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M Trucksis

Publications and source records attributed to M Trucksis.

At least 19 recordsLinked to original sources
PubMed2000

Vibrio cholerae ACE stimulates Ca(2+)-dependent Cl(-)/HCO(3)(-) secretion in T84 cells in vitro.

ACE, accessory cholera enterotoxin, the third enterotoxin in Vibrio cholerae, has been reported to increase short-circuit current (I(sc)) in rabbit ileum and to cause fluid secretion in ligated rabbit ileal loops. We studied the ACE-induced change in I(sc) and potential difference (PD) in T84 monolayers mounted in modified Ussing chambers, an in vitro model of a Cl(-) secretory cell. ACE added to the apical surface alone stimulated a rapid increase in I(sc) and PD that was concentration dependent and immediately reversed when the toxin was removed. Ion replacement studies established that the current was dependent on Cl(-) and HCO(3)(-). ACE acted synergistically with the Ca(2+)-dependent acetylcholine analog, carbachol, to stimulate secretion in T84 monolayers. In contrast, the secretory response to cAMP or cGMP agonists was not enhanced by ACE. The ACE-stimulated secretion was dependent on extracellular and intracellular Ca(2+) but was not associated with an increase in intracellular cyclic nucleotides. We conclude that the mechanism of secretion by ACE involves Ca(2+) as a second messenger and that this toxin stimulates a novel Ca(2+)-dependent synergy.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2000

Transformation and transposition of the genome of Mycobacterium marinum.

OBJECTIVE: To develop and evaluate protocols for genetic manipulations (transformation and transposition) of the fish pathogen, Mycobacterium marinum. SAMPLE POPULATION: Isolates of M. marinum obtained from fish and humans. PROCEDURE: Electrocompetent cells were prepared from isolates of M. marinum grown to various growth phases at several temperatures and with or without the addition of ethionamide or cycloheximide. Mycobacterial cells were transformed by electroporation with a replicative Escherichia coli-mycobacteria shuttle vector (pYUB18) as well as suicide vectors (pYUB285 and pUS252) that carried transposable elements (IS1096 and IS6110, respectively). Mutants from both isolates of M. marinum were recovered on 7H10 agar plates supplemented with kanamycin. Transformation and transposition efficiencies for various protocols were compared. Southern hybridization analysis was performed on mycobacterial mutants to confirm transposition events. RESULTS: Competent cells prepared at room temperature (23-25 C) from organisms in late-exponential growth phase yielded higher transposition efficiency, compared with cells prepared at 4 C or from organisms in early- or mid-exponential growth phase. Naturally developing kanamycin-resistant colonies of M. marinum were not detected. Only the IS1096-derived transposition was able to efficiently mutate M. marinum. Southern hybridization of M. marinum mutants revealed random integration of IS 1096 into the M. marinum genome. CONCLUSIONS: Transposition and transformation efficiencies were comparable, suggesting that the limiting factor in transposition is the transformation step. Most of the experiments resulted in transposition of IS1096; however, better approaches are needed to improve transposition efficiency.

Animals↗
PubMed1999

Pathogenicity of Mycobacterium fortuitum and Mycobacterium smegmatis to goldfish, Carassius auratus.

Despite the ubiquitous presence of atypical mycobacteria in the environment and the potential risk of infection in humans and animals, the pathogenesis of diseases caused by infection with atypical mycobacteria has been poorly characterized. In this study, goldfish, Carassius auratus were infected either with the rapidly growing fish pathogen, Mycobacterium fortuitum or with another rapidly growing mycobacteria, Mycobacterium smegmatis. Bacterial persistence and pathological host response to mycobacterial infection in the goldfish are described. Mycobacteria were recovered from a high percentage of inoculated fish that developed a characteristic chronic granulomatous response similar to that associated with natural mycobacterial infection. Both M. fortuitum and M. smegmatis were pathogenic to fish. Fish infected with M. smegmatis ATCC 19420 showed the highest level of giant cell recruitment compared to fish inoculated with M. smegmatis mc(2)155 and M. fortuitum. Of the three strains of mycobacteria examined, M. smegmatis ATCC 19420 was the most virulent strain to goldfish followed by M. fortuitum and M. smegmatis mc(2)155, respectively.

Animals↗
PubMed1999

Clonal diversity of Chilean isolates of enterohemorrhagic Escherichia coli from patients with hemolytic-uremic syndrome, asymptomatic subjects, animal reservoirs, and food products.

To determine clonal relationship among Chilean enterohemorrhagic Escherichia coli (EHEC) strains from different sources (clinical infections, animal reservoirs, and food), 54 EHEC isolates (44 of E. coli O157, 5 of E. coli O111, and 5 of E. coli O26) were characterized for virulence genes by colony blot hybridization and by pulsed-field gel electrophoresis (PFGE). By colony blotting, 12 different genotypes were identified among the 44 E. coli O157 isolates analyzed, of which the genetic profile stx1+ stx2+ hly+ eae+ was the most prevalent. All human O157 strains that were associated with sporadic cases of hemolytic-uremic syndrome (HUS) carried both the stx1 and stx2 toxin-encoding genes and were eaeA positive. Only 9 of 13 isolates from human controls were stx1+ stx2+, and 8 carried the eaeA gene. Comparison of profiles obtained by PFGE of XbaI-digested genomic DNA showed a great diversity among the E. coli O157 isolates, with 37 different profiles among 39 isolates analyzed. Cluster analysis of PFGE profiles showed a wide distribution of clinical isolates obtained from HUS cases and asymptomatic individuals and a clonal relationship among O157 isolates obtained from HUS cases and pigs. Analysis of virulence genes showed that a correlation exists among strains with the genotype stx1+ stx2+ eae+ and pathogenic potential. A larger difference in the PFGE restriction patterns was observed among the EHEC strains of serogroups O26 and O111. These results indicate that several different EHEC clones circulate in Chile and suggest that pigs are an important animal reservoir for human infections by EHEC. Guidelines have been proposed for better practices in the slaughter of animals in Chile.

Animals↗
PubMed1999

Superoxide production in phagocytes obtained from Mycobacterium marinum-stimulated goldfish (Carassius auratus) that were exposed to copper.

OBJECTIVE: To investigate the effects of copper exposure and recovery from copper toxicosis on the nonspecific immune response in Mycobacterium marinum-inoculated goldfish. ANIMALS: Goldfish (Carassius auratus) with a mean weight of 33.5 g. PROCEDURE: Superoxide (O2-) production was measured in fish 2 to 6 weeks after injection with phosphate-buffered saline (PBS) solution or M marinum (10(2) to 10(7) colony-forming units [CFU]/fish). Then, paired groups of fish were injected with PBS solution or 10(4) CFU of M marinum and exposed to copper (100 microg/L) for 7 days or for 4 days with 3 days of recovery. One paired group not exposed 14 days later to copper served as control fish. Phagocyte production of O2-was measured by use of the nitroblue tetrazolium reduction assay. Inflammation and bacterial colony counts were determined by use of routine histologic and microbiologic procedures. RESULTS: Superoxide production achieved a maximal response 2 to 4 weeks after M marinum inoculation. Compared with control fish, O2- production increased in the groups exposed to copper but then decreased in the exposed groups that were allowed to recover. Superoxide response and peritoneal inflammation were greater in M marinum-inoculated groups than in non-inoculated groups. CONCLUSIONS: Copper exposure and inoculation with M marinum increased O2- production, whereas recovery after exposure decreased O2- production, even in fish that were immunostimulated by M marinum. CLINICAL RELEVANCE: When the antimicrobial oxidative response is suppressed after copper exposure, steps should be taken to avoid imposing additional stress and minimize the possibility of resurgent or secondary pathogenic infections.

Animals↗
PubMed1998

The Vibrio cholerae genome contains two unique circular chromosomes.

Vibrio cholerae, the etiologic agent of the diarrheal disease cholera, is a Gram-negative bacterium that belongs to the gamma subdivision of the family Proteobacteriaceae. The physical map of the genome has been reported, and the genome has been described as a single 3.2-Mb chromosome [Majumder, R., et al. (1996) J. Bacteriol. 178, 1105-1112]. By using pulsed-field gel electrophoresis of genomic DNA immobilized in agarose plugs and digested with the restriction enzymes I-CeuI, SfiI, and NotI, we have also constructed the physical map of V. cholerae. Our analysis estimates the size of the genome at 4.0 Mb, 25% larger than the physical map reported by others. Our most notable finding is, however, that the V. cholerae chromosome appears to be not the single chromosome reported but two unique and separate circular megareplicons.

Chromosome Mapping↗
PubMed1998

Goldfish, Carassius auratus, a novel animal model for the study of Mycobacterium marinum pathogenesis.

We have developed an animal model for studying mycobacterial pathogenesis using Mycobacterium marinum and the goldfish, Carassius auratus. Goldfish are injected intraperitoneally with doses between 10(2) and 10(9) CFU of M. marinum organisms. Depending on the dose of M. marinum organisms administered, an acute or chronic disease is produced. The acute disease is characterized by systemic mycobacterial infection, severe peritonitis, tissue necrosis, and a short median survival time. The chronic disease is characterized by granuloma formation in all organs and survival of animals to the end point of the experiment (56 days). Colony counts in organ homogenates showed recovery of mycobacteria from a high percentage of inoculated animals. We believe this well-characterized animal model will be useful for studying mycobacterial pathogenesis.

Acute Disease↗
PubMed1997

Disseminated cutaneous zoster and aseptic meningitis in a previously healthy patient.

A previously healthy, 37-year-old immunocompetent man presented with disseminated cutaneous zoster and aseptic meningitis. Varicella zoster virus DNA was recovered from the cerebrospinal fluid (CSF) by the polymerase chain reaction. Cytological evaluation of the CSF revealed 'reactive, highly atypical lymphocytosis'. The patient fully recovered after treatment with aciclovir.

Acyclovir↗
PubMed1997

Identification of mycobacteria infecting fish to the species level using polymerase chain reaction and restriction enzyme analysis.

An assay is described utilizing PCR technology for a rapid diagnostic test to identify fish infection with Mycobacterium marinum, M. fortuitum and M. chelonae. A 924 bp DNA fragment from a highly conserved area of the mycobacterial 16S rRNA gene was amplified using mycobacteria genus-specific primers and digested with restriction enzymes (BanI and ApaI). This examination yielded unique restriction patterns for each mycobacterial specie enabling identification of mycobacteria infecting fish to the species level. The protocol can be applied to purified DNA, a simple colony preparation or infected fish tissue. This protocol can be completed in 1-2 days.

Animals↗
PubMed1997

Production of Vibrio cholerae accessory cholera enterotoxin (Ace) in the yeast Pichia pastoris.

Accessory cholera enterotoxin (Ace) is a recently identified toxin of Vibrio cholerae. Preliminary studies using crude toxin extracts in animal models indicate that Ace increases transcellular ion transport, which is proposed to contribute to diarrhea in cholera. The lack of purified toxin has hindered elucidation of the mechanism of action of Ace. In this study, ace was cloned and was expressed in and secreted by the methylotrophic yeast Pichia pastoris. Secreted toxin constituted 50% of the total supernatant protein from Pichia pastoris. Presumed monomer and dimer forms with molecular masses of 9 and 18 kDa, respectively, were observed. The 18-kDa form predominated. Biological activity was assayed by studying ion fluxes across epithelial membranes in Ussing chambers. Among the characteristics of Ace was the unusual property of staining with silver but not Coomassie blue stain. To our knowledge this is the first report of a biologically active bacterial toxin produced with the P. pastoris system. The purified protein may now be used in studies of the mechanism of action of Ace in physiologic systems.

Cholera Toxin↗
PubMed1997

Cloning and characterization of the Bacteroides fragilis metalloprotease toxin gene.

Strains of Bacteroides fragilis that produce a ca. 20-kDa heat-labile protein toxin (termed B. fragilis toxin [BFT]) have been associated with diarrheal disease of animals and humans. BFT alters the morphology of intestinal epithelial cells both in vitro and in vivo and stimulates secretion in ligated intestinal segments of rats, rabbits, and lambs. Previous genetic and biochemical data indicated that BFT was a metalloprotease which hydrolyzed G (monomeric) actin, gelatin, and azocoll in vitro. In this paper, the cloning and sequencing of the entire B. fragilis toxin gene (bft) from enterotoxigenic B. fragilis (ETBF) 86-5443-2-2 is reported. The bft gene from this ETBF strain consists of one open reading frame of 1,191 nucleotides encoding a predicted 397-residue holotoxin with a calculated molecular weight of 44,493. Comparison of the predicted BFT protein sequence with the N-terminal amino acid sequence of purified BFT indicates that BFT is most probably synthesized by ETBF strains as a preproprotein. These data predict that BFT is processed to yield a biologically active toxin of 186 residues with a molecular mass of 20.7 kDa which is secreted into the culture supernatant. Analysis of the holotoxin sequence predicts a 20-residue amphipathic region at the carboxy terminus of BFT. Thus, in addition to the metalloprotease activity of BFT, the prediction of an amphipathic domain suggests that oligomerization of BFT may permit membrane insertion of the toxin with creation of a transmembrane pore. Comparison of the sequences available for the bft genes from ETBF 86-5443-2-2 and VPI 13784 revealed two regions of reduced homology. Hybridization of oligonucleotide probes specific for each bft to toxigenic B.fragilis strains revealed that 51 and 49% of toxigenic strains contained the 86-5433-2-2 and VPI 13784 bft genes, respectively. No toxigenic strain hybridized with both probes. We propose that these two subtypes of bft be termed bft-1 (VPI 13784) and bft-2 (86-5433-2-2).

Amino Acid Sequence↗
PubMed1996

Quinolone resistance mutations in topoisomerase IV: relationship to the flqA locus and genetic evidence that topoisomerase IV is the primary target and DNA gyrase is the secondary target of fluoroquinolones in Staphylococcus aureus.

Mutations in the flqA (formerly ofx/cfx) resistance locus of Staphylococcus aureus were previously shown to be common after first-step selections for resistance to ciprofloxacin and ofloxacin and to map on the S. aureus chromosome distinctly from gyrA, gyrB, and norA.grlA and grlB, the genes for the topoisomerase IV of S. aureus, were identified from a genomic lambda library on a common KpnI fragment, and grlB hybridized specifically with the chromosomal SmaI A fragment, which contains the flqA locus. Amplification of grlA sequences (codons 1 to 251) by PCRs from nine independent single-step flqA mutants, one multistep mutant, and the parent strain identified mutations encoding a change from Ser to Phe at position 80 in four mutants, a novel change from Ala to either Glu or Pro at position 116 in three mutants, and no change in three mutants. In the multistep mutant, another resistance locus, flqC, was mapped by transformation to the chromosomal SmaI G fragment by linkage to omega(ch::Tn551)1051 (58%) and nov (97.9%), which encodes resistance to novobiocin. This fragment contains the gyrA gene, and flqC mutants had a mutation in gyrA encoding a change from Ser to Leu at position 84, a change previously found in resistant clinical isolates. In genetic outcrosses, the flqC (gyrA) mutation expressed resistance only in flqA mutants, including those with both types of grla mutations. The silent mutant allele of gyrA was present in a flqA background and expressed resistance only upon introduction of a grlA mutation. At fourfold the MIC of ciprofloxacin, the bactericidal activity of ciprofloxacin was reduced in a grlA mutant and was abolished in gyrA grlA double mutants. These findings provide direct genetic evidence that topoisomerase IV is the primary target of current fluoroquinolones in S. aureus and that this effect may result from the greater sensitivity of topoisomerase IV relative to that of DNA gyrase to these agents. Furthermore, resistance from an altered DNA gyrase requires resistant topoisomerase IV for its expression.

Anti-Infective Agents↗
PubMed1994

Quinolone resistance mediated by norA: physiologic characterization and relationship to flqB, a quinolone resistance locus on the Staphylococcus aureus chromosome.

We identified a quinolone resistance locus, flqB, linked to transposon insertion omega 1108 and fus on the SmaI D fragment of the Staphylococcus aureus NCTC 8325 chromosome, the same fragment that contains the norA gene. S. aureus norA cloned from flqB and flqB+ strains in Escherichia coli differed only in a single nucleotide in the putative promoter region. There was no detectable change in the number of copies of norA on the chromosomes of flqB strains, but they had increased levels of norA transcripts. Cloned norA produced resistance to norfloxacin and other hydrophilic quinolones and reduced norfloxacin accumulation in intact cells that was energy dependent, suggesting active drug efflux as the mechanism of resistance. Drug efflux was studied by measurement of norfloxacin uptake into everted inner membrane vesicles prepared from norA-containing E. coli cells. Vesicles exhibited norfloxacin uptake after the addition of lactate or NADH, and this uptake was abolished by carbonyl cyanide m-chlorophenylhydrazone and nigericin but not valinomycin, indicating that it was linked to the pH gradient across the cell membrane. Norfloxacin uptake into vesicles was also saturable, with an apparent Km of 6 microM, a concentration between those that inhibit the growth of flqB and flqB+ S. aureus cells, indicating that drug uptake is mediated by a carrier with a high apparent affinity for norfloxacin. Ciprofloxacin and ofloxacin competitively inhibited norfloxacin uptake into vesicles. Reserpine, which inhibits the multidrug efflux mediated by the bmr gene of bacillus subtilis, which is similar to norA, abolished norfloxacin uptake into vesicles as well as the norfloxacin resistance of an flqB mutant, suggesting a potential means for circumventing quinolone resistance as a result of drug efflux in S. aureus. These findings indicate that the chromosomal flqB resistance locus is associated with increased levels of expression of norA and strongly suggest that the NorA protein itself functions as a drug transporter that is coupled to the proton gradient across the cell membrane.

Amino Acid Sequence↗
PubMed1993

Accessory cholera enterotoxin (Ace), the third toxin of a Vibrio cholerae virulence cassette.

Vibrio cholerae causes the potentially lethal disease cholera through the elaboration of the intestinal secretogen cholera toxin. A second toxin of V. cholerae, Zot, decreases intestinal tissue resistance by modifying intercellular tight junctions. In this report, a third toxin of V. cholerae, Ace (accessory cholera enterotoxin), is described. Ace increases short-circuit current in Ussing chambers and causes fluid secretion in ligated rabbit ileal loops. The predicted protein sequence of Ace shows striking similarity to eukaryotic ion-transporting ATPases, including the product of the cystic fibrosis gene. The gene encoding Ace is located immediately upstream of the genes encoding Zot and cholera toxin. The ctx, zot, and ace genes, which are located on a dynamic sector of the chromosome, comprise a V. cholerae "virulence cassette."

Amino Acid Sequence↗
PubMed1991

A novel locus conferring fluoroquinolone resistance in Staphylococcus aureus.

Fluoroquinolones such as ciprofloxacin and ofloxacin are potent antimicrobial agents that antagonize the A subunit of DNA gyrase. We selected and mapped a novel fluoroquinolone resistance gene on the Staphylococcus aureus chromosome. Resistant mutants were selected with ciprofloxacin or ofloxacin and were uniformly localized to the A fragment of chromosomal DNA digested with SmaI and arrayed by pulsed-field gel electrophoresis. Several mutants (cfxB, ofxC) were genetically mapped between the thr and trp loci in the A fragment. A majority of A fragment fluoroquinolone resistance mutations were associated with reduced susceptibility to novobiocin, an antagonist of the B subunit of DNA gyrase. Two genes previously associated with fluoroquinolone resistance, the gyrA gene of DNA gyrase and the norA gene (associated with decreased drug accumulation), were localized to the G and D fragments, respectively. Thus, the fluoroquinolone resistance mutations in the A fragment are distinct from previously identified fluoroquinolone resistance mutations in gyrA and norA. Whether mutations in the A fragment after a second topoisomerase or another gene controlling supercoiling or affect drug permeation is unknown.

Anti-Infective Agents↗
PubMed1981

Identification and localization of a gene that specifies production of Escherichia coli DNA topoisomerase I.

A gene that specifies production of Escherichia coli DNA topoisomerase I (omega protein) was identified with the aid of a radioimmunoassay for this protein. E. coli DNA topoisomerase I was produced by Salmonella typhimurium merodiploids that harbored E. coli plasmid F' 123, but not by strains that lost this plasmid. Analysis of strains with spontaneous deletions of F' 123 showed that the gene, topA, required for production of the E. coli omega protein was between the trp operon and the cysB gene. Deletions that eliminated topA also eliminated the supX gene. We suggest that topA is the structural gene of E. coli DNA topoisomerase I and that topA is identical to supX.

Chromosome Mapping↗