prick tested with the isolated preparation at a concentration of 0.2 mg/ml. A possible explanation for the discrepancy observed between RAST and skin test results is discussed. Concanavalin A also precipitated with D. farinae extracts but the material isolated with this lectin did not react with tridacnin and reacted weakly in the RAST with sera from mite-allergic subjects. We suggest that the high molecular weight component(s) which react with tridacnin may be useful for immunotherapy of house dust mite allergy.

Allergens↗
PubMed2005

Harmful delayed effects of exogenous isolation enzymes on isolated human islets: relevance to clinical transplantation.

The isolation process exposes human pancreatic islets to exogenous isolation enzymes. Exposure to these enzymes, as a result of intraductal injection in the pancreas or simple contact of islets with enzyme components, causes internalization into the islet cells of enzymes and their by-products. Human islets exposed to Liberase-HI exhibit a decreased insulin secretory ability that correlates with the time of exposure. This phenomenon is paralleled by increased expression of adhesion molecules (CD106 and CD62p) and activation of apoptotic pathways (Bax and Bcl-2) in islet cells. Increased functional impairment is also observed after islet transplantation in diabetic immunodeficient mice. Experimental exposure of islet grafts to exogenous isolation enzymes causes intense inflammation (CD11b positive cells) at the transplant site and it was associated with sickness behavior and eventually death of mouse recipients. The extent of these adverse effects likely deceives the standard qualitative protocols currently in use to assess islet quality in vitro. Reducing the secondary effects of exogenous isolation enzymes on isolated human islets may be crucial to enhance the quality of islets as tissue grafts.

Animals↗
PubMed1976

Further studies of twitching Streptococcus sanguis isolated from the human throat. Isolation of strains with a new antigen.

alpha-haemolytic streptococci, classified as Streptococcus sanguis, with spreading growth due to twitching mobility were isolated from 41% of 121 swab cultures from the human pharynx. Sixty-five out of 70 isolates with spreading growth belonged to Lancefield's group H, while 5 isolates constituted a new serological entity. None of 159 non-spreading alpha-haemolytic streptococci isolated from the same cultures possessed either of these antigens. An examination of strains of types I, II and I/II described by Washburn et al., in the group H antisera employed in the present study indicated that type I and type I/II differ from one another in the degree of cross-reactivity with anti-type II serum. (The type II antigen was not demonstrated in any of the isolated strains). We support the opinion that type II should not be considered as S. sanguis.

Antigens, Bacterial↗
PubMed1982

Method for isolation of gardnerella vaginalis (Haemophilus vaginalis). Characterization of isolates by gas chromatography.

A method for the isolation of Gardnerella vaginalis (Haemophilus vaginalis) is presented. Bacteria isolated from 48-hour cultures grown on human blood agar were identified by means of beta-hemolysis, colony morphology, sensitivity to antimicrobial agents, oxydase and catalase reactions. Thirty-eight clinical isolates and one test strain were examined for fatty acid composition. Hexadecanoic (16:0), octadecenoic (18:1) and octadecanoic (18:0) were the major fatty acids. Also present, but in minor quantities, were myristic (14:0), hexadecenoic (16:1) and octadecadienoic (18:2) acids. Only insignificant differences between isolates could be detected. No hydroxy fatty acids commonly found in gram-negative bacteria were encountered. Gas chromatographic analysis of G. vaginalis revealed a characteristic and relatively simple pattern. The results support the use of the isolation method, which provides conditions highly selective for G. vaginalis.

Chromatography, Gas↗
PubMed1997

[Isolation of verotoxin-producing Escherichia coli from cattle and characterization of the isolated strains].

Attempts to isolate Verotoxin-producing Escherichia coli (VTEC) from 53 dead baby cattle were made during a period from January 1989 to July 1991. From 6 cattle of 5 farms, VTECs were isolated. Further examination of cattle from 2 farms with dead baby cattle and from 1 farm with an outbreak of diarrhea among baby cattle, from 8 of 26 cattle with diarrhea and from 9 of 58 healthy cattle, VTECs were isolated. Several serotypes which caused food poisoning in Japan as well as in the US and Europe were included in the isolated strains. VTECs were also isolated from several cattle imported from the US suggesting that these VTECs were from outside the country together with the imported cattle.

Animals↗
PubMed1974

Characterization of Klebsiella isolates from natural receiving waters and comparison with human isolates.

Two hundred sixty-six strains of Klebsiella pneumoniae isolated from natural water sources in geographically diverse areas (Florida, Massachusetts, and Oregon) were analyzed to determine the serotype, biochemical, virulence, and antimicrobial susceptibility differences between these natural strains and human Klebsiella isolates. Sixty of 72 defined serotypes were found among 210 typable strains. Geographic patterns were present, but in general were not pronounced among serotypes. Reactions with 28 biochemical tests showed percentage responses which were very similar to the summaries of primarily human Klebsiella isolates (as reported by Edwards and Ewing, 1972) and that represented diverse geographic sampling. Virulence studies in representative strains showed no geographic variability and little difference from comparable hospital patient-obtained isolates. In contrast to human hospital isolates, strains demonstrated 90% or greater susceptibility to all antibiotics except ampicillin and carbenicillin; and in further contrast, there was little multiple antibiotic resistance beyond that with ampicillin and carbenicillin.

Ampicillin↗
PubMed2006

Isolation and antimicrobial susceptibilities of Chlamydial isolates from Western barred bandicoots.

A range of species of Chlamydiales have previously been detected in a variety of Australian marsupials, including koalas and western barred bandicoots. Thirty-seven ocular, urogenital, or nasal swabs were obtained from 21 wild western barred bandicoots. Chlamydia culture and antibiotic susceptibility testing were performed for cycloheximide-treated HEp-2 cells in 96-well microtiter plates. Chlamydia spp. were isolated from 11 specimens from 9 (42.8%) bandicoots. All isolates were identified as Chlamydiales by conventional PCR with 16S and 23S rRNA gene primers specific to Chlamydiales and were confirmed to be Chlamydia pneumoniae by a C. pneumoniae-specific ompA-based real-time PCR assay and 16S rRNA and 23S rRNA gene signature sequence analyses. The MICs of azithromycin, doxycycline, ciprofloxacin, and enrofloxacin for 10 C. pneumoniae isolates from these bandicoots ranged from 0.015 to 1 microg/ml, 0.25 to 1 microg/ml, 0.25 to 2 microg/ml, and 0.25 to 0.5 microg/ml, respectively. The MICs at which 90% of isolates were inhibited and the minimal bactericidal concentrations were within the ranges reported previously for human isolates of C. pneumoniae.

Animals↗
PubMed1982

Haemophilus influenzae: comparison of respiratory tract isolates with genitourinary tract isolates.

Haemophilus influenzae isolates recovered from the genitourinary (GU) tract were shown to have a significantly different biotype distribution compared with respiratory tract isolates. Biotype IV strains were recovered more commonly from the GU tract, and most strains were non-serotypable. Antibiotic-susceptible strains isolated from the GU tract more frequently harbored plasmids of less than 10 megadaltons than did antibiotic-susceptible respiratory tract strains. One 2.8-megadalton plasmid resident in a GU tract isolate and one 1.8-megadalton plasmid resident in a respiratory tract isolate were shown to be related to the small ampicillin resistance plasmids previously described in H. influenzae, Haemophilus parainfluenzae, Haemophilus ducreyi, and Neisseria gonorrhoeae. This supports the suggestion that these ampicillin resistance plasmids originated by transposition or recombination of the ampicillin transposon (TnA) with cryptic endogenous Haemophilus plasmids.

Anti-Bacterial Agents↗
PubMed1987

Comparative studies on use of fresh and frozen peripheral blood lymphocyte specimens for isolation of human immunodeficiency virus and effects of cell lysis on isolation efficiency.

Heparinized blood specimens (n = 44) and frozen peripheral blood lymphocyte (PBL) specimens (n = 42) were used to evaluate the effects of lysis on human immunodeficiency virus (HIV) isolation. In the two respective groups, 17 and 27 specimens were HIV antibody positive. In the first group there were 8 and in the second group there were 25 that were symptomatic and were classified as indicating an acquired immunodeficiency syndrome-related condition or a pre-acquired immunodeficiency syndrome-related condition by the Centers for Disease Control definition. One-half of the cells from each specimen were frozen and thawed three times before cocultivation with uninfected lymphocytes, and the isolation rates from whole and lysed cells were compared. HIV was isolated from 15 (88%) of 17 fresh specimens and from 24 (89%) of 27 frozen PBLs from HIV antibody-positive patients, and lysis had no overall effect on the isolation rate, which suggested that frozen PBLs were as suitable as fresh blood for HIV isolation attempts and that it was not necessary to maintain cell integrity when submitting PBL samples. Of 21 asymptomatic patients, 20 were culture positive, and of 23 symptomatic patients, 19 were culture positive. Specimens from the 42 antibody-negative individuals were culture negative.

Freezing↗
PubMed1989

Comparison of lipopolysaccharides from Brazilian purpuric fever isolates and conjunctivitis isolates of Haemophilus influenzae biogroup aegyptius. Brazilian Purpuric Fever Study Group.

Haemophilus influenzae biogroup aegyptius (H. aegyptius) has been identified as the etiologic agent of the recently described disease Brazilian purpuric fever (BPF). Although there is heterogeneity among the strains associated with conjunctivitis, isolates from patients with BPF appear to be derived from a single clone. The clinical presentation of BPF suggests that bacterial lipopolysaccharides (LPS) are involved in its pathogenesis. We prepared LPS from H. influenzae biogroup aegyptius and found them to be similar to H. influenzae type b LPS in apparent size (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), biological activities, and fatty acid composition. We compared LPS from BPF clone isolates with LPS from non-BPF clone isolates in tests of Limulus lysate activation, spleen cell mitogenesis, promotion of neutrophil adherence to LPS-treated endothelial cells, and the dermal Shwartzman reaction. In none of these activities were LPS from the BPF clone isolates more potent. Because LPS shed from growing bacteria may be involved in the pathogenesis of purpura, we also measured the rate at which LPS were released into culture medium during bacterial growth and found no significant difference between BPF clone and non-BPF clone isolates.

Chemical Phenomena↗
PubMed1989

Isolation and biochemical characterization of Haemophilus species isolated simultaneously from the oropharyngeal and anogenital areas.

Several reports have described the high frequency of pharyngeal isolation of Haemophilus species. Few studies have compared the simultaneous isolation rate of this species in the oropharyngeal and anogenital areas. Using two selective media, heart infusion agar (HIA) supplemented with 5% defibrinated rabbit blood, 1% IsoVitaleX, and either bacitracin alone (100 micrograms/ml) or bacitracin (5 micrograms/ml), vancomycin (3 micrograms/ml), and polymyxin B (1 microgram/ml), we isolated Haemophilus species in both areas in 89 of 399 (22.2%) patients consulting a sexually transmitted disease clinic. Of those, 56 were males and 33 were females. We recovered Haemophilus species in the oropharyngeal area in 384 patients (96%), while rectal and genital areas were colonized in 48 (12.0%) and 55 (13.8%) patients, respectively (both areas were colonized in 14 patients). Haemophilus parainfluenzae was isolated almost twice as often in the anogenital area as was H. influenzae. H. influenzae biotypes II and III and H. haemolyticus were the more prevalent XV-requiring haemophili isolated from the oropharynx, while H. influenzae biotype IV was more prevalent in the anogenital area. H. parainfluenzae biotypes I, II, and III were more prevalent in the oropharynx, while biotypes I and II were more prevalent in the anogenital area.

Anal Canal↗
PubMed1998

Pre-AIDS era isolates of Pneumocystis carinii f. sp. hominis: high genotype similarity with contemporary isolates.

Isolates of Pneumocystis carinii f. sp. hominis were examined from six individuals who died of P. carinii pneumonia between 1968 and 1981 and who had underlying immunodeficiencies which were not due to human immunodeficiency virus infection. DNA sequence variation was analyzed in the genes encoding the mitochondrial large subunit rRNA (mt LSU rRNA), the internal transcribed spacer (ITS) regions of the nuclear rRNA, the arom locus, and the mitochondrial small subunit rRNA. No major variations were observed when these isolates were compared to isolates from HIV-infected individuals. A small number of minor differences were detected. A new position at which variation occurred in the mt LSU rRNA was observed in one sample. Three new ITS sequence types were identified. A total of nine different ITS sequence types were found in the six samples. Mixed infection with different ITS sequence types of P. carinii f. sp. hominis was observed in four of the six samples. The ITS locus was the most informative of the four loci for distinguishing among the isolates of P. carinii f. sp. hominis. The data suggest that isolates of P. carinii f. sp. hominis from before the AIDS pandemic are genetically very similar to those currently found in HIV-infected individuals.

AIDS-Related Opportunistic Infections↗
PubMed2002

Detection of seg, seh, and sei genes in Staphylococcus aureus isolates and determination of the enterotoxin productivities of S. aureus isolates Harboring seg, seh, or sei genes.

To investigate the distribution of staphylococcal enterotoxin (SE) A to I (SEA to SEI) genes (sea to sei) in Staphylococcus aureus, 146 isolates obtained in Japan from humans involved in and samples from food poisoning outbreaks, healthy humans, cows with mastitis, and bovine raw milk were analyzed by multiplex PCR. One hundred thirteen (77.4%) S. aureus isolates were found to be positive for one or more se genes. The se genotype was classified into 14 genotypes. seg and sei coexisted in the same S. aureus strain. The newly developed sandwich enzyme-linked immunosorbent assay showed that most seh-harboring S. aureus isolates were able to produce a significant amount of SEH. However, most of the S. aureus isolates harboring seg and about 60% of the isolates harboring sei did not produce a detectable level of SEG or SEI, while reverse transcription-PCR analysis proved that the mRNAs of SEG and SEI were transcribed in S. aureus strains harboring seg and sei genes. These results suggest the importance of quantitative assessment of SEG and SEI production in foods in order to clarify the relationship between these new SEs and food poisoning.

Animals↗
PubMed2003

Evolution of sporadic isolates of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals and their similarities to isolates of community-acquired MRSA.

Forty-one methicillin-resistant Staphylococcus aureus (MRSA) hospital isolates that clearly differed from the six major pandemic clones of MRSA in pulsed-field gel electrophoresis type, mecA and Tn554 polymorphism, and epidemic behavior were selected from an international strain collection for more detailed characterization. SpaA typing, multilocus sequence typing, and SCCmec (staphylococcal cassette chromosome mec) typing demonstrated extensive diversity among these sporadic isolates both in genetic background and also in the structure of the associated SCCmec elements. Nevertheless, the isolates could be grouped into restricted clonal complexes by using the BURST (i.e., based upon related sequence types) program algorithm, which predicted that most sporadic MRSA isolates evolved from pandemic MRSA clones. Several of the sporadic MRSA resembled community-acquired MRSA isolates in properties that included a relatively limited multiresistance pattern, faster growth rates, diversity of genetic backgrounds, and a frequent association with SCCmec type IV.

Algorithms↗
PubMed1991

Normal postnatal androgen production and action in isolated micropenis and isolated hypospadias.

To try and find out if a defect in androgen biosynthesis or action could be responsible for the incomplete virilisation seen in boys with isolated hypospadias and isolated micropenis, androgen receptor binding was studied in genital skin fibroblasts established from 18 boys with isolated micropenis and 19 boys with isolated hypospadias. The production of gonadotrophins and testosterone was also measured in the boys with micropenis. There was no evidence of gonadotrophin deficiency, or of a defect in testosterone biosynthesis in the boys with micropenis, and there was no evidence of a quantitative or qualitative defect of androgen binding in either group. These isolated abnormalities may be the result of transient defects in androgen synthesis or action, or both, during a critical phase of embryogenesis.

Adolescent↗
PubMed1989

Isolation and partial characterization of plasmids found in three Halobacterium volcanii isolates.

Three new isolates of Halobacterium volcanii were screened for the presence of plasmids. Each of the different isolates was found to contain one plasmid. These plasmids do not show any homology to each other, nor to the previously isolated plasmid pHV2. Partial restriction maps of these plasmids were determined. One of the plasmids contains chromosomal repetitive sequences as judged by the existence of homologous sequences in the chromosomal DNA of the three isolates. Using the protoplast fusion technique, we showed that at least one of the newly isolated plasmids is compatible with pHV2.

Cloning, Molecular↗