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PubMed2004

DNA amplification and rearrangements in archival Salmonella enterica serovar Typhimurium LT2 cultures.

Variations in genome size and gene order were observed in archival Salmonella enterica serovar Typhimurium cultures stored for over 40 years. In one strain, microarray analysis revealed a large, stable amplification. PCR analysis of the same strain revealed a genomic duplication that underwent a translocation. Other strains had smaller duplications and deletions. These results demonstrate that storage in stabs over time at room temperature not only allows for further bacterial growth but also may produce an environment that selects for a variety of mutations, including genomic rearrangements.

Biological Specimen Banks↗
PubMed1997

Gordona terrae central nervous system infection in an immunocompetent patient.

The bacterial genus Gordona includes seven species of mycolic acid-containing actinomycetes which are cultured from environmental sources and rarely from clinical samples. They have been implicated in primary pulmonary diseases, nosocomial wound infections, and central nervous system infections in two immunocompromised patients. We isolated Gordona terrae from the cerebrospinal fluid sample of an immunocompetent patient with meningitis and multiple brain abscesses and detected Gordona terrae DNA in the abscesses. The outcome was good at the 4-year follow-up, after prolonged treatment with trimethoprim-sulfamethoxazole. Phenotypic identification of this isolate was confirmed by analysis of the 16S rRNA gene sequence, which shared 100% homology with that of G. terrae reference strains. Physicians and clinical microbiologists must be aware of the occurrence of Gordona species infection not only among immunocompromised patients but among all patients. Accurate identification of Gordona species may be accomplished by molecular techniques.

Actinomycetales↗
PubMed1997

Biodiversity of Pneumocystis carinii hominis: typing with different DNA regions.

The purpose of this study was to identify the most useful gene for the detection of biodiversity of Pneumocystis carinii hominis isolates and to compare samples from French and Italian subjects. We studied 20 bronchoalveolar lavage fluid specimens from 20 human immunodeficiency virus-infected patients (10 French and 10 Italian patients) with Pneumocystis carinii pneumonia by DNA sequencing of the thymidylate synthase (TS), 5S rRNA, large-subunit mitochondrial rRNA (mt LSU rRNA), and internal transcribed spacer (ITS1 and ITS2) genes. Thirteen of the 20 sequenced samples had the prototype TS gene sequence. Fourteen of the 20 samples showed the prototype sequence of the 5S rRNA gene, and 6 had variant sequences of the 5S rRNA gene. The mt LSU rRNA gene was sequenced for 18 of the 20 samples; all sequences were different from the prototype sequence and were classified into four groups. Thirteen of the 20 ITS1 and ITS2 sequences were analyzed, and all the sequences were found to be different from the prototype sequence and were classified into 10 groups. The internal transcribed spacer regions thus appear to be the most discriminatory region of DNA for analysis of the biodiversity of P. carinii hominis isolates.

AIDS-Related Opportunistic Infections↗
PubMed1997

Clinical and molecular study of Corynebacterium diphtheriae systemic infections in France. Coryne Study Group.

Diphtheria is a disease with a long history that almost completely disappeared from developed countries. In addition, until 1987, systemic infections involving Corynebacterium diphtheriae were rare. However, in 1990, an epidemic occurred in Russia. These two circumstances have provided the stimulus to gain insight into the situation in France. In fact, between 1987 and 1993, a total of 59 C. diphtheriae strains were isolated. Epidemiological data were collected for patients from whom 40 strains were isolated from normally sterile sites, including 34 from blood cultures, and half of the bacteremic patients developed endocarditis. Osteoarticular involvement was noted in 11 of these 40 patients, including 5 bacteremic patients. The fatality rate following bacteremia was 36%, despite specific antibiotic treatment (beta-lactams and aminoglycosides). The mean age of the participants was 38 years, with half of the patients subsisting under low socioeconomic conditions and suffering from homelessness or alcoholism. Apparently, the skin turned out to be the major route of transmission in this reemerging disease. Eighty-eight percent of the isolates belonged to the C. diphtheriae biotype mitis. These were found predominantly in the Paris area, and most were of the same ribotype. Those isolates originating from the overseas territories (Guyana and New Caledonia) belonged to C. diphtheriae biotype gravis. No strains were positive for the tox gene by PCR. This study attests to the persistent circulation in France of C. diphtheriae in the form of systemic infections. The matter is especially significant since these strains are nontoxigenic and are of a unique ribotype. The strains are, however, sensitive to most antibiotics, although 20% are rifampin resistant.

Adolescent↗
PubMed1997

Identification of Serpulina species associated with porcine colitis by biochemical analysis and PCR.

A PCR system for the detection and identification of group IV spirochetes (Serpulina pilosicoli) was designed to complement biochemical tests, e.g., the hippurate hydrolysis and beta-glucosidase tests, and to verify the accuracy of a previously proposed biochemical classification system. The PCR assay was based on amplification of a segment of the 16S rRNA gene. Both primers were constructed to selectively amplify the 16S rRNA gene of Serpulina pilosicoli. All analyzed Serpulina strains exhibiting the capacity to hydrolyze hippurate and lacking beta-glucosidase activity, including the type strain for spirochetal diarrhea, P43, were amplified with the PCR system. All other tested strains, including type and field strains of different phenotypes of Serpulina species, as well as Salmonella species, Campylobacter species, and Escherichia coli strains, were negative in the assay. Among the tested strains were 18 Scottish field isolates originating from the mucosae of pigs with colitis. A simple classification scheme, suitable for routine classification of porcine intestinal spirochetes, is also presented. The scheme is based on hemolysis, indole production, and the hippurate hydrolysis test.

Animals↗
PubMed1997

Identification and characterization of Serpulina pilosicoli isolates recovered from the blood of critically ill patients.

The phenotypic and genetic characteristics of spirochetes isolated from the blood of one U.S. and six French patients with severe clinical disease or impaired immunity were examined. All spirochetes were anaerobic, weakly beta-hemolytic, positive for hippurate hydrolysis, and negative for beta-glucosidase activity. Cell lengths ranged from 4 to 8 microm, and each isolate had between 8 and 12 periplasmic flagella per cell. These features were consistent with the spirochetes' being Serpulina pilosicoli, the agent of intestinal spirochetosis. All isolates were positive in a PCR assay amplifying a portion of the S. pilosicoli 16S rRNA gene, and they all grouped with fecal isolates of S. pilosicoli in multilocus enzyme electrophoresis (MLEE). The blood isolates could be differentiated from each other by MLEE, although the U.S. and two French isolates were closely related. Apparently S. pilosicoli may translocate from the large intestine to establish spirochetemia. The clinical significance of this finding remains uncertain and requires further investigation.

Bacteremia↗
PubMed1998

Molecular typing of Borrelia burgdorferi sensu lato by randomly amplified polymorphic DNA fingerprinting analysis.

To study whether pathogenic clusters of Borrelia burgdorferi sensu lato strains occur, we typed 136 isolates, cultured from specimens from patients (n = 49) with various clinical entities and from ticks (n = 83) or dogs (n = 4) from different geographic regions, by randomly amplified polymorphic DNA (RAPD) fingerprinting with four arbitrary primers. The RAPD patterns were reproducible up to the 95% similarity level as shown in duplicate experiments. In these experiments the purified DNAs prepared on different days, from different colonies, and after various passages were used as templates. With an intergroup difference of 55%, the 136 strains could be divided into seven genetic clusters. Six clusters comprised and corresponded to the established species B. burgdorferi sensu stricto (n = 23), Borrelia garinii (n = 39), Borrelia afzelii (n = 59), Borrelia japonica (n = 1), Borrelia valaisiana (n = 12), and genomic group DN127 (n = 1). One strain from a patient with erythema migrans (EM) did not belong to any of the species or genomic groups known up to now. The RAPD types of B. burgdorferi sensu stricto, B. garinii, and B. afzelii isolates, which may give rise to human Lyme borreliosis (LB), were associated with their geographic origins. A high degree of genetic diversity was observed among the 39 B. garinii strains, and six subgroups could be recognized. One of these comprised eight isolates from patients with disseminated LB only and no tick isolates. B. afzelii strains from patients with EM or acrodermatitis chronica atrophicans were not clustered in particular branches. Our study showed that RAPD analysis is a powerful tool for discriminating different Borrelia species as well as Borrelia isolates within species.

Animals↗
PubMed1998

Molecular evidence that a distinct Vibrio cholerae O1 biotype El Tor strain in Calcutta may have spread to the African continent.

We present molecular evidence that a distinct genotype of Vibrio cholerae O1 which appeared in Calcutta, India, in September 1993 and which is characterized by a unique ribotype that is not found in the standardized ribotyping scheme of V. cholerae and that shows a specific pulsed-field gel electrophoresis profile may have spread to the west African country of Guinea-Bissau where it was responsible for an epidemic of cholera which began in October 1994 and continued into 1996.

Bacterial Typing Techniques↗
PubMed1998

16S rRNA sequence diversity in Mycobacterium celatum strains caused by presence of two different copies of 16S rRNA gene.

Direct sequencing of the 16S rRNA gene (16S rDNA) of Mycobacterium celatum isolates showed ambiguities, suggesting heterogeneity. Cloned 16S rDNA yielded two copies of the gene, which differed by insertion of a thymine at position 214 and by additional mismatches. Restriction fragment length polymorphism analysis confirmed the presence of two copies of 16S rDNA within the bacterial chromosome.

Base Sequence↗
PubMed1998

Experimental transmission of Ehrlichia equi to horses through naturally infected ticks (Ixodes pacificus) from Northern California.

We report the experimental transmission of Ehrlichia equi from naturally infected Ixodes pacificus ticks to horses. Three weeks after exposure to ticks, two of three horses developed clinical signs compatible with E. equi infection, while one horse remained asymptomatic. 16S rRNA gene PCR of blood leukocyte lysates was positive for all horses at various time points; two horses seroconverted. The 16S rRNA gene sequences amplified from tick-exposed horses showed more than 99% homology to corresponding fragments of the 16S rRNA genes of E. equi, Ehrlichia phagocytophila, and the human granulocytic ehrlichiosis agent.

Animals↗
PubMed1998

Facklamia ignava sp. nov., isolated from human clinical specimens.

Two strains of a hitherto-undescribed gram-positive, catalase-negative coccus isolated from human sources were characterized by phenotypic and molecular taxonomic methods. Comparative 16S rRNA gene sequencing studies demonstrated that the unknown strains are genealogically identical and constitute a new line close to, but distinct from, Facklamia hominis. The unknown bacterium was readily distinguished from F. hominis by biochemical tests and electrophoretic analysis of whole-cell proteins. On the basis of phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as Facklamia ignava sp. nov. The type strain of Facklamia ignava is CCUG 37419.

Aged↗
PubMed1998

Outbreak of Staphylococcus schleiferi wound infections: strain characterization by randomly amplified polymorphic DNA analysis, PCR ribotyping, conventional ribotyping, and pulsed-field gel electrophoresis.

Within a 1-year period, six surgical-site infections (SSI) caused by Staphylococcus schleiferi were observed in the department of cardiac surgery of Ignatius Hospital, Breda, The Netherlands. Since outbreaks caused by this species of coagulase-negative staphylococci have not been described before, an extensive environmental survey and a case control study were performed in combination with molecular typing of the causative microorganism in order to identify potential sources of infection. Variability, as detected by four different genotyping methods (random amplification of polymorphic DNA [RAPD], conventional and PCR-mediated ribotyping, and pulsed-field gel electrophoresis [PFGE] of DNA macro restriction fragments), appeared to be limited both among the clinical isolates and among several control strains obtained from various unrelated sources. Among unrelated strains, RAPD and PCR-mediated ribotyping identified two types only, whereas seven different types were identified in a relatively concordant manner by conventional ribotyping and PFGE. The latter two procedures proved to be the most useful tools for tracking the epidemiology of S. schleiferi. Four of the outbreak-related strains were identical by both methods, and two isolates showed limited differences. In the search for a potential source of S. schleiferi infection, two slightly different PFGE types were encountered on several occasions in the nose of a single surgeon. These strains were, however, clearly different from the outbreak type. In contrast, S. schleiferi cultures remained negative for two persons identified on the basis of case control analysis. It was demonstrated that SSI caused by S. schleiferi had a clinical impact for patients comparable to that of a wound infection caused by Staphylococcus aureus. This report describes the first well-documented outbreak of S. schleiferi infection. A source of the outbreak was not detected.

Aged↗
PubMed1998

Canine intestinal spirochetes consist of Serpulina pilosicoli and a newly identified group provisionally designated "Serpulina canis" sp. nov.

The spirochetes inhabiting the large intestines of humans and animals consist of a diverse group of related organisms. Intestinal spirochetosis caused by Serpulina pilosicoli is a newly recognized enteric disease of human beings and animals with potential public health significance. The purpose of this study was to determine the species identity of canine intestinal spirochetes by comparing 30 isolates obtained from dogs in Australia (n = 25) and the United States (n = 5) with reference strains representing Serpulina species and Brachyspira aalborgi, by phenotypic and genetically based typing methods. All of the canine isolates were indole negative and produced a weak beta-hemolysis when cultured anaerobically on agar medium containing blood. Four isolates were identified as S. pilosicoli by 16S rRNA-specific PCR assays, rRNA gene restriction fragment length polymorphism or ribotyping, and multilocus enzyme electrophoresis. The remaining 26 isolates formed a cluster related to porcine Serpulina innocens as determined by multilocus enzyme electrophoresis but had a unique ribotype pattern. The data suggested the existence of a novel Serpulina species, provisionally designated "Serpulina canis," colonizing the intestines of dogs.

Animals↗
PubMed1998

Genotypic characterization of Salmonella enteritidis phage types by plasmid analysis, ribotyping, and pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) was used to resolve XbaI and SpeI macrorestriction fragments from 60 defined phage type (PT) reference strains of Salmonella enteritidis. The level of discrimination was compared to that afforded by plasmid profile analysis and ribotyping. Twenty-eight distinct XbaI pulsed-field profiles (PFPs) were observed, although a single type, PFP X1, predominated. Absence of the 57-kb spv-associated fragment was observed for three PT reference strains, and the profile was designated PFP X1A. The XbaI macrorestriction profiles of a further four PT reference strains were altered by the presence of plasmid-associated bands. Twenty-six SpeI-generated PFPs (plus one subtype) were observed for the same strains. No SpeI fragment corresponding to the 38-MDa serovar-specific plasmid was detected. The distribution of XbaI and SpeI profiles did not always correspond, producing a total of 32 combined PFPs for the 60 PT reference strains. This compared with a total of 18 different plasmid profiles and three PvuII ribotypes generated by the same strains. The results of this study indicate that PFGE may offer an improved level of discrimination over other genotypic typing methods for the epidemiological typing of S. enteritidis.

Bacterial Typing Techniques↗
PubMed1999

Differentiation of two biovars of Ureaplasma urealyticum based on the 16S-23S rRNA intergenic spacer region.

The 16S-23S rRNA intergenic spacer regions of 14 strains representing the 14 serovars of Ureaplasma urealyticum were amplified by PCR and sequenced for genetic differentiation between the two biovars Parvo and T960. Although the spacer region of the Parvo and T960 biovars comprised 302 nucleotides and lacked spacer tRNA genes, 15 nucleotides were different between the two biovars. The four nucleotide sequences of the 16S-23S rRNA intergenic spacer region of serovars 1, 3, 6, and 14 in the Parvo biovar were found to be identical. Similarly, the 10 nucleotide sequences of the 16S-23S rRNA intergenic spacer region of serovars 2, 4, 5, and 7 to 13 in the T960 biovar were found to be identical. The nucleotide sequence of the T960 biovar contains multiple restriction sites for restriction endonuclease SspI, which allows differentiation of the T960 biovar from the Parvo biovar.

Base Sequence↗
PubMed1997

Nucleosome binding by the polymerase I transactivator upstream binding factor displaces linker histone H1.

Upstream binding factor (UBF) is a vertebrate RNA polymerase I transcription factor that can bend and wrap DNA. To investigate UBF's likely role as an architectural protein of rRNA genes organized in chromatin, we tested UBF's ability to bind rRNA gene enhancers assembled into nucleosome cores (DNA plus core histones) and nucleosomes (DNA plus core histones plus histone H1). UBF bound with low affinity to nucleosome cores formed with enhancer DNA probes of 162 bp. However, on nucleosome cores which contained approximately 60 bp of additional linker DNA, UBF bound with high affinity similar to its binding to naked DNA, forming a ternary DNA-core histone-UBF complex. UBF could be stripped from ternary complexes with competitor DNA to liberate nucleosome cores, rather than free DNA, suggesting that UBF binding to nucleosome cores does not displace the core histones H2A, H2B, H3, and H4. DNase I, micrococcal nuclease, and exonuclease III footprinting suggests that UBF and histone H1 interact with DNA on both sides flanking the histone octamer. Footprinting shows that UBF outcompetes histone H1 for binding to a nucleosome core and will displace, if not dissociate, H1 from its binding site on a preassembled nucleosome. These data suggest that UBF may act to prevent or reverse the assembly of transcriptionally inactive chromatin structures catalyzed by linker histone binding.

Animals↗