Uptake of 3H-Tetracycline by Resistant and Sensitive Escherichia coli

The uptakes of (3)H-tetracycline by 12 tetracycline-sensitive and 24 tetracycline-resistant Escherichia coli hospital cultures were found to be 270 and 75 nmoles of tetracycline per milliliter of cell water per 20 min, respectively. This confirms reports by other investigators who, by using only one or two cultures, suggested a relationship between tetracycline uptake and tetracycline resistance. However, minimum inhibitory concentrations of tetracycline for the cultures bore no relation to the tetracycline uptake values, suggesting that loss of tetracycline uptake may not be the primary cause of resistance. In addition there were three resistant cultures with uptake values greater than 140 and two sensitive cultures with uptakes lower than 180, raising the question of how these tetracycline-resistant cultures could grow with tetracycline at concentrations nearly as high as those found to inhibit growth of sensitive organisms. Of the tetracycline-resistant cultures, 15 were able to transfer tetracycline resistance to a recipient organism and 9 were not. Two of the cultures transferred TC-resistance to a recipient with no modification-restriction system (E. coli C) but did not transfer resistance to a recipient with a known modification-restriction system (E. coli K-12).     

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.     

Inhibition of L-lysine uptake by alpha-methyl lysine in Escherichia coli and Bacillus sphaericus

alpha-Methyl lysine was investigated as a potential inhibitor of lysine transport in Escherichia coli and Bacillus sphaericus. At equimolar concentrations, no inhibition was observed in either organism, but at 10X and 100X the lysine concentration, alpha-methyl lysine caused a 20-50% reduction in the initial rate of lysine uptake in both bacteria. A similar inhibitory effect was observed with epsilon-N-methyl lysine on lysine uptake in B. sphaericus, but not in E. coli. alpha-Methyl lysine had a reduced effect on ornithine uptake and no effect on arginine transport in either bacterium.     

Tetracycline resistance genes from Bacillus plasmid pAB124 confer decreased accumulation of the antibiotic in Bacillus subtilis but not in Escherichia coli

Expression of tetracycline resistance by genes originating in the Bacillus plasmid pAB124 was examined in both Bacillus subtilis and Escherichia coli host cells. Expression of resistance in B. subtilis by genes from pAB124 was inducible and associated with decreased accumulation of the antibiotic. A fragment of pAB124 carrying the genes coding for tetracycline resistance was cloned into the E. coli plasmid RSF2124. The cloned fragment conferred a low level of resistance in E. coli, but this was not associated with decreased uptake of tetracycline and was not inducible.     

Changes in Acholeplasma laidlawii plasma membrane functions during development of resistance to ciprofloxacin and tetracycline]

The role of transport activity of Acholeplasma laidlawii plasmatic membrane in the development of resistance to ciprofloxacin and tetracycline was investigated. It was shown that one of the important steps of resistance development in acholeplasms is a complex of adaptation metabolic reactions providing limited antibiotic accumulation by the cells. In the case of ciprofloxacin resistance metabolism changes concerning transport systems took place before mutations in target genes. Development of tetracycline resistance of mollecutes after incubation in the medium with enhancing antibiotic concentrations and not connected with the presence of tet(M) determinant was demonstrated for the first time.     

Light-induced, carrier-mediated transport of tetracycline by Rhodopseudomonas sphaeroides.

Tetracycline accumulation by the phototrophic bacterium Rhodopseudomonas sphaeroides has been studied, using the fluorescence properties of the antibiotic and measuring uptake of [7- 3H]tetracycline. Accumulation was carrier mediated, with a Km of approximately 300 micronM. Efflux also appeared to be carried mediated, with a Km of 25 mM. Chlorotetracycline competitively inhibited tetracycline transport. The transport was energy dependent. Efflux occurred during the influx process, and an energy-requiring steady state was reached when influx balanced efflux. Transport was inhibited by metabolic inhibitors such as antimycin A, cyanide, and iodoacetate. Proton conductors such as carbonylcyanide m-chlorophenyl hydrazone were strongly inhibitory. Efflux was not energy dependent. Efflux is partially blocked by mercuric ions and completely blocked by an external pH of 9 to 11. Although efflux rates increased continuously with lowering of the pH, influx rates have a sharp maximum at pH 7.     

Effect of Escherichia coli endotoxin on ascorbic acid transport in isolated adrenocortical cells

The active uptake of ascorbic acid by isolated rat adrenocortical cells increases with ascorbic acid concentration, depends on time and calcium, and is inhibited by ACTH concentrations required for maximal steroidogenesis. Lipopolysaccharide of Escherichia coli 0111:B4 modifies the ascorbic acid uptake in a calcium-dependent manner. At low calcium concentrations, lipopolysaccharide exerts a stimulatory effect on ascorbic acid transport and at high concentrations lipopolysaccharide produces a dose-dependent inhibitory effect. This inhibition of the ascorbic acid transport by the endotoxin can alter the ascorbic acid accumulation in the adrenal gland during endotoxin shock.     

Antibiotic resistance among coliforms and Pseudomonas spp. from bodies of water around Port Harcourt, Nigeria

Samples from municipal waste water, the Bonny River estuary and wells in and around Port Harcourt were examined for bacteriological quality over a 9 month period. A total of 157 Pseudomonas spp., 133 Escherichia coli and 282 other coliforms were isolated and tested for the incidence of resistance to 10 antibiotics. All of the Pseudomonas spp. were resistant to at least one of the antibiotics while 96.2% were resistant to two or more. Most (83.5%) of the E. coli and other coliforms (91.8%) were resistant to at least one antibiotic. All strains were susceptible to gentamicin. Minimal inhibitory concentrations of ampicillin and tetracycline for E. coli ranged from 6.25 to 50 and 6.25 to 12.5 micrograms/ml, respectively. Minimal inhibitory concentrations of ampicillin and tetracycline were 1000 and 25 micrograms/ml for the Pseudomonas strains. The high incidence of bacterial resistance to antibiotics is discussed in relation to the widespread use of antibiotics, and possible public health implications.     

Inhibition of isoleucyl-transfer ribonucleic acid synthetase in Echerichia coli by pseudomonic acid

The mode of action of the antibiotic pseudomonic acid has been studied in Escherichia coli. Pseudomonic acid strongly inhibits protein and RNA synthesis in vivo. The antibiotic had no effect on highly purified DNA-dependent RNA polymerase and showed only a weak inhibitory effect on a poly(U)-directed polyphenylalanine-forming ribosomal preparation. Chloramphenicol reversed inhibition of RNA synthesis in vivo. Pseudomonic acid had little effect on RNA synthesis in a regulatory mutant, E. coli B AS19 RC(rel), whereas protein synthesis was strongly inhibited. In pseudomonic acid-treated cells, increased concentrations of ppGpp, pppGpp and ATP were observed, but the GTP pool size decreased, suggesting that inhibition of RNA synthesis is a consequence of the stringent control mechanism imposed by pseudomonic acid-induced deprivation of an amino acid. Of the 20 common amino acids, only isoleucine reversed the inhibitory effect in vivo. The antibiotic was found to be a powerful inhibitor of isoleucyl-tRNA synthetase both in vivo and in vitro. Of seven other tRNA synthetases assayed, only a weak inhibitory effect on phenylalanyl-tRNA synthetase was observed; this presumably accounted for the weak effect on polyphenylalanine formation in a ribosomal preparation. Pseudomonic acid also significantly de-repressed threonine deaminase and transaminase B activity, but not dihydroxyacid dehydratase (isoleucine-biosynthetic enzymes) by decreasing the supply of aminoacylated tRNA(Ile). Pseudomonic acid is the second naturally occurring inhibitor of bacterial isoleucyl-tRNA synthetase to be discovered, furanomycin being the first.     

β-lactams and florfenicol antibiotics remain bioactive in soils while ciprofloxacin, neomycin, and tetracycline are neutralized

It is generally assumed that antibiotic residues in soils select for antibiotic-resistant bacteria. This assumption was tested by separately adding 10 different antibiotics (≥200 ppm) to three soil-water slurries (silt-loam, sand-loam, and sand; 20% soil [wt/vol]) and incubating mixtures for 24 h at room temperature. The antibiotic activity of the resultant supernatant was assessed by culturing a sensitive Escherichia coli strain in the filter-sterilized supernatant augmented with Luria-Bertani broth. We found striking differences in the abilities of supernatants to suppress growth of the indicator E. coli. Ampicillin, cephalothin, cefoxitin, ceftiofur, and florfenicol supernatants completely inhibited growth while bacterial growth was uninhibited in the presence of neomycin, tetracycline, and ciprofloxacin supernatants. High-performance liquid chromatography (HPLC) analysis demonstrated that cefoxitin and florfenicol were almost completely retained in the supernatants, whereas tetracycline and ciprofloxacin were mostly removed. Antibiotic dissipation in soil, presumably dominated by adsorption mechanisms, was sufficient to neutralize 200 ppm of tetracycline; this concentration is considerably higher than reported contamination levels. Soil pellets from the tetracycline slurries were resuspended in a minimal volume of medium to maximize the interaction between bacteria and soil particles, but sensitive bacteria were still unaffected by tetracycline (P = 0.6). Thus, residual antibiotics in soil do not necessarily exert a selective pressure, and the degree to which the pharmaceutical remains bioactive depends on the antibiotic. Efforts to control antibiotic contamination would be better directed toward compounds that retain biological activity in soils (e.g., cephalosporins and florfenicol) because these are the antibiotics that could exert a selective pressure in the environment.     

Cation transport in yeast

1. The distribution of azide added to suspensions of bakers' yeast was studied under various conditions. The recovery of azide was estimated in the volume of water into which low concentrations of electrolytes can readily diffuse (anion space). Considerable azide disappeared from this anion space. 2. The incomplete recovery of azide in the anion space is due to its uptake by the cells. This uptake occurs against a concentration gradient at 0 degrees C., and is attributed to binding of azide by cell constituents. 3. Confirmatory evidence is presented that one such constituent is the K carrier in the cell membrane. The azide inhibition of K transport is not mediated by inhibition of cytochrome oxidase in the mitochondria. 4. From the amount of combined azide and the experimentally determined dissociation constant of the K carrier-inhibitor complex, the maximum value for the concentration of this carrier is calculated as 0.1 microM/gm. yeast. 5. The addition of glucose and PO(4) causes a secondary K uptake which is not azide-sensitive and is clearly distinct from the primary, azide-sensitive mechanism. 6. The existence of a separate carrier responsible for Na extrusion is reconsidered. It is concluded that present evidence does not necessitate the assumption that such a carrier is active in yeast.     

Antibiotic resistance among coliforms and Pseudomonas spp. from bodies of water around Port Harcourt, Nigeria

Samples from municipal waste water, the Bonny River estuary and wells in and around Port Harcourt were examined for bacteriological quality over a 9 month period. A total of 157 Pseudomonas spp., 133 Escherichia coli and 282 other coliforms were isolated and tested for the incidence of resistance to 10 antibiotics. All of the Pseudomonas spp. were resistant to at least one of the antibiotics while 96.2% were resistant to two or more. Most (83.5%) of the E. coli and other coliforms (91.8%) were resistant to at least one antibiotic. All strains were susceptible to gentamicin. Minimal inhibitory concentrations of ampicillin and tetracycline for E. coli ranged from 6.25 to 50 and 6.25 to 12.5 μg/ml, respectively. Minimal inhibitory concentrations of ampicillin and tetracycline were 1000 and 25 μg/ml for the Pseudomonas strains. The high incidence of bacterial resistance to antibiotics is discussed in relation to the widespread use of antibiotics, and possible public health implications.     

Regulation of intracellular calcium in chick embryo fibroblast: calcium uptake by the microsomal fraction.

The total membrane fraction of a chick embryo fibroblast (CEF) homogenate accumulates calcium in an energy-dependent manner. This activity can be dissociated into azide-sensitive and azide-insensitive components. The azide-sensitive component of calcium uptake is believed to represent mitochondrial calcium uptake. The azide-insensitive component of calcium uptake is enhanced by the presence of a calcium trapping agent such as oxalate, and cannot utilize, ADP, inorganic phosphate and a Krebs cycle substrate to support uptake. The distribution of the azide-insensitive calcium uptake in subcellular fractions suggests that this uptake occurs in other than mitochondrial membranes. The membranes most likely to contribute to the azide-insensitive component of calcium uptake are the endoplasmic reticulum and plasma membrane. A microsomal preparation from CEF cells is essentially devoid of the azide-sensitive calcium uptake activity. This microsomal activity is similar in characteristics to the sarcoplasmic reticulum of skeletal muscle. However the specific activity of CEF microsomal calcium uptake system is much less than that found in the skeletal muscle system. The transport of calcium by these membranes provide a mechanism for the regulation of cytosol calcium levels and may play a role in the control of movement and growth of cultured cells.     

Evidence for common binding sites for ferrichrome compounds and bacteriophage phi 80 in the cell envelope of Escherichia coli.

Mutants ton A and ton B of Escherichia coli K12, known to be resistant to bacteriophage phi80, were found to be insensitive as well to albomycin, an analogue of the specific siderochrome ferrichrome. Ferrichrome at micromolar concentrations strongly inhibited plaque production by phi80. Preincubation with ferrichrome did not inactivate the phage. At a concentration at which ferrichrome allowed 90% inhibition of plaque formation, the chromium analogue of ferrichrome showed no detectable activity. Similarly, ethylenediaminetetraacetic acid, ferrichrome A, and certain siderochromes structurally distinct from ferrichrome, such as ferrioxamine B, schizokinen, citrate, and enterobactin, did not show detectable inhibitory activity. However, rhodotorulic acid showed moderate activity. A host range mutant of phi80, phi80h, was also inhibited by ferrichrome, as was a hybrid of phage lambda possessing the host range of phi80. However, phage lambdacI- and a hybrid of phi80 possessing the host range of lambda were not affected by ferrichrome. Finally, ferrichrome and chromic deferriferrichrome were shown to inhibit adsorption of phi80 to sensitive cells, ferrichrome giving 50% inhibition of adsorption at a minimal concentration of 8 nM. It is suggested that a component of the ferrichrome uptake system may reside in the outer membrane of E. coli K12 and may also function as a component of the receptor site for bacteriophage phi80, and that ferrichrome inhibition of the phage represents a competition for this common site.     

Thymidine uptake and utilization in Escherichia coli: a new gene controlling nucleoside transport.

A commonly used strain of Escherichia coli K-12 was shown to be deficient in the transport of a number of nucleosides, including thymidine. Thymidine incorporation was unaffected. Strain AB2497 exhibited a strikingly lower thymidine pulse-label incorporation at low (less than 1 mug/ml) thymidine concentrations than do many other strains. The deficiency appeared to be due to mutation in a single gene. This gene, which we designated nup (for nucleoside uptake), is located at 10 to 13 min on the E. coli linkage map. In nup+ strains, the transport of a given nucleoside was relatively insensitive to large excesses of other nucleosides but was competitively inhibited by the same nucleoside. Mutants deficient inthymidine kinase are deficient in thymidine uptake but normal in deoxyadenosine uptake. A two-step model for nucleoside transport is presented in which the first step, utilizing the nup gene product, is a nonspecific translocation of nucleoside to the interior of the cell. In the second step, the individual nucleosides are modified by cellular enzymes (e.g., nucleosides kinases) facilitate accumulation.     

The effect of the antibiotic edeine on tRNA interaction with A, P and E sites of Escherichia coli 70S ribosomes

Edeine inhibits poly(U)-dependent binding of tRNAPhe to the P and A sites simultaneously, both on 30S subunits and 70S ribosomes. Hence, edeine cannot be considered as antibiotic, "complementary" to tetracycline for selective adsorption of tRNA only to the P or to the A site. Further, edeine decreases the affinity constant of tRNAPhe for the P-site by more than two orders of magnitude, no matter poly(U) is present or not. Neither edeine nor tetracycline affect interaction of deacylated tRNAPhe with the E-site of E. coli 70S ribosomes.     

Uptake and release of tetracycline by cultured carrot cells

Summary The nature of tetracycline uptake by carrot cell suspension cultures is described. Tetracycline enters the cells by diffusion and the intracellular level of the antibiotic increases with the amount added. Exposure of carrot cells to high levels of tetracycline for a limited time (24 hr) followed by the removal of the drug and the resuspension of the cells in drug-free medium does not affect cell growth and has no inhibitory effect on protein synthesis (¹⁴C-leucine incorporation).     

Influence of tetracycline exposure on tetracycline resistance and the carriage of tetracycline resistance genes within commensal Escherichia coli populations

AIMS: To assess the influence of incremental tetracycline exposure on the genetic basis of tetracycline resistance within faecal Escherichia coli. METHODS AND RESULTS: Through the adoption of a novel combination of multiple breakpoint selection, phenotypic characterization and the application of a polymerase chain reaction based gene identification system it proved possible to monitor the influence of antibiotic exposure on resistance gene possession. Using tetracycline as a case study a clear hierarchy was revealed between tet genes, strongly influenced by host antimicrobial exposure history. CONCLUSIONS: The antimicrobial exposure regime under which an animal is produced affects both the identity and magnitude of resistance gene possession of a selected bacterial population within its enteric microflora. Among the ramifications associated with such resistance gene selection is the degree of resistance conferred and the carriage of linked resistance determinants. This selection is applied by exposure to antibiotic concentrations well below recognized minimum inhibitory tetracycline concentration breakpoints widely adopted to characterize bacterial 'susceptibility'. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms the ability of minimal antibiotic exposure to select for the continued persistence of resistance genes within the enteric microflora. It is clearly demonstrated that different antimicrobial regimes select for different resistance genes, the implications of which are discussed.     

Diversity of tet resistance genes in tetracycline-resistant bacteria isolated from a swine lagoon with low antibiotic impact

Tetracycline resistance has been extensively studied and shown to be widespread. A number of previous studies have clearly demonstrated that a variety of tetracycline resistance genes are present in swine fecal material, treatment lagoons, and the environments surrounding concentrated animal feeding operations (CAFOs). The diversity of tetracycline resistance within a swine lagoon located at a CAFO that used only bacitricin methylene disalicylate as an antibiotic was evaluated by screening 85 tetracycline-resistant isolates for the presence of 18 different genes by performing PCR with primers that target tetracycline efflux genes of Gram-negative bacteria and ribosomal protection proteins. In addition, partial 16S rRNA sequences from each of these isolates were sequenced to determine the identity of these isolates. Of the 85 isolates examined, 17 may represent potential novel species based on BLAST results. Greater than 50% of the isolates (48 out of 85) were found to not contain targeted tet efflux genes. Though minimum inhibitory concentrations ranged widely (16 - >256 mg/L), these values did not give an indication of the tet genes present. Ten new genera were identified that contain at least one tet efflux gene. Five other genera possessed tet efflux genes that were not found in these organisms previously. Interestingly, none of the isolates possessed any of the selected ribosomal protection protein genes. Though tetracycline resistance was found in bacteria isolated from a swine CAFO lagoon, it appears that the limited antibiotic use at this CAFO might have impacted the presence and diversity of tetracycline resistance genes.