Crude Bacterial Lysates Research Articles

Molecular epidemiology of resistance to trimethoprim in enterobacteria isolated in a Parisian hospital

Between January, 1981 and December, 1984, 419 strains of enterobacteria isolated from patients at the Hôpital Saint-Joseph were studied for (1) the level of resistance to trimethoprim (Tp) by determination of minimal inhibitory concentration (MIC), (2) transferability of this resistance by conjugation into Escherichia coli, (3) plasmid content of wild-type strains and transconjugants by agarose gel electrophoresis of crude bacterial lysates and by incompatibility grouping, and (4) type of dihydrofolate reductase (DHFR) by colony hybridization with probes specific for DHFR types I and II. Tp resistance was defined as MIC greater than or equal to 4 micrograms/ml and high-level resistance by MIC greater than or equal to 1000 micrograms/ml. Amongst the strains studied, 90% were resistant to high levels of Tp, while 10% had low-level resistance to Tp was detected in 180 strains corresponding to 185 plasmids. In the vast majority of the plasmids, resistance to Tp was associated with resistance to sulphonamide (94%), streptomycin (75-90%), ampicillin (75-90%) and chloramphenicol (65-80%). Plasmids conferring resistance to Tp were often large, most (84%) ranging in size from 90 to 175 Kb. They belonged to six different incompatibility groups and Inc6-C was the most prevalent (34 to 75%). The study of the distribution of the dfr genes by colony hybridization in 183 transconjugants and 89 strains with non-transferable Tp resistance revealed the presence of dfrI genes in most of these strains (48 and 53%, respectively). DHFR of types I and II were found in only 3% of the transconjugants, but in 15% of the strains with non-transferable resistance. DHFR of other types were found equally (15%) in strains with transferable and non-transferable resistance. The high incidence of the type I enzyme among the Tp-resistant strains probably results from the integration of transposon Tn7 into the chromosome or into a non-transferable plasmid.

Transposable resistance to trimethoprim and 0/129 in Vibrio cholerae.

Vibrio cholerae biotype el tor strain BM2508, resistant to trimethoprim, 0/129, streptomycin and spectinomycin was isolated from the faeces of a child with severe diarrhoea. Resistance to trimethoprim and 0/129 was due to a dihydrofolate reductase type I and resistance to streptomycin-spectinomycin to a 3'',9-aminoglycoside-aminocyclitol adenylyltransferase. The resistance genes were not transferable to Escherichia coli and, as inferred from ultracentrifugation in cesium chloride-ethidium bromide and agarose gel electrophoresis of crude bacterial lysates, were located on the chromosome. The resistance genes were transposed to multiple sites of plasmids belonging to incompatibility groups 6-C and P, introduced in BM2508 and were subsequently transferred to E. coli (rec-), Salmonella typhimurium, V. cholerae and V. parahaemolyticus strains where they re-transposed into the chromosome. Analysis of plasmid DNA from the transconjugants by agarose gel electrophoresis following digestion with HindIII and by Southern hybridization using a ColEl::Tn7 probe indicated the presence of a 14-kilobase transposon, Tn1527, closely related to Tn7. The emergence of Tn1527 in V. cholerae may lead to prophylactic and therapeutic failures due to trimethoprim resistance and to bacterial misidentification because of cross resistance to 0/129.

Structural relationship between the genes encoding 3′-aminoglycoside phosphotransferases in Campylobacter and in gram-positive cocci

Campylobacter coli strain BM2509 resistant to ampicillin, chloramphenicol,erythromycin, kanamycin, spectinomycin, streptomycin and tetracycline was isolated from a patient with hospital-acquired diarrhoea. Resistance to kanamycin has not been thus far described in Campylobacter. Phosphocellulose paper-binding assay indicated that resistance to kanamycin and structurally related antibiotics in strain BM2509 was due to synthesis of a 3′-aminoglycoside phosphotransferase of type III (APH(3′)-III), an enzyme so far confined to Gram-positive cocci. The kanamycin and tetracycline resistances were transferable en bloc by conjugation to C. fetus but not to Escherichia coli. Analysis by agarose gel electrophoresis of crude bacterial lysates revealed the presence, in BM2509 and in the transconjugants, of a plasmid, pIP1433, with a size of 47.2 kilobases (Kb). Strain BM2509 also harboured a 4.5-Kb cryptic plasmid. DNA annealing studies indicated a close structural relationship between the kanamycin resistance gene of C. coli BM2509 and that representative of this type of resistance determinant in Gram-positive cocci. These results indicate that emergence of resistance to kanamycin in Campylobacter is due to acquisition in vivo of a gene or a plasmid from Gram-positive bacteria.

Pathogenesis of Shigella diarrhea. IX. Simplified high yield purification of Shigella toxin and characterization of subunit composition and function by the use of subunit-specific monoclonal and polyclonal antibodies.

A simple purification scheme for shigella cytotoxin was devised, resulting in high yields (approximately 50%) and a 1,300-fold increase in specific activity compared with the initial crude bacterial cell lysate. The purified toxin was enterotoxic in ligated rabbit ileal loops and neurotoxic when injected into the peritoneal cavity of mice. Measurement of specific activity of cytotoxin and enterotoxin demonstrated that these two toxicities copurify during the fractionation procedure. On sodium dodecyl sulfate gel electrophoresis, the toxin migrated as two polypeptide subunits, an A subunit of 32,000 mol wt and a B subunit of 6,500 mol wt. Chemical cross-linking experiments demonstrate that the toxin is a complex consisting of one A and five B subunits with a molecular weight of 64,000. Polyclonal rabbit anti-toxin and anti-subunit B antisera were produced as well as subunit-specific mouse monoclonal antibodies. All antibodies preincubated with toxin neutralized cytotoxic effects in HeLa cell monolayers. In contrast, only A subunit-specific antibodies were able to neutralize toxin prebound to the HeLa cell surface. Antibody to the B subunit also inhibited binding of 125I-labeled toxin to these cells by 94% or more. These data demonstrate that the B subunit is involved in shigella toxin binding to the cell surface.

Hepatitis B virus core antigen: synthesis in Escherichia coli and application in diagnosis.

Fragments of hepatitis B virus DNA cloned in plasmid pBR322 carrying the gene for the viral core antigen have been placed under the control of the lac promoter of Escherichia coli. Several of the new recombinants direct higher levels of synthesis of the antigen, but the degree of enhancement varies with the different structures of the plasmids and hence the mRNAs produced. The antigen in crude bacterial lysates is a satisfactory diagnostic reagent for antibodies to the core antigen in serum samples.

Method for obtaining more-accurate covalently closed circular plasmid-to-chromosome ratios from bacterial lysates by dye-buoyant density centrifugation

A method that gives high recovery of deoxyribonucleic acid (DNA) from crude bacterial lysates using ethidium bromide-cesium chloride density gradient centrifugation is presented. After Pronase digestion and shearing of the lysate, essentially 100% recovery of chromosomal DNA and a reproducible recovery of covalently closed circular (CCC) plasmid DNA is obtained for a specific plasmid in a given strain. This method should be useful for comparing the CCC plasmid/chromosome ratio of various plasmid-host combinations.