Transmission of the R-plasmids and expression of their genes in Proteus mirabilis]

Resistance markers to individual antibiotics are transmitted in E. coli with the same frequency in the shape of a uniform linkage group, and in Proteus mirabilis--with a different one. Possibly in Proteus mirabilis plasmide R6 dissociated, this being expressed in a different incidence of transconjugates, characterized by a different set of antibiotic resistance genes. Tetracyclin resistance gene can be transmitted in P. mirabilis without being bound with other resistance determinants and with the functioning Tra-operon. The expression of individual antibiotic resistance genes of plasmide R6 in P. mirabilis differed, i.e. tetracycline resistance was inducible, and to kanamycin and chloramphenicol--constitutional. The level of expression of the gene controlling the tetracycline resistance was in noninduced condition in P. mirabilis, lower than in E. coli, P. mirabilis containing no R-factor possessed an inducible resistance mechanism to tetracycline, as in case of P. mirabilis strains containing R-factors.     

Resistance of Escherichia coli to tetracyclines. Changes in permeability to tetracyclines in Escherichia coli bearing transferable resistance factors

1. When strains of Escherichia coli, bearing transferable factors for resistance to the tetracyclines (R-factors), and previously cultured in the absence of the tetracyclines, are grown for 15–30min. in a low, subinhibitory, concentration (10μg./ml.) of oxytetracycline or tetracycline, there is a rapid and striking increase in resistance to oxytetracycline or tetracycline, this being associated with a marked fall in the absorption of the drug by the cells. 2. Very short preincubation (1min.) with oxytetracycline, followed by growth for 15–30min. in drug-free medium, produces a marked fall in the absorption of the drug by the resistant cells. Preincubation for 30min. with very low concentrations (0·05μg./ml.) of oxytetracycline produces a similar effect. 3. β-Apo-oxytetracycline, which has very little antibacterial activity, also induces a decreased absorption of oxytetracycline. 4. The ability to exclude oxytetracycline is retained by preincubated resistant cells after growth for 2hr. in drug-free medium. However, after growth for 16hr. in drug-free medium, the cells absorb oxytetracycline freely. 5. Chloramphenicol and proflavine inhibit the adaptive decrease in tetracycline absorption. 5-Fluorouracil has only a slight effect. 6. Spheroplasts prepared from resistant cells show an impaired response to preincubation with tetracycline, compared with intact cells. 7. The relevance of these results to the probable mechanism of tetracycline resistance in R-factor-bearing E. coli is discussed.     

Characterization of a novel tetracycline resistance that functions only in aerobically grown Escherichia coli.

A tetracycline resistance (Tcr) gene that was found originally on two Bacteroides plasmids (pBF4 and pCP1) confers tetracycline resistance on Escherichia coli, but only when it is grown aerobically. Using maxicells, we have identified a 44-kilodalton protein which is encoded by the region that carries the Tcr gene and which may be the Tcr gene product. Localization experiments indicate that this 44-kilodalton protein is cytoplasmic. To determine whether the tetracycline resistance gene is expressed under anaerobic conditions, we have constructed a protein fusion between the Tcr gene and lacZ. In strains of E. coli carrying the fusion, beta-galactosidase activity was the same when the cells were grown under anaerobic conditions as when the cells were grown under aerobic conditions. This indicates that the tetracycline resistance gene product is made under anaerobic conditions but does not work. The failure of the Tcr protein to function under anaerobic conditions was not due to a requirement for function of the anaerobic electron transport system, because neither nitrate nor fumarate added to anaerobic media restored tetracycline resistance. Inhibition of the aerobic electron transport system with potassium cyanide did not prevent growth on tetracycline of cells containing the Tcr gene. A heme-deficient mutant, E. coli SHSP19, which carries the Tcr gene, was still resistant to tetracycline even when grown in heme-free medium. These results indicate that functioning of the Tcr gene product is not dependent on the aerobic electron transport system. Thus the requirement for aerobic conditions appears to reflect a requirement for oxygen. Spent medium from an E. coli strain carrying the Tcr gene, which was grown in medium containing tetracycline (50 micrograms/ml), did not inhibit growth of a tetracycline-susceptible strain of E. coli. Thus, the Tcr gene product may be detoxifying tetracycline.     

Resistance of Escherichia coli to penicillins. 8. Physiology of a class II ampicillin-resistant mutant

Class II ampicillin-resistant mutants of Escherichia coli are defined as having a twofold increase in penicillinase-mediated ampicillin resistance when determined by colony formation tests on plates. In this paper, one class II mutant has been compared to its parent strain. In liquid medium, the mutant was less resistant than the parent strain both in the absence and in the presence of R1 and R-factor mediating penicillinase activity. The penicillinase activity was found to be almost completely bound to the cells in the parent strain, whereas it was excreted to a great extent in the class II mutant strain. In liquid medium, resistance was well correlated to the cell-bound penicillinase activity, whereas the excreted penicillinases were also of great importance for survival on ampicillin plates. The mutant also had a changed resistance to a great number of other antibacterial drugs. The mutant was found to be more sensitive than the parent strain to osmotic shock, especially when treated with ethylenediaminetetraacetic acid or washed with sodium ions. However, the osmotic stability was restored by the presence of 1 mm Mg(2+) ions. The class II mutant was more sensitive than the parent strain to sodium cholate, and it adsorbed the phages T4 and T3-1 at a slower rate than did the parent strain. The two strains adsorbed T6 at the same rate. The class II phenotype could be gradually reversed by increasing concentrations of divalent cations. The pleiotropic changes in the phenotype are apparently unrelated to the specific targets for the antibacterial agents tested. They are secondary consequences of a cell envelope mutation. The findings indicate that the class II mutation mediates a structural change in the lipopolysaccharide of the cell envelope.     

Formation of plasmid pBR322 oligomers as depending on a tetracycline concentration

Escherichia coli K12 strains containing the plasmid pBR322 often show varying contents of plasmid oligomers, in which the monomer units are arranged in tandem. When the concentration of the plasmid-selective antibiotic tetracycline in the medium becomes increased selection of cells containing largely higher oligomers occurs. The number of monomer units organized in the oligomers increases with tetracycline concentration. recA- mutants are unable to generate oligomers under the same conditions and show lower tetracycline resistance. This observations suggest a selective advantage of oligomer containing cells in the presence of tetracycline as a result of higher gene dosage. But E. coli cells transformed with monomers, dimers, trimers, as well as tetramers of pBR322 are characterized by roughly the same plasmid DNA content as well as plasmid coded beta-lactamase and resistance to tetracycline.     

Fluctuations in Escherichia coli O-serotypes in Pigs Throughout Life in the Presence and Absence of Antibiotic Treatment

A detailed study, throughout life, of the coliform and Escherichia coli gut flora of two pigs, is presented. One pig was given oral tetracycline for 3 d on two separate occasions; the other pig was not treated and housed separately. The fluctuations in numbers of coliforms, O-serotypes of E. coli , the occurrence and persistence of antibiotic resistance, and their interrelations, are analysed for both animals. Oral tetracycline profoundly affected the proportion of antibiotic sensitive and resistant E. coli , and, by selecting for R-factor bearing O-serotypes, limited the number of O-serotypes isolated from individual faceal specimens. Specific O-serotypes exhibited marked differences in their ability to persist in the gut of the control pig. The R-factor bearing O-serotypes, selected by tetracycline treatment, also showed differences in ability to persist after treatment indicating that properties other than the possession of R-factors, decide colonizing ability. The size of the colony sample for serotype analysis is discussed.     

Uptake of tetracycline by membrane preparations from Escherichia coli

1. A membrane fraction from Escherichia coli has been prepared essentially free from ribosomes by treatment of the membranes with Triton X-100 at 0 degrees C followed by differential centrifugation. 2. The ribosome-free membrane vesicles absorbed tetracycline by a reversible temperature-dependent process with an apparent K(m) of 0.029mm at pH7.5 and 37 degrees C. 3. The absorption process was negligible below 25 degrees C and had an optimum at 40 degrees C; a pH optimum at 7.5 was observed. 4. The absorption of tetracycline was strongly inhibited by EDTA and ATP; ADP inhibited less strongly and AMP had no effect. 5. There was no significant difference in the rates or extent of uptake of tetracycline by membranes prepared from tetracycline-sensitive and tetracycline-resistant, R-factor-bearing E. coli.     

Active accumulation of tetracycline by Escherichia coli

1. At low concentrations of tetracycline (10mug/ml) net accumulation of the drug by Escherichia coli cells ceased after 7-10min. 2. At higher concentrations of tetracycline (>30mug/ml) the period of net accumulation of the drug was significantly extended. 3. The efflux of tetracycline from E. coli cells transferred from medium containing 10mug of tetracycline/ml to drug-free medium was a rapid temperature-dependent process and was accelerated by 2,4-dinitrophenol. 4. As the concentration of tetracycline in the preloading phase was increased, the rate of subsequent efflux of the drug progressively declined. The efflux of drug from cells preloaded in medium containing 200mug of tetracycline/ml was negligible, although efflux was readily provoked by 2,4-dinitrophenol, by N-ethylmaleimide or by omission of glucose from the medium. 5. The initial rate of uptake of tetracycline by E. coli cells was linearly proportional to the concentration of tetracycline in the medium up to the maximum concentration of drug obtainable under the experimental conditions used (400mug/ml, 0.83mm). 6. Although N-ethylmaleimide strongly inhibited the accumulation of tetracycline by E. coli, no evidence was obtained for the direct involvement of thiol groups in the transport process. It was concluded that N-ethylmaleimide inhibited accumulation by interruption of the energy supply of the cells. 7. Osmotic shock of E. coli cells did not significantly affect the influx of tetracycline, but promoted both efflux of tetracycline and cell lysis in cells treated with a high concentration of tetracycline. 8. A study of the distribution of tetracycline among the subcellular fractions of penicillin-induced spheroplasts preincubated with various concentrations of tetracycline indicated that 60-70% of the accumulated tetracycline was in the high-speed supernatant fraction. Sephadex chromatography showed that the tetracycline of this fraction was present as the free drug. Sephadex chromatography of a detergent extract of the membrane fraction, however, indicated that a significant proportion of the tetracycline radioactivity of this fraction was apparently bound to some macromolecular component. 9. Cellulose phosphate paper chromatography of cold-acid extracts of spheroplasts preloaded with tetracycline indicated that the accumulated drug was chemically unchanged. 10. Membrane preparations isolated from osmotically lysed penicillin-induced spheroplasts showed a temperature-dependent binding of tetracycline that was not energy-dependent and was not inhibited by N-ethylmaleimide. The binding process was stimulated by omitting Mg(2+) from the medium, but conversely was profoundly inhibited by EDTA. 11. The relevance of these findings to the probable mechanism of active tetracycline accumulation by E. coli is discussed.     

Expression of the resistance genes of plasmid RP4 in Escherichia coli cells grown by continuous cultivation

The resistance to tetracycline decreased in Escherichia coli C600 cells containing plasmid RP4 and grown under the conditions of continuous cultivation. The population of cells containing plasmid RP4 is heterogeneous in the trait of tetracycline resistance, and most cells cannot grow in a selective medium with tetracycline at a concentration of 20 micrograms/ml. The decreased resistance to tetracycline was most pronounced for a glucose-limited chemostat culture and also in the presence of two plasmids, RP4 and pBS94 , in the cells. No decrease was found in the resistance to other drugs determined by plasmid genes.     

R-plasmid transfer in a wastewater treatment plant.

Enteric bacteria have been examined for their ability to transfer antibiotic resistance in a wastewater treatment plant. Resistant Salmonella enteritidis, Proteus mirabilis, and Escherichia coli were isolated from clinical specimens and primary sewage effluent. Resistance to ampicillin, chloramphenicol, streptomycin, sulfadiazine, and tetracycline was demonstrated by spread plate and tube dilution techniques. Plasmid mediation of resistance was shown by ethidium bromide curing, agarose gel electrophoresis, and direct cell transfer. Each donor was mated with susceptible E. coli and Shigella sonnei. Mating pairs (and recipient controls) were suspended in unchlorinated primary effluent that had been filtered and autoclaved. Suspensions were added to membrane diffusion chambers which were then placed in the primary and secondary setting tanks of the wastewater treatment plant. Resistant recombinants were detected by replica plating nutrient agar master plates onto xylose lysine desoxycholate agar plates that contained per milliliter of medium 10 micrograms of ampicillin, 30 micrograms of chloramphenicol, 10 micrograms of streptomycin, 100 micrograms of sulfadiazine, or 30 micrograms of tetracycline. Mean transfer frequencies for laboratory matings were 2.1 X 10(-3). In situ matings for primary and secondary settling resulted in frequencies of 4.9 X 10(-5) and 7.5 X 10(-5), respectively. These values suggest that a significant level of resistance transfer occurs in wastewater treatment plants in the absence of antibiotics as selective agents.     

Novel aerobic tetracycline resistance gene that chemically modifies tetracycline.

A tetracycline resistance gene that was found originally on the Bacteroides plasmid pBF4 confers resistance on Escherichia coli but only when cells are growing aerobically. When E. coli EM24 carrying this aerobic tetracycline resistance (*Tcr) gene is grown in medium containing tetracycline, the resulting spent medium is no longer toxic to tetracycline-sensitive (Tcs) E. coli EM24 (B.S. Speer and A.A. Salyers, J. Bacteriol. 170: 1423-1429, 1988). To determine whether the *Tcr gene product modified tetracycline, we characterized the material resulting from incubation of E. coli (*Tcr) with tetracycline. When [7-3H(N)]tetracycline was added to cultures of E. coli (*Tcr), at least 90% of the label was recovered in the extracellular fluid. Therefore, tetracycline was not being sequestered by the cells. The labeled material behaved similarly to tetracycline with respect to solubility in various organic solvents. However, the UV-visible light spectrum had a single peak at 258 nm, whereas the tetracycline spectrum had a peak at 364 nm. The labeled material also had a faster migration rate than did tetracycline on thin-layer plates in a solvent system of butanol-methanol-10% citric acid (4:1:2, vol/vol/vol) and was separable from tetracycline by reverse-phase high-pressure liquid chromatography, using an acetronitrile-0.1% trifluoroacetic acid solvent system. These results demonstrate that the *Tcr gene product chemically modifies tetracycline. The *Tcr gene is the first example of a chemically modifying tetracycline resistance mechanism.     

Molecular cloning and characterization of the recA gene of Pseudomonas aeruginosa PAO.

The recA gene of Pseudomonas aeruginosa PAO has been isolated and introduced into Escherichia coli K-12. Resistance to killing by UV irradiation was restored in several RecA-E. coli K-12 hosts by the P. aeruginosa gene, as was resistance to methyl methanesulfonate. Recombination proficiency was also restored, as measured by HfrH-mediated conjugation and by the ability to propagate Fec-phage lambda derivatives. The cloned P. aeruginosa recA gene restored both spontaneous and mitomycin C-stimulated induction of lambda prophage in lysogens of a recA strain of E. coli K-12.     

R-Factor-Mediated Resistance to Tetracycline in Proteus mirabilis

The expression of R-factor-mediated resistance to tetracycline has been compared in Proteus mirabilis and Escherichia coli. Resistance to a range of concentrations of tetracycline was significantly lower in P. mirabilis than in E. coli in both induced and repressed states. Indirect evidence showed that conditions which result in a marked increase in the level of resistance of P. mirabilis harboring the R factor NR1 to chloramphenicol, streptomycin, and spectinomycin due to an amplification in the number of copies of r-determinants per cell do not detectably increase the level of resistance to tetracycline. Tetracycline resistance was inducible in early stationary-phase P. mirabilis NR1 although not after 5 h in this state. Double isotope labeling of control and tetracycline-induced P. mirabilis NR1 in early stationary phase revealed isotopic enrichment of certain peaks in extracts from induced cells subjected to polyacrylamide gel electrophoresis.     

Dual mode of energy coupling by the oxyanion-translocating ArsB protein.

The arsA and arsB genes of the ars operon of R-factor R773 confer arsenite resistance in Escherichia coli by coding for an anion-translocating ATPase. Arsenite resistance and the in vivo energetics of arsenite transport were compared in cells expressing the arsA and arsB genes and those expressing just the arsB gene. Cells expressing the arsB gene exhibited intermediate arsenite resistance compared with cells expressing both the arsA and arsB genes. Both types of cells exhibited energy-dependent arsenite exclusion. Exclusion of 73AsO2- from cells expressing only the arsB gene was coupled to electrochemical energy, while in cells expressing both genes, transport was coupled to chemical energy, most likely ATP. These results suggest that the Ars anion transport system can be either an obligatory ATP-coupled primary pump or a secondary carrier coupled to the proton motive force, depending on the subunit composition of the transport complex.     

Role of outer membrane barrier in efflux-mediated tetracycline resistance of Escherichia coli.

Accumulation of tetracycline in Escherichia coli was studied to determine its permeation pathway and to provide a basis for understanding efflux-mediated resistance. Passage of tetracycline across the outer membrane appeared to occur preferentially via the porin OmpF, with tetracycline in its magnesium-bound form. Rapid efflux of magnesium-chelated tetracycline from the periplasm was observed. In E. coli cells that do not contain exogenous tetracycline resistance genes, the steady-state level of tetracycline accumulation was decreased when porins were absent or when the fraction of Mg(2+)-chelated tetracycline was small. This is best explained by assuming the presence of a low-level endogenous active efflux system that bypasses the outer membrane barrier. When influx of tetracycline is slowed, this efflux is able to reduce the accumulation of tetracycline in the cytoplasm. In contrast, we found no evidence of a special outer membrane bypass mechanism for high-level efflux via the Tet protein, which is an inner membrane efflux pump coded for by exogenous tetA genes. Fractionation and equilibrium density gradient centrifugation experiments showed that the Tet protein is not localized to regions of inner and outer membrane adhesion. Furthermore, a high concentration of tetracycline was found in the compartment that rapidly equilibrated with the medium, most probably the periplasm, of Tet-containing E. coli cells, and the level of tetracycline accumulation in Tet-containing cells was not diminished by the mutational loss of the OmpF porin. These results suggest that the Tet protein, in contrast to the endogenous efflux system(s), pumps magnesium-chelated tetracycline into the periplasm. A quantitative model of tetracycline fluxes in E. coli cells of various types is presented.     

Reduced expression of Tn 10-mediated tetracycline resistance in Escherichia coli containing more than one copy of the transposon

In Escherichia coli a single copy of Tn10 confers high-level resistance to tetracycline. Resistance itself results from expression of three distinct mechanisms which normally act together (Shales et al., 1980). In cells containing two copies of Tn10, the level of resistance to tetracycline was reduced. This was not due to overproduction of the repressor which controls the resistance genes, because strains diploid for an operator-constitutive allele of Tn10 also exhibited reduced expression of resistance. The negative gene dosage effect resulted from decreased expression of two mechanisms (1 and 2) consequent on enhanced expression of the third mechanism. The net result of increasing the copy number was a decrease in resistance because mechanism 3 was less efficient than mechanisms 1 and 2 in protecting the cell against tetracycline. The DNA sequence responsible for the reduced expression of resistance was contained in the internal BglII fragment of Tn10. This sequence, which is probably unique to Tn10, may encode the protein which mediates mechanism 3. Elevated levels of this protein probably cause decreased expression of mechanisms 1 and 2.     

The antimicrobial susceptibility of Escherichia coli isolated in eastern area of Romania. A surveillance study

To evaluate the resistance trends for Escherichia coli isolates during 1993-1999 period in Eastern Romania to: ampicillin (A), ampicillin-sulbactam (A/S), ceftazidime (CAZ), cefotaxime (CTA), ceftriaxone (CRO), aztreonam (AZT), ofloxacin (OF), ciprofloxacin (CIP), tetracycline (T) and chloramphenicol (C). We tested 2012 clinical isolates obtained from faeces and urine. MICs were determined by a dilution method in Mueller-Hinton agar (NCCLS guidelines). Resistance rates were analyzed using the NCCLS breakpoints for the fully susceptible category (moderately susceptible strains were classified as resistant). No significant differences were observed in susceptibility of E. coli to ampicillin and ampicillin/sulbactam in the last years. The high percentage of resistant isolates was observed in 1995 for ampicillin (89.7%). Higher incidences of resistance were detected for A, A/S, T; the addition of sulbactam restored A susceptibility only for a small percent. CAZ, CTA, CRO, AZT, OF and CIP resistance among E. coli isolates was progressively increased in the last period.     

Second-site suppressor mutations for the serine 202 to phenylalanine substitution within the interdomain loop of the tetracycline efflux protein Tet(C)

The serine 202 to phenylalanine substitution within the cytoplasmic interdomain loop of Tet(C) greatly reduces tetracycline resistance and efflux activity (Saraceni-Richards, C. A., and Levy, S. B. (2000) J. Biol. Chem. 275, 6101-6106). Second-site suppressor mutations were identified following hydroxylamine and nitrosoguanidine mutagenesis. Three mutations, L11F in transmembrane 1 (TM1), A213T in the central interdomain loop, and A270V in cytoplasmic loop 8-9, restored a wild type level of resistance and an active efflux activity in Escherichia coli cells bearing the mutant tet(C) gene. The Tet S202F protein with the additional A270V mutation was expressed in amounts comparable with the original mutant, whereas L11F and A213T Tet(C) protein mutants were overexpressed. Introduction of each single mutation into the wild type tet(C) gene by site-directed mutagenesis did not alter tetracycline resistance or efflux activity. These secondary mutations may restore resistance by promoting a conformational change in the protein to accommodate the S202F mutation. The data demonstrate an interaction of the interdomain loop with other distant regions of the protein and support a role of the interdomain loop in mediating tetracycline resistance.     

A study on the preservation of the beating rhythm of single myocardial cells in vitro

Single myocardial cells from fetal mouse heart beat spontaneously in monolayer culture. In standard medium they maintained constant beating rates for at least 5 h at 37 °C. When the beats of single myocardial cells were stopped for a short time by treatment with EGTA, or slowed down by incubating the cells in medium of low pH, the original beating rates could be restored by replacing the cells in the original medium. The same procedure also restored the rates after they had been disturbed by incubating the cells in medium of low sodium and high potassium ion content. Moreover, the original beating rates could be restored after keeping the cells at 10 °C for 22–24 h, but not after keeping them at 37 °C for 22–24 h.     

Effect of β-Lactamase Location in Escherichia coli on Penicillin Synergy

Resistance to ampicillin in Escherichia coli is due generally to the presence of a beta-lactamase (penicillinase). Resistant strains have been found to fall into two groups: those with high-level resistance (1,000 mug/ml or greater) and those with low-level resistance (8 to 250 mug/ml). Most of the high-level resistant organisms posses beta-lactamases whose synthesis is episomally mediated. These strains release penicillinase from the cell when they are subjected to osmotic shock. Low-level resistant strains do not release the enzyme with osmotic shock. High-level resistant strains are not susceptible to the synergistic action of a penicillinase-resistant penicillin with ampicillin. Seventy eight per cent of low-level resistant strains are susceptible to the synergistic action of ampicillin and oxacillin. The two types of beta-lactamases are similar in regard to most properties; both enzymes are subject to competitive inhibition by penicillinase-resistant penicillins. The difference in location in the cell might explain why only some strains of E. coli are susceptible to the synergistic action of penicillin combinations.