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Publications and source records attributed to V Vincent.
Linear plasmids were found in Mycobacterium xenopi, M. branderi, and M. celatum. These elements represented a wide size range (from 20 to 320 kb), had Streptomyces-like terminal structures, and defined five hybridization groups. Cross-hybridization and common restriction fragment length polymorphism patterns of plasmids from the different species suggest either genetic exchange or a common ancestry of the species.
Different molecular typing methods including restriction fragment length polymorphism (RFLP) analysis with the major polymorphic tandem repeat (MPTR) probe and the IS1652 probe, pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and PCR restriction analysis of the hsp-65 gene (PRA) were applied to clinical and water isolates of Mycobacterium kansasii. RFLP with the MPTR probe, PRA, PFGE, and AFLP analysis revealed five homogeneous clusters which appeared to be subspecies. RFLP with the MPTR probe and PRA gave patterns specific for each cluster, whereas PFGE and AFLP analysis gave polymorphic patterns. IS1652 was present in two of the five clusters and provided polymorphic patterns for one cluster only. The two IS1652-positive clusters were Accuprobe negative (Accuprobe test; Gen-Probe Inc.), and only two other clusters were Accuprobe positive. A PCR test based on the detection of a species-specific fragment (M. Yang, B.C. Ross, and B. Dwyer, J. Clin. Microbiol. 31:2769-2772, 1993) was positive for all M. kansasii strains. This PCR test is an accurate, rapid, and specific M. kansasii identification test. No subspecies was particularly more virulent, because all clusters contained clinical strains, from AIDS patients and non-AIDS patients, and environmental strains.
One hundred ninety-six Mycobacterium avium isolates from blood samples recovered from 93 AIDS patients for several months were typed by serotyping, by IS1245 restriction fragment length polymorphism (RFLP) analysis and in some cases RFLP analysis with plasmids pVT2 and pLR7 as probes, and by pulsed-field gel electrophoresis (PFGE). PCR typing of single colonies was also used to detect polyclonal infections. Strains belonged mainly to serotypes 1, 4, and 8. pVT2- and pLR7-related plasmids were detected in strains from 49% of the patients. The IS1245 RFLP and PFGE analyses showed a 96.8% diversity of the M. avium strains from the 93 patients. The vast majority (95.2%) of infections were monoclonal, indicating that recent infection is unlikely, even at an advanced stage of AIDS. For one patient, sequential isolates gave divergent patterns of sensitivity and resistance to clarithromycin, but all were identified as the initial clone. RFLP analysis and PCR typing of single colonies allowed for the detection of three polyclonal infections during the bacteriological follow-up. Among strains from patients whose samples were positive by culture after treatment for 2 to 15 months, 97.4% were the same as the initial strain. In conclusion, relapses and failures were mostly due to the initial strain. These relapses and failures resulted either from the selection of resistant mutants or the reappearance of sensitive strains, suggesting the persistence of nonsterilized tissue reservoirs.
Based on cultural and biochemical tests, a total of 84 strains (72 clinical and 12 environmental isolates from the Caribbean Isles, Europe, and the Indian subcontinent) were identified as members of the Mycobacterium avium complex (MAC). They were further characterized with MAC, M. avium, and M. intracellulare probes of the AccuProbe system, and this was followed by selective amplification of DT6 and DT1 sequences. Seventy isolates gave concordant results; 63 were identified as M. avium, 5 were identified as M. intracellulare, and 24 remained untypeable by both methods. Fourteen isolates gave discrepant results, as they were DT1 positive but gave negative results by the M. intracellulare AccuProbe test. Consequently, a detailed molecular analysis of all DT1-positive isolates (14 discrepant strains plus 5 M. intracellulare strains) was performed by PCR-restriction analysis (PRA) of the hsp65 gene and 16S rRNA gene sequencing. The results confirmed the reported heterogeneity of M. intracellulare, as only 6 of 19 isolates (32%) gave PRA results compatible with published M. intracellulare profiles while the rest of the isolates were grouped in four previously unpublished profiles. 16S rRNA gene sequencing showed that only 8 of 19 isolates (42%) were related to M. intracellulare IWGMT 90247 (EMBL accession no. X88917), the rest being related to MCRO19 (EMBL accession no. X93030) and MIWGTMR10 (EMBL accession no. X88915). In conclusion, we have characterized a significant number of MAC isolates which were not identified by the AccuProbe test, PRA, or 16S rRNA sequencing. However, all of them were identifiable by DT1-DT6 PCR (they were DT6 negative and DT1 positive) and could be tentatively identified as M. intracellulare based on previously published observations. It is noteworthy that the majority of such isolates (14 of 19) were from the Indian subcontinent, with 12 of 14 being environmental isolates. Our study confirms the marked heterogeneity of M. intracellulare isolates and shows the utility of in-house DT1 PCR to detect this group of isolates, which would otherwise have been missed by the AccuProbe system in a routine clinical microbiology laboratory.
A ligation-mediated PCR (LMPCR) method for the amplification of sequences flanking the IS6110 of the Mycobacterium tuberculosis complex has been developed. The method uses one primer specific for IS6110 and a second specific for a linker ligated to SalI-restricted genomic DNA. LMPCR is a rapid screening method, valuable for the fingerprinting of M. tuberculosis complex strains.
In addition to a long form of 591 amino acids (aa), two other forms of PRL receptor (PRLR), differing in the length of their cytoplasmic domains, have been identified in the rat. The Nb2 form, lacking 198 aa in the cytoplasmic domain, is able to transmit a lactogenic signal similar to the long form, whereas the short form of 291 aa is inactive. The ability of PRL to activate the promoter of the beta-casein gene or the lactogenic hormone responsive element fused to the luciferase reporter was assessed in Chinese hamster ovary cells or 293 fibroblasts transiently transfected with PRLR cDNAs. The function of the short form was examined after cotransfection of both the long and short forms. These results clearly show that the short form acts as a dominant negative inhibitor through the formation of inactive heterodimers, resulting in an inhibition of Janus kinase 2 (JAK2) activation. The present study also investigates the possible participation of cytoplasmic receptors in the signal transduction pathway, using cotransfection experiments and a new approach that selectively determines the contribution of cytoplasmic receptors in the process of signal transduction. We cotransfected Chinese hamster ovary cells with two cDNA constructs: a cytoplasmic (soluble) form of the receptor with a deleted signal peptide (delta-19), which is unable to bind PRL, and a functionally inactive receptor mutant (lacking box 1), which is anchored in the plasma membrane and able to bind PRL. This approach has allowed us to show that delta-19, lacking expression at the plasma membrane, can transduce the hormonal message, at least to a limited extent (up to 30% of wild type efficiency), providing that association/activation occurs with a PRL-PRLR complex initiated at the cell surface level; box 1 of the cytoplasmic form is necessary to rescue this partial transcriptional activity of the inactive mutant. This partial recovery is also parallel to the partial activation of JAK2, indicating that the signal transduction pathway implicated JAK2. Our results provide evidence that heterodimerization of receptors can be implicated either in the positive or in negative activation of gene transcription.
Three hundred and twenty Laparoscopic Adjustable Silicone Gastric Banding (LASGB) procedures have been performed in our institution from September, 1993 to January, 1997 for the treatment of morbid obesity. Before the first human operation a new prototype of the silicone band for laparoscopic use has been devised and a new surgical protocol developed in a preliminary animal lab programme. All the patients were morbidly obese (92-200 kg). The conversion rate to laparotomy has been very low (1.25%). Early complications have been rare. Pouch dilatation and/or stomach slippage has been the only significant late complication. The rate of this complication has been considerably improved by reducing the pouch volume and by putting more gastro-gastric sutures. The post-operative weight loss of LASGB has been comparable with our series of VBG and ASGB.
INTRODUCTION: Mesotherapy is an alternative medical process defined by the intradermal injection of small amounts of pharmacological substances. It has been associated with the development of mycobacterial infections. CASE REPORT: A 80-year-old woman presented with a Mycobacterium bovis infection of the face following mesotherapy. Genome analysis of the mycobacterial strain isolated from a skin lesion using pulse-field gel electrophoresis demonstrated the presence of a vaccinal strain identical to the one employed by the same physician to vaccinate a child two hours before. DISCUSSION: Given the severity of mycobacterial infections following mesotherapy and given the lack of evidence about its efficacy, mesotherapy should not be performed on the face. Standardisation of aseptic measures in the daily medical practice could prevent such infectious complications.
The A subunit of DNA gyrase in Mycobacterium leprae, unlike its counterpart in Mycobacterium tuberculosis, is produced by protein splicing as its gene, gyrA, harbors a 1260-bp in-frame insertion encoding an intein, a putative homing endonuclease. Analysis of the gyrA locus from different mycobacterial species revealed the presence of inteins in Mycobacterium flavescens, Mycobacterium gordonae and Mycobacterium kansasii but not in 10 other pathogenic or saprophytic mycobacteria. In all four cases where intein coding sequences were found, they were localized in the same position in gyrA, immediately downstream of the codon for the key active-site residue Tyr-130. The intein products were similar, but not identical, in sequence and the splice junctions displayed all the features found in other polypeptides known to be produced by protein splicing from a precursor protein. Paired motifs, found in homing endonucleases encoded by some group I RNA introns, and inteins showing endonuclease activity, were present in the gyrA inteins as were other intein-specific signatures. Some strains of M. flavescens, M. gordonae, and M. kansasii were shown by PCR analysis to have inteinless gyrA genes, in contrast to the situation in M. leprae where all the isolates possessed insertions in gyrA. Sequencing of the corresponding regions revealed that, although the GyrA protein sequence was conserved, the nucleotide sequences differed in gyrA genes with and without inteins, suggesting that the homing endonuclease displays sequence specificity.
A new, well-documented case of Mycobacterium shimoidei infection is reported. This bacterium was isolated from a patient with preexisting pulmonary lesions who developed a tuberculosis-like cavity in the lung. Isolation of fewer than ten strains, not epidemiologically related, has been reported worldwide; all originated from human pulmonary samples. Identification of the strain described here was first accomplished by 16S rDNA direct sequencing after in vitro amplification. This method allows rapid identification of unusual bacteria. The identification was confirmed by specific phenotypical tests and by chromatographic methods.
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MICs of clarithromycin were determined by the Etest method for thirty clinical strains of Mycobacterium avium complex (MAC) and compared with MICs results as determined by the reference agar dilution method. Agreement (within +/- 1 log2 dilution) between the Etest and the reference method was 70% for susceptible strains and 100% for resistant strains. No major errors resulting in misclassification in susceptibility or resistance categories were detected for the Etest MIC method. It is suggested that the Etest is easy to perform and is an accurate method for determining susceptibility of MAC strains to clarithromycin.
During previous cooperative numerical taxonomic studies of slowly growing mycobacteria, the International Working Group on Mycobacterial Taxonomy described a number of strains whose taxonomic status was ambiguous. A new study of DNA, RNA, and proteins from 66 of these organisms was performed to correlate their properties with phenotypic clustering behavior; the results of this study permitted 51 of the strains studied to be assigned to known species. The methods used to characterize the semantides included nucleotide sequencing and assessment of levels of semantide relatedness by affinity binding techniques, including whole DNA-DNA hybridization, probe hybridization, and antibody binding. There was good overall agreement between the phenotypic and chemotaxonomic clusters and the groups of organisms identified by semantide analyses. Our results supported the conclusion that we should continue to rely on polyphasic taxonomy to provide satisfactory systematic resolution of members of the genus Mycobacterium. We identified no single 16S rRNA interstrain nucleotide sequence difference value that unequivocally defined species boundaries. DNA-DNA hybridization remains the gold standard, but common resources are needed to permit DNA-DNA hybridization analyses to be made available to laboratories that are not prepared to use this technology. One of the large novel clusters which we studied corresponds to the recently described species Mycobacterium interjectum, a pathogen that resembles the nonpathogen Mycobacterium gordonae phenotypically. We also identified strains that appear to represent ribovars of Mycobacterium intracellulare which do not react with the commercial diagnostic probes that are currently used for identification of this species. Other branches or clusters consisted of too few strains to permit a decision about their taxonomic status to be made.
An insertion sequence (IS) of Mycobacterium xenopi has been isolated and sequenced. This 1323 bp element, designated IS1395, is present in up to 18 copies in the M. xenopi genome and may be harboured in an M. xenopi extrachromosomal element. It encodes a putative transposase of 415 amino acids which displays sequence homology to the Staphylococcus aureus IS256 family. Members of this class of elements have been described in the genus Mycobacterium-for example IS1081 is present in the M. tuberculosis complex, IS1245-IS1311 in M. avium, and IS6120 in M. smegmatis; these elements exhibit an 89%, 45% and 16% amino acid identity with IS1395, respectively. Investigation of the host range of IS1395 by Southern blot analysis revealed additional IS1395-related repeated sequences in M. gordonae and M. celatum. Moreover, IS1395 represents a useful epidemiological tool for M. xenopi strain typing as it provides a diversity of restriction fragment length polymorphism patterns.
Selective amplification of a 187-bp fragment within the DT6 sequence using the AV6 and AV7 primers for Mycobacterium avium and of a 666-bp fragment within the DT1 sequence of Mycobacterium intracellulare using the IN38 and IN41 primers was performed for 69 clinical isolates identified as M. avium complex by conventional methods. The results were compared in parallel with results with commercial M. avium and M. intracellulare probes. A positive response to either of the two PCRs or M. avium-M. intracellulare AccuProbes constituted positive detection as M. avium complex; this cumulative detection limit was 94.2% for PCR, compared with 90% for AccuProbe. Concordance, on the other hand, was considered an identical species identification using either DT1 PCR and the M. intracellulare probe or DT6 and DT1 PCRs are inexpensive and at least equally sensitive, in-house options to the AccuProbe system for species identification of M. avium and M. intracellulare.
A Mycobacterium avium typing method based on PCR amplification of genomic sequences located between the recently described repetitive elements IS1245 and IS1311 was developed. This method was applied to a set of epidemiologically related and unrelated strains and compared with restriction fragment length polymorphism analysis with IS1245 as the probe. This PCR typing consists of a rapid and simple technique, providing a reproducible M. avium characterization as discriminant as restriction fragment length polymorphism analysis.
Typing of the glycopeptidolipid antigens performed by thin layer chromatography on 59 Mycobacterium avium-intracellulare (MAC) strains isolated in Italy from AIDS patients showed that the most frequent types were 1, 4, 3, 8, and 21 (24, 19, 14, 14 and 8% of the strains, respectively). Among non-AIDS patients, types 1, 4 and 8 were also frequently found. The antimicrobial susceptibility tested in agar and/or liquid media to a panel of drugs indicated in clofazimine and rifabutin effective agents against both AIDS and non-AIDS strains. The data obtained show that MAC type distribution in Italy appears to be different from that reported for other countries. No major differences in drug susceptibility between AIDS and non-AIDS related strains were found.
Four multiply disabled, profoundly retarded young adults received two series of instructional activities designed to teach them to use handguiding to communicate basic needs and intents. All had failed in previous attempts to learn to communicate using augmentative devices and strategies. Caregivers and staff served as partners in communication. Three of the four subjects were successful in learning some basic communication skills using the strategy of having a partner and handguiding as a technique.