Frequency of R Factor-mediated Multiple Drug Resistance in Klebsiella and Aerobacter

A comparative study was done on the transfer frequency of R factors from 90 strains of multiple drug-resistant Aerobacter and 81 strains of Klebsiella to Escherichia coli CSH-2 (F(-), met(-), pro(-), Nal-r). The most common resistance patterns for the Aerobacter isolants were ampicillin streptomycin chloramphenicol tetracycline and ampicillin streptomycin chloramphenicol tetracycline kanamycin neomycin; for the Klebsiella isolants, the most common resistance pattern was ampicillin kanamycin streptomycin tetracycline chloramphenicol neomycin. R factors were isolated from 14.1% of the Aerobacter strains; 61.5% of these R factors harbored R determinants for ampicillin streptomycin tetracycline. R factors were isolated from 79.1% of the Klebsiella strains; four R factors were isolated with significant frequency; streptomycin chloramphenicol kanamycin neomycin, 37.5%; ampicillin streptomycin tetracycline kanamycin neomycin, 14.1%; ampicillin streptomycin tetracycline, 12.5%; and streptomycin chloramphenicol tetracycline, 12.5%.Chloramphenicol, kanamycin, and neomycin resistance was rarely transferred from the Aerobacter strains, although over 50% of the clinical isolants possessed resistance to these antibiotics. In contrast, over 75% of the Klebsiella strains transferred resistance to chloramphenicol, kanamycin, neomycin. Highest frequency of transferred resistance to individual drugs in the Aerobacter strains was to streptomycin (14.8%), whereas in the Klebsiella group resistance to four drugs was transferred at a very high frequency: streptomycin (80.8%), chloramphenicol (78.5%), kanamycin (76.4%), and neomycin (75.9%).     

Modification of in vitro metabolism of T-2 toxin by esterase inhibitors.

A shuttle vector that can replicate in both Streptococcus spp. and Escherichia coli has been constructed by joining the E. coli plasmid pACYC184 (chloramphenicol and tetracycline resistance) to the streptococcal plasmid pGB305 (erythromycin resistance). The resulting chimeric plasmid is designated pSA3 (chloramphenicol, erythromycin, and tetracycline resistance) and has seven unique restriction sites: EcoRI, EcoRV, BamHI, SalI, XbaI, NruI, and SphI. Molecular cloning into the EcoRI or EcoRV site results in inactivation of chloramphenicol resistance, and cloning into the BamHI, SalI, or SphI site results in inactivation of tetracycline resistance in E. coli. pSA3 was transformed and was stable in Streptococcus sanguis and Streptococcus mutans in the presence of erythromycin. We have used pSA3 to construct a library of the S. mutans GS5 genome in E. coli, and expression of surface antigens in this heterologous host has been confirmed with S. mutans antiserum. A previously cloned determinant that specifies streptokinase was subcloned into pSA3, and this recombinant plasmid was stable in the presence of a selective pressure and expressed streptokinase activity in E. coli, S. sanguis (Challis), and S. mutans.     

Amplifiable resistance to tetracycline, chloramphenicol, and other antibiotics in Escherichia coli: involvement of a non-plasmid-determined efflux of tetracycline.

Increasing levels of resistance to tetracycline and to a number of other unrelated antibiotics, including chloramphenicol, beta-lactams, puromycin, and nalidixic acid, occurred in Escherichia coli after 50 to 200 generations of growth in the presence of subinhibitory concentrations of tetracycline or chloramphenicol. In the absence of selective pressure, resistances fell to low levels within 100 generations of growth. This amplification of resistance was observed in laboratory and naturally occurring E. coli strains as well as in polA and recA strains. With the exception of previously identified cmlA and cmlB mutations, tetracycline or chloramphenicol resistances were not P1 transducible. Coincident with the emergence of resistance was the appearance of a previously cryptic energy-dependent efflux system for tetracycline. The expression of resistance phenotypes and the tetracycline efflux system were temperature sensitive at 42 degrees C.     

Specificity and mechanism of tetracycline resistance in a multiple drug resistant strain of Escherichia coli

Izaki, Kazuo (University of Tokyo, Tokyo, Japan), Kan Kiuchi, and Kei Arima. Specificity and mechanism of tetracycline resistance in a multiple drug resistant strain of Escherichia coli. J. Bacteriol. 91:628-633. 1966.-A decrease in the uptake of tetracycline occurred concurrently with a rise in the level of resistance of a multiple drug resistant strain of Escherichia coli grown in the presence of tetracycline. Although the strain was also resistant to streptomycin and chloramphenicol, growth in the presence of these two antibiotics did not influence the uptake of tetracycline. The induction of resistance, or decreased uptake of tetracycline, was dependent on growth of the organism in the presence of the drug. Decreased uptake of tetracycline could not be induced in a sensitive strain of the same organism under conditions suitable for induction of the resistant strain. The decrease in accumulating power of the resistant organism cultured in the presence of tetracycline does not appear to be due to selection of a resistant strain from cultures containing both resistant and sensitive strains.     

A spontaneous point mutation in the single 23S rRNA gene of the thermophilic arachaeon Sulfolobus acidocaldarius confers multiple drug resistance.

Development of transformable vectors for thermophilic archaea requires the characterization of appropriate selectable marker genes. Many antibiotic inhibitors of protein biosynthesis are known to bind to rRNA; therefore, we screened 14 for their capacity to inhibit growth of the thermophilic archaeon Sulfolobus acidocaldarius. Carbomycin, celesticetin, chloramphenicol, puromycin, sparsomycin, tetracycline, and thiostrepton all inhibited growth by different degrees. Spontaneous drug-resistant mutants were isolated from plates containing celesticetin or chloramphenicol. Six mutants from each plate exhibited a C-2585-to-U transition in the peptidyl transferase loop of 23S rRNA (corresponding to C-2452 in Escherichia coli 23S rRNA). The single-site mutation also conferred resistance to carbomycin. The mutated 23S rRNA gene provides a potentially useful and dominant marker for a thermophilic archael vector.     

Increased epidermal growth factor receptor in multidrug-resistant human neuroblastoma cells

Multidrug-resistant human neuroblastoma cell lines obtained by selection with vincristine or actinomycin D from two independent clonal lines, SH-SY5Y and MC-IXC, have 3- to 30-fold more cell surface epidermal growth factor (EGF) receptors than the drug-sensitive parental cells as indicated by EGF binding assays and immunoprecipitation, affinity-labeling, and phosphorylation studies. Reversion to drug sensitivity in one line was accompanied by a return to the parental level of EGF receptor. SH-EP cells, a clone derived from the same neuroblastoma cell line as SH-SY5Y but which displays melanocyte rather than neuronal lineage markers, also express significantly more EGF receptor than SH-SY5Y cells. By nucleic acid hybridization analysis with a molecularly cloned probe, increased receptor level in multidrug-resistant cells was shown to be the result of higher levels of EGF receptor mRNA in drug-resistant than in drug-sensitive cells. The increased steady state amount of specific RNA did not result from amplification of receptor-encoding genes. A small difference was observed in the electrophoretic mobility under denaturing conditions of EGF receptor immunoprecipitated from drug-resistant and drug-sensitive cells. Quantitative and qualitative modulation of the EGF receptor might reflect alterations in the transformation and/or differentiation phenotype of the resistant cells or might result from unknown selective pressures associated with the development of multidrug resistance.     

Characterisation of the binding of virginiamycin S to Escherichia coli ribosomes.

Virginiamycin S is an inhibitor of protein synthesis in vivo. In this paper we show by equilibrium dialysis that it binds specifically to the 50-S subunit of Escherichia coli ribosomes, with one binding site per subunit. This binding is not altered by the presence of chloramphenicol, tetracycline or puromycin but is competed for by erythromycin. Using the splitting-reconstitution method, it could be demonstrated that protein L16 is absolutely required for the binding of virginiamycin S to the 50-S subunit.     

Chloramphenicol stimulation of lysogeny by lambda regulatory mutants.

Inhibition of protein synthesis in Escherichia coli by amino acid starvation or chloramphenicol addition increases the frequency of lysogeny by lambda phage two- to fourfold. Lambda cIII mutants, which normally lysogenize at very low frequencies, lysogenize at very high frequencies in the presence of chloramphenicol.     

Increased epidermal growth factor receptor in an estrogen-responsive,adriamycin-resistant MCF-7 cell line

We examined the expression of the estrogen and epidermal growth factor (EGF) receptors in a drug-resistant subline of MCF-7 cells in order to study potential alterations in hormone dependence or in the growth factor pathway that could be related to the development of drug resistance in human breast cancer. The drug-resistant subline was derived from MCF-7 cells by selection with Adriamycin in the presence of the P-giycoprotein antagonist, verapamil, to prevent acquisition of the classical multidrug resistance phenotype. The Adriamycin-resistant cells retain estrogen-binding, estrogen-responsive monolayer growth, and estrogen-dependent tumorigenesis. Estrogen-binding studies demonstrate 1.4 × 10⁶ sites per cell with unaltered affinity when compared to parental MCF-7 cells, which have 2.7 × 10⁵ sites per cell. An increase in expression of EGF receptor, eight to 12-fold, occurred early in the selection for drug resistance, and appears to be unrelated to verapamil exposure, since cells maintained in Adriamycin without verapamil also have increased EGF receptor expression. Partially drug-sensitive revertants carried a verapamil, but out of Adriamycin, demonstrate a decline in EGF receptor expression. We postulate that activation of growth factor pathways in drug-resistant cells may enhance mechanisms of drug resistance, or provide mitogenic stimuli for cells to recover after damage by drug exposure. © 1993 Wiley-Liss, Inc.     

Replication of lambda plasmid in amino acid-starved strains of Escherichia coli.

We found that lambda plasmid replication, as measured by the increase in plasmid content per bacterial mass, proceeds for hours in an amino acid-starved, relaxed mutant of Escherichia coli K-12, whereas is inhibited in its wild-type stringent partner. Replication of lambda plasmid in amino acid-starved, relaxed cells reveals absolute lambda O dependence and is not inhibited by chloramphenicol at 200 micrograms/ml. The replication also occurs in wild-type cells treated with chloramphenicol. We conclude that lambda plasmid replication is under stringent control, probably as a result of the action of ppGpp, the signal for the stringent response, on RNA polymerase.     

Multiple antibiotic susceptibility associated with inactivation of the prc gene.

A Tn5 insertion which led to increased susceptibility to multiple drugs, including tetracycline, chloramphenicol, nalidixic acid, erythromycin, spectinomycin, norfloxacin, and novobiocin, was identified in Escherichia coli. Cloning and sequence studies showed that the insertion was in the previously identified prc gene at min 40.4. The prc product is known to function as a protease linked to processing of penicillin-binding protein 3 and lambda repressor and when absent to allow some leakage of periplasmic constituents. Complementation studies with the prc gene on plasmids showed complete recovery of parental levels of susceptibility to all drugs except chloramphenicol, with which only partial reversion to wild-type levels was observed.     

Mechanisms of antibiotic resistance determined by resistance-transfer factors

Unowsky, Joel (Northwestern University Medical School, Chicago, Ill.), and Martin Rachmeler. Mechanisms of antibiotic resistance determined by resistance-transfer factors. J. Bacteriol. 92:358-365. 1966.-This study was concerned with the mechanism of expression of drug resistance carried by resistance-transfer (R) factors of two types: fi(-) (negative fertility inhibition) and fi(+) (positive fertility inhibition). The levels of drug resistance determined by R factors used in this study were similar to those reported by other investigators. A new finding was that Escherichia coli carrying the fi(-) episome was resistant to 150 to 200 mug/ml of streptomycin. The growth kinetics of R factor-containing cells were similar in the presence or absence of streptomycin, chloramphenicol, and tetracycline, but a period of adaptation was necessary before cells began exponential growth in the presence of tetracycline. By use of radioactive antibiotics, it was shown that cells containing the fi(-) episome were impermeable to tetracycline and streptomycin, whereas cells containing the fi(+) episome were impermeable only to chloramphenicol. Cell-free extracts from fi(+) and fi(-) cells were sensitive to the antibiotics tested in the polyuridylic acid-stimulated incorporation of phenylalanine into protein.     

Direct transfer of coliphage lambda DNA from Escherichia coli to cellular slime mold Dictyostelium discoideum

Dictyostelium discoideum myxamoebae were cultured with Escherichia coli cells infected with lambda phage in the presence of chloramphenicol. After eliminating the uningested bacteria by repeated centrifugation in a Percoll gradient, we examined the myxamoeba cytoplasm (not the food vacuole) for the presence of phage DNA. A significant amount of DNA extracted from the myxamoebae was hybridizable with purified phage lambda DNA, and capable of forming phage particles when packaged in vitro with phage lambda proteins. The EcoRI restriction maps of the phages recovered from the plaques were identical to that of the infecting phage. These results strongly suggest that phage DNA molecules were taken up by the cellular slime mold cells and that at least some fraction existed in intact form.     

Characteristics of intracellular proteolysis in the cells of Bacillus subtilis

The rate of total protein degradation down to acid-soluble products in the B. subtilis cells growing on a minimal medium is about 4--5% per hour. Under amino acid deficiency the rate of proteolysis depends on the allelic state of the relA gene, so that in the rel+ cells it increases two-fold, while in the rel- cells it remains low. Elimination of NH4+, PO43- and Mg2+ from the culture medium or an addition of NaN3 (8 mM) or 2,4-dinitrophenol (2 mM) results in 1.5--2.0-fold stimulation of proteolysis independently of the relA gene. In all cases studied the rate of proteolysis decreases after addition of chloramphenicol (100 micrograms/ml). It is proposed that chloramphenicol decreases the intracellular concentration of ppGpp, which is believed to exert pleiotropic alterations of cellular metabolism under condition of growth limitation. Quite different is the case of degradation of anomalous proteins synthesized in the presence of the lysine analog--S-2-aminoethylcystein. Degradation of anomalous proteins proceeds very rapidly (about 70% per hour); chloramphenicol (100 micrograms/ml) decreases the rate of proteolysis only two-fold. It was found that tetracycline (100 micrograms/ml) effectively inhibits the degradation of anomalous proteins. This activity of tetracycline was not observed in the presence of 50 mM of Mg2+ and seems to be dependent on the capacity of the antibiotic to form complexes with bivalent cations. These results reveal common features of control of proteolysis in the cells of B. subtilis and E. coli.     

Evidence for a New Endonuclease Synthesized by λ Bacteriophage

Infection of nonlysogenic Escherichia coli CR34(S) (Thy(-)) with bacteriophage lambda C(I)857 resulted in the formation of twisted circular double-stranded phage deoxyribonucleic acid (DNA; species I). When such infected bacteria were incubated in the absence of thymine, there was a significant decrease in the amount of species I DNA after 60 min of incubation. A similar loss of species I lambda DNA during incubation in a thymine-deficient medium was also observed after infection of the endonuclease I-deficient strain, E. coli 1100(S) (Thy(-)). This destruction of twisted, circular lambda DNA in thymine-deprived cells did not occur in the presence of chloramphenicol nor in lysogenic E. coli CR34 carrying a noninducible lambda prophage. It is therefore concluded that the endonuclease which attacks this circular configuration of lambda DNA is newly synthesized after infection and is directed by the phage chromosome.     

Identification of a second tetracycline-inducible polypeptide encoded by Tn10.

Three Tn10 polypeptides were detected by analyzing the proteins synthesized in ultraviolet light-irradiated Escherichia coli cells after infection with lambda::Tn10. One of these polypeptides was the previously identified 36,000-dalton TET polypeptide. The other two had approximate sizes of 25,000 and 13,000 daltons. The syntheses of both the TET polypeptide and the 25,000-dalton polypeptide were inducible by tetracycline in lambda-immune hosts. Similarly, the synthesis of the TET polypeptide was inducible in nonimmune hosts. However, the synthesis of the 25,000-dalton polypeptide was constitutive in nonimmune hosts. An amber mutation in a gene required for tetracycline resistance on lambda::Tn10 was isolated that eliminated the synthesis of the TET polypeptide in sup+ hosts but not the synthesis of the 25,000-dalton or the 13,000-dalton polypeptides. The expression of tetracycline resistance from wild-type Tn10 was found to be anomalous in E. coli strains carrying the amber suppressors supD, supE, and supF. In general, strains containing these nonsense suppressors were less resistant to tetracycline.     

Cloning, expression in Escherichia coli and nucleotide sequence of a tetracycline-resistance gene from Streptomyces rimosus

Determinants of tetracycline resistance have been cloned from two different tetracycline-producing industrial strains of Streptomyces into Streptomyces lividans using the plasmid vector pUT206. Three plasmids, pUT250 and pUT260 with a 9.5 and a 7.5 kb insert respectively of Streptomyces rimosus DNA, and pUT270 with a 14.0 kb insert of Streptomyces aureofaciens DNA, conferring resistance to tetracycline, have been isolated. By in vitro sub-cloning, a similar fragment of 2.45 kb containing the tetracycline resistance gene (tet347) was further localized on these plasmids. The S. rimosus gene has been cloned into Escherichia coli and expressed under the control of lambda pL or Lpp promoters. Differential protein extraction of E. coli cells revealed the presence of an additional membrane-embedded protein in tetracycline-resistant cells. On the basis of available restriction endonuclease maps, the tet347 gene is probably identical to the tetB gene from S. rimosus recently identified by T. Ohnuki and co-workers as responsible for the reduced accumulation of tetracycline. The nucleotide sequence of a 2052 bp DNA fragment containing the TcR structural gene from S. rimosus has been determined. The amino acid sequence of the tet347 protein (Mr35818) deduced from the nucleotide sequence shows a limited but significant homology to other characterized tetracycline transport acting determinants from pathogenic bacteria.     

Some effects of chlorambucil on nuclear protein phosphorylation in the yoshida ascites sarcoma

The ability of nuclei, isolated from Yoshida ascites sarcoma cells, to phosphorylate nuclear proteins in the presence of [γ-³²P] ATP has been investigated. Comparisons were made between a strain sensitive to the effects of the alkylating agent, chlorambucil, with a corresponding resistant strain both before and after drug-treatment of tumour-bearing animals.There was no gross quantitative differences between the drug-sensitive and -resistant untreated cells but treatment resulted in increased levels in the sensitive strain.Qualitative differences were seen before treatment in the phosphorylation pattern of the tris-saline-soluble nuclear sap fraction. The high molecular weight species in the fraction from sensitive cells showed phosphorylations which were absent, or present at very low levels, in the corresponding fraction from drug-resistant cells.Changes were observed in the tris-saline-soluble and non-histone protein fractions from drug-sensitive cells following treatment of tumour-bearing animals. Only minor alterations in patterns of phosphorylation were seen in fractions from drug-resistant cells.