Detection of a chloramphenicol efflux system in Escherichia coli isolated from poultry carcass.

An active chloramphenicol efflux system was demonstrated in a multiresistant E. coli isolated from poultry carcass. The effect of different concentrations of chloramphenicol on the original strain and on the plasmid-cured strain was determined in the presence and in the absence of CCCP, an uncoupler of the proton-motive force. Minimal inhibitory concentration (MIC) was lower in the presence of CCCP in the original strain. The plasmid-cured strain displayed lower resistance for chloramphenicol than the wild type, but the MIC was not affected by CCCP. The combined results indicate a plasmid encoded energy dependent resistance mechanism. 3H-chloramphenicol accumulation within the cells was measured by scintillation counting. The uptake or the efflux of 3H-chloramphenicol was influenced by CCCP in the original strain, but not in the plasmid-cured strain. More than one chloramphenicol resistance mechanism may exist in this strain. E. coli is an important commensal or pathogen that inhabits the gastrointestinal tracts of humans and animals, so a plasmid encoded active drug resistance mechanism can be a potential source of horizontal transfer of resistance.

Secretion of the virulence-associated Campylobacter invasion antigens from Campylobacter jejuni requires a stimulatory signal.

Campylobacter jejuni are a common cause of human diarrheal illness. Previous work has demonstrated that C. jejuni synthesize a novel set of proteins upon coculturing with epithelial cells, some of which are secreted. The secreted proteins have been collectively referred to as Campylobacter invasion antigens (Cia proteins). Metabolic labeling experiments revealed that Cia protein synthesis and secretion are separable and that secretion is the rate-limiting step of these processes. Additional work indicated that Cia protein synthesis is induced in response to bile salts and various eukaryotic host cell components. Host cell components also can induce Cia protein secretion. Culturing C. jejuni on plates supplemented with the bile salt deoxycholate retarded the inhibitory effect of chloramphenicol on C. jejuni invasion, as judged by the gentamicin-protection assay. These data suggest that the coordinate expression of the genes encoding the Cia proteins is subject to environmental regulation.

Inactivation of chloramphenicol by O-phosphorylation. A novel resistance mechanism in Streptomyces venezuelae ISP5230, a chloramphenicol producer.

Plasmid pJV4, containing a 2.4-kilobase pair insert of genomic DNA from the chloramphenicol (Cm) producer Streptomyces venezuelae ISP5230, confers resistance when introduced by transformation into the Cm-sensitive host Streptomyces lividans M252 (Mosher, R. H. Ranade, N. P., Schrempf, H., and Vining, L. C. (1990) J. Gen. Microbiol. 136, 293-301). Transformants rapidly metabolized Cm to one major product, which was isolated and purified by reversed phase chromatography. The metabolite was identified by nuclear magnetic resonance spectroscopy and mass spectrometry as 3'-O-phospho-Cm, and was shown to have negligible inhibitory activity against Cm-sensitive Micrococcus luteus. The nucleotide sequence of the S. venezuelae DNA insert in pJV4 contains an open reading frame (ORF) that encodes a polypeptide (19 kDa) with a consensus motif at its NH2 terminus corresponding to a nucleotide-binding amino acid sequence (motif A or P-loop; Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 1, 945-951). When a recombinant vector containing this ORF as a 1.6-kilobase pair SmaI-SmaI fragment was used to transform S. lividans M252, uniformly Cm-resistant transformants were obtained. A strain of S. lividans transformed by a vector in which the ORF had been disrupted by an internal deletion yielded clones that were unable to phosphorylate Cm, and exhibited normal susceptibility to the antibiotic. The results implicate the product of the ORF from S. venezuelae as an enzymic effector of Cm resistance in the producing organism by 3'-O-phosphorylation. We suggest the trivial name chloramphenicol 3'-O-phosphotransferase for the enzyme.

[The course of typhoid fever in levomycetin resistance of the causative agent of the disease]. / O techenii briushnogo tifa pri rezistentnosti vozbuditelia bolezni k levomitsetinu.

Kept under observation was a patient aged 21 years, who had come from India, from whose blood taken at day 45 typhoid fever a causative agent S. typhi resistant to chloramphenicol, ampicillin, polymyxin was isolated. The condition presented with fever of long duration (60 days), apparent intoxication, jaundice, development of appendicular symptoms and intestinal hemorrhage continueing for 10 days. Treatment with chloramphenicol, ampicillin, gentamicin, furazolidone appeared to be ineffective. Detoxicational and hemostatic therapies were tried. The patient resumed his health.

Interactions in vitro between agents used to treat melioidosis.

Combinations of antimicrobial agents are usually recommended for the treatment of melioidosis. In Thailand, the conventional treatment regimen for Pseudomonas pseudomallei infections is a combination of chloramphenicol, doxycycline and cotrimoxazole. We have consistently observed antagonism between these agents during routine disc susceptibility testing. Interactions between these antimicrobials were studied further by a chequerboard microdilution method, using five clinical isolates of P. pseudomallei. Both trimethoprim and sulphamethoxazole antagonised the bacteriostatic activity of chloramphenicol and doxycycline. The combination of trimethoprim and sulphamethoxazole was synergistic, but not bactericidal. Bacteriostatic drugs antagonised the bactericidal activity of ceftazidime, which is undergoing clinical trials in severe melioidosis. These findings may be of therapeutic relevance.

[The functioning of mammalian mitochondria injected into fish embryos]. / Funktsionirovanie mitokhondrii mlekopitaiushchikh, in''etsirovannykh v zarodyshi ryb.

The possibility of mammalian mitochondria functioning in fish embryos has been studied. Suspension of mitochondria isolated from the mouse fibroblast B-82/cap (chloramphenicol-resistant) and B-82 (chloramphenicol sensitive) cell cultures, were injected into the fertilized loach eggs. These embryos with an artificially increased number of mouse mitochondria developed and lived till the larval stages. Activity of cytochrome oxidase in these embryos was 1.5-2 times that in the control several hours after the injection, decreased during development and reached the control level by the gastrula stage. If these embryos with artificially increased number of mouse mitochondria were incubated in presence of chloramphenicol, only embryos that contained mitochondria from chloramphenicol-resistant cells survived, thus suggesting that the injected mitochondria do not degrade but are preserved and function in the cytoplasm of developing loach embryos.

[Presence of genetic exchange in Crithidia oncopelti (Trypanosomatidae) studied with drug resistance markers]. / Issledovanie nalichiia geneticheskogo obmena u Crithidia oncopelti (Trypanosomatidae) s pomoshch'iu markerov lekarstvennoi rezistentnosti.

Strains resistant to 100 mkg/ml of cycloheximide (ChxR100) and to 2.5 mg/ml of chloramphenicol (CapR2.5) were used in studies for the presence of genetic interchange in C. oncopelti. Phenotypes of these strains were stable on the nonselective medium and specific. Strains ChxR100 and CapR2.5 were mixed in equal proportions, cultivated during 7 days and tested. Under consecutive effect of inhibitors the culture of strains mixture and clones isolated from it exhibited double resistance. Phenotypes of recombinant clones were stable on the nonselective medium during, at the minimum, 70 to 90 cell generations. At the same conditions control cultures of parental strains and clones did not exhibit resistance that excludes the possibility of origin of cells resistant to both antibiotics from Crithidia of one of the parental strains as a result of secondary mutation. Thus, the results obtained show convincingly the presence of genetic interchange between flagellates of ChxR100 and CapR2.5 strains.

[Cloning of the regulator gene of Erwinia carotovora repressing pectate lyase ptlA gene expression]. / Klonirovanie gena-reguliatora Erwinia carotovora, podavliaiushchego ékspressiiu gena pektatliazy ptlA.

Pectate lyase synthesis in the cells of Erwinia carotovora ELA 49 is induced by polypectate. This suggested that the Erwinia chromosomes carried a regulator gene responsible for negative regulation of the pectate lyase gene expression. In the present study the regulator gene controlling expression of one of the pectate lyase structural genes was cloned and designated as ptlA gene. For this purpose a genetic system with the tester plasmid pPc624 as the main element was constructed. The tester plasmid contained cat gene (resistance to chloramphenicol) controlled by the promotor of the ptlA gene cloned on vector pPD620. Plasmid pPC624 was maintained in the E. coli cells in a number of 1-2 copies and transferred resistance to chloramphenicol in concentrations up to 100 micrograms/ml to the cells. The E. carotovora cells containing pPC624 were sensitive to chloramphenicol in media containing no inductor (sodium polypectate). In media with the inductor they were resistant to chloramphenicol. Therefore, plasmid pPC624 proved to be a suitable system for testing the regulator gene product. The E. coli cells containing plasmid pPC624 were transformed by the hybrid Ptl+ plasmids identified in the clonotheque of the Erwinia DNA EcoRI fragments. The E. coli cotransformants were characterized by chloramphenicol sensitivity which provided a conclusion that the regulator ptlR gene controlling the ptlA gene expression was localized on the DNA EcoRI fragment (7.3 kb) containing the pectate lyase ptlA and ptlB genes. Deletion analysis showed that the investigated genes were localized in the EcoRI fragment (7.3 kb) of the E. carotovora chromosomal DNA in the following order: ptlA--ptlB--ptlR.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA damage induced by chloramphenicol and nitroso-chloramphenicol: protection by N-acetylcysteine.

We have previously demonstrated that nitroso-chloramphenicol (NO-CAP) in small concentrations causes the hydrolysis of isolated double stranded DNA in vitro and this action is blocked by sulfhydryl groups. The present study was designed to assess damage to isolated DNA as well as intact cells DNA and examine the protective effect of N-acetylcysteine (NAC). Using alkaline sucrose gradient sedimentation and the alkali elution technique of Kohn we were able to demonstrate DNA damage in Raji cells as well as phytohemagglutinin stimulated human lymphocytes after exposure to NO-CAP. Damage could be totally blocked by NAC. In preliminary studies we also observed that NAC protects bone marrow cells from the growth-inhibitory effects of chloramphenicol and thiamphenicol.

The effect of nitroso-chloramphenicol on mitochondrial DNA polymerase activity.

We studied the effects of nitroso-chloramphenicol, chloramphenicol, amino-chloramphenicol, and thiamphenicol on the activity of mitochondrial DNA polymerase of rat liver. 3H-thymidine triphosphate incorporation into DNA was used to measure the DNA polymerase activity in the mitochondrial matrix fraction. This fraction was in the supernatant of sonicated mitochondria obtained by ultracentrifugation. Under standard experimental conditions, thymidine triphosphate incorporation was time dependent up to 10 minutes. This activity was enhanced by beta-mercaptoethanol and was blocked by the known polymerase inhibitors ethidium bromide and 2',3'-dideoxythymidine 5'-triphosphate. Chloramphenicol and its analogues, amino-chloramphenicol and thiamphenicol, did not have a significant effect on the polymerase activity, whereas nitroso-chloramphenicol was inhibitory. The degree of inhibition was dependent on the experimental conditions. Thus, in the absence of beta-mercaptoethanol, nitroso-chloramphenicol caused inhibition; however, in its presence, there was no significant inhibitory effect. Under similar conditions, the addition of dithiothreitol also provided partial protection. On the other hand, the inhibition by nitroso-chloramphenicol was significantly enhanced with its preincubation in the mitochondrial matrix fraction before the addition of nucleotides and DNA; thus after 40 minutes of preincubation, nitroso-chloramphenicol at a concentration of 200 mumol/L gave 53% inhibition, and produced total inhibition at 600 mumol/L. The addition of NADH or NADPH to the preincubation medium produced substantial protection against nitroso-chloramphenicol, whereas nicotinamide-adenine dinucleotide had no effect. These results suggest that mitochondrial DNA polymerase may be a target for nitroso-chloramphenicol action. The potentiation of that action by preincubation and the protection against it by NADH and NADPH suggest the involvement of intermediate metabolic steps for maximal inhibition.

[Insertion of transposon Tn9 into the spinal (Escherichia coli-Saccharomyces cerevisiae) plasmids and the expression of the prokaryotic gene of chloramphenicol resistance in yeast cells]. / Vkliuchenie transpozona Tn9 v chelnochnye (Escherichia coli-Saccharomyces cerevisiae) plazmidy i ékspressiia prokarioticheskogo gena khloramfenikolustoichivosti v kletkakh drozhzhei.

Transposon Tn9 carrying camr gene which controls resistance to chloramphenicol has been introduced in vivo (in cells of Escherichia coli) into two chimeric shuttle plasmids pYF91 and YEp13. These plasmids consist of the different parts of the E. coli plasmid pBR322, the yeast 2mkm DNA plasmid and the yeast LEU2 structural gene. The plasmidis able to autonomously replicate in both yeast and bacterial cells. A recipient yeast strain carrying cams and leu2 markers was constructed to study the functional expression of the prokaryotic camr gene in eukaryotic yeast cells. The chimeric plasmids pYF91::Tn9 and YEp13::Tn9 were introduced into the yeast and bacterial recipient strains by transformation. The camr LEU2 yeast transformants were isolated. They were genetically unstable when grown on non-selective medium and they simultaneously lost camr and LEU2 markers with a frequency of 10 to 30%. The E. coli transformants were genetically stable under nonselective conditions and they maintain all plasmid markers. The chimeric plasmid pYF91::Tn9 was isolated from the yeast transformants and reintroduced into the cams leuB bacterial strain by transformation. The camr LEUB transformants were obtained. All these data confirm the possibility of the expression of the prokaryotic camr gene in yeast cells and present evidence for introduction of transposon Tn9 into chimeric plasmids.

[Rapid methods of detecting enzymatic resistance to ampicillin and chloramphenicol in Haemophilus influenzae]. / Méthodes rapides de détection de la résistance enzymatique a l'ampicilline et au chloramphénicol chez Haemophilus influenzae.

Several methods used for the detection of beta-lactamase activity in Haemophilus influenzae are described. The rapid iodemetric, acidimetric, and chromogenic cephalosporin techniques are specific tests for the presence of beta-lactamase. The Gots test can also be used for the detection of enzymatic resistance to ampicillin and chloramphenicol.

Mechanism of antibiotic resistance in Mycobacterium intracellulare.

The mechanism of resistance of Mycobacterium intracellulare strain 103 and other clinical isolates to a variety of drugs including aminoglycoside and peptide antibiotics was investigated. Enzymatic inactivation of aminoglycoside and peptide antibiotics could not be demonstrated. Ribosomes of the strain were found to be sensitive to the antibiotics. The levels of resistance of strain 103 and other clinical isolates decreased dramatically when the culture medium was changed from Dubos agar to Tween 80-containing agar. These results suggest that a permeability barrier is the reason for naturally occurring resistance in M. intracellulare.

[Intergeneric bacterial conjugation in crosses of Vibrio cholerae biotype proteus X Serratia marcescens]. / Mezrodovaia kon"iugatsiia bakterii pri skreshchivaniiakh Vibrio cholerae biotip proteus X Serratia marcescens.

Intergeneric conjugants were obtained in crosses of bacteria Vibrio cholerae biotype proteus (donor) x Serratia marcescens. The study of exconjugants demonstrates the following characteristics: 1. The majority of clones isolated possess some morphological characters of the donor (colourlessness and transparency of colonies) which gradually disappear during successive transfers and return to the phenotype of recipients (red colour of colonies); 2. Exconjugants acquire a plasmid factor of the fertility of vibrios (P-factor) and may transmit it to other cells; 3. The majority of exconjugants are agglutinated by immune sera of both donor and recipient; 4. The factor of streptomycine resistance is transmitted from the donor to a recipient. Conjugants acquire streptomycine resistance from the donor and laevomycetine (chloramphenicol) resistance from the recipient and can grow on a nutrient medium containing both antibiotics; 5. The conjugants isolated show a great diversity in a number of characters and, supposedly, form a genetically heterogenous group. A great part of exconjugants is characterized by a slow growth, some of them being not viable and unable to survive during transfers. In connection with instability of conjugants, we suppose that the exongenome is not incorporated into the chromosome of the recipient; more likely, it exists in a form of self-replicating duplex, or is connected with a plasmid genome.

[Analysis of insertions of the determinant of drug resistance to kanamycin and chloramphenicol into bacteriophage lambda att80]. / Analiz vnedrenii determinant lekarstvennoi ustoichivosti k kanamitsinu i khloramfenikolu v bakteriofag lambda att80.

The insertion sites of elements Tn9 and Tn601 which determine chloramphenicol and kanamycin resistance have been detected restriction analysis. The functioning of transposons i.e. their stability or instability, has been found to influence the specificity of their insertions into the genome of lambda att80 bacteriophage. During transposition from stable integration sites both transposons are inserted into the regions of the lambda att80 bacteriophage genome, definite for each transposon. However, during transposition from the site of unstable integration both determinants of drug resistance are inserted into different regions of the phage genome.