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Mechanism of High-level Resistance to Chloramphenicol in Different Escherichia coli Variants
Abstract
SUMMARY Mutants resistant to high levels of chloramphenicol can be obtained in Escherichia coli B by one or two mutational events. All of I44 high-level resistant mutant clones examined were powerful inactivators of the drug. Growth of this kind of mutants in nutrient media containing chloramphenicol IOO ,ug./ml. or more depended on the inoculum size, the composition of the medium, and the concentration of the drug. No growth was observed with lactose as sole energy source unless the organisms had been previously induced for P-galactosidase production. With a strain of Escherichia coli K 12 an entirely different type of resistant mutants occurred. High-level resistant derivatives were obtainable only through several serial mutations. None of 36 high-level resistant mutants was able to inactivate the drug. Growth of these bacteria was extremely slow, even in the absence of drug. This resistance was due to a decreased rate of permeation, which probably was non-specific and concerned many species of micromolecules.
... Organisms resistant by virtue of resistance factors that were transferable were inactivators, whereas this was not the case when resistance was induced by serial transfers in graded concentrations of the drug. This distinction has later been denied (Sompolinsky and Samra 1968). Other workers from Japan have demonstrated inactivation of Cm by a new mechanism . ...
... (2) E. coli B and E. coli B 11 are a Cm-susceptible strain and its inactivating mutant, respectively (Sompolinsky and Salnra 1968). (3) E. coli K 12 (W 945) and a Cm-tolerant derivative (Sompolinsky and Samra 1968) will be designated E. coli K 12 and E. coli K 1211, respectively. (4) E. coli A 460 is a non-reverting acetate-requiring mutant of E. coli K 12. ...
... After growth in defined medium without Cm, the strains of E. coli gave a negligible reaction. For routine examination of susceptibility to Cm, Rosco sensitivitv tablets (Denmark) were used Media and Chemicals Used: Analytical Methods (Sompolinsky et al. 1957). A sterile zone of ...
Two derivative strains of Escherichia coli with high-level resistance to chloramphenicol, one carrying an episomal resistance factor and the other a chromosomal mutant, were both shown to be potent inactivators of the drug. When 1 mM chloramphenicol was added to an exponential culture in minimal medium, growth was halted until 85–90% of the drug was inactivated by acylation. At this state the drug was essentially monoacylated. During and after growth, esterification of the second alcoholic group occurred, though at a slower rate. Arylamines, in amounts up to 10% of chloramphenicol equivalents, were demonstrated in the growth medium after 1–3 days' incubation.With an acetateless mutant of Escherichia coli K12, carrying a resistance factor, it was shown that 5–6 moles of acetate was consumed for every mole of chloramphenicol acylated.Inactivation of chloramphenicol by Gram-negative organisms from infections in hospitalized patients was also examined. Among 103 strains susceptible to chloramphenicol, none produced considerable amounts of chloramphenicol esters. The same was the case with 14 resistant strains of Pseudomonas. Of 134 other resistant organisms examined, including strains of Escherichia, Proteus, Klebsiella, Salmonella, and Shigella, 133 were producers of chloramphenicol esters, and in most cases the drug was partly or entirely diacylated.
... This remarkable characteristic may be attributable to the presence of neuropathies due to peripheral arterial disease, which in more severe cases can culminate in the removal of the affected limb. 3 In the United States, infected foot ulcers in patients with diabetes are the leading cause of non-traumatic amputations. In line with this, in 2014, the prevalence of 29.1 million people with DM in the USA was verified, representing about 9.1% of the total population. ...
It is necessary to know the resistance profile of Staphylococcus aureus to better control diabetic foot ulcer infections, to establish rational antibiotic therapy, and to avoid the development of resistant strains. This cross-sectional study evaluated the clinical parameters , virulence, and antimicrobial resistance profiles of S aureus in patients with diabetic foot disease admitted to a public hospital. S aureus strains were identified in patients with diabetes with amputation indication. Infected tissue samples were collected, microbes were isolated and identified. The microbial resistance profile was determined. Samples were also analyzed for biofilm formation and other virulence markers. The 34 individuals examined were mostly men, black, aged 60 years on average, and generally had a low income and education level. Most individuals had type 2 diabetes, and the mean time since diagnosis was 13.9 years. On an SF-36 (the Medical Outcomes Study 36-item short-form health survey) quality-of-life questionnaire, 75% of individuals obtained a score equal to 0 for physical impairment. S aureus specimens from 17 patients were isolated, corresponding to 50% of samples. Five isolates were classified as methicillin-resistant S aureus (MRSA). Molecular typing revealed that 20% of MRSA strains were SCCmec type V and 80% were type I. All isolates were sensitive to doxycycline; 61.5% were resistant to erythromycin, 38.5% to cefoxitin, 30.7% to clinda-mycin and ciprofloxacin, 23% to meropenem, 15.3% to gentamicin, 38.5% to oxacillin, and 7.7% (one strain) to vancomycin. Regarding biofilm production, 53% of samples were able to produce biofilms, and 84.6% had icaA and/or icaD genes. Additionally, the following enterotoxin genes were identified in the isolates: seb, sec, seg, and sei (5.9%, 5.9%, 11.8%, and 23.9%, respectively) and agr types 1 (5.9%) and 2 (11.8%). Genotypic evaluation made it possible to understand the pathogenicity of S aureus strains isolated from the diabetic foot; laboratory tests can assist in the monitoring of patients with systemic involvement.
Chloramphenicol (CM)-resistant mutants of Streptococcus lactis strain ML3 were obtained either as a consequence of continuous transfer of the bacteria in broth containing increasing amounts of CM or by selecting for high-level resistant derivatives after mutagenic treatment of the bacteria. Some CM-resistant cells obtained by the first method were also resistant to the homologous bacteriophage. Cells trained to grow in the presence of CM developed resistance to some heterologous attacking phages but not to phage ml3. Mutants selected for phage resistance were not resistant to CM. There appear to be two different loci for CM resistance on the bacterial chromosome: the one for high-level resistance is associated with the phage-resistance locus and the other is independent of it. A concentration of CM (280 μg/ml) that was bacteriostatic for ML3 inhibited the intracellular growth of ml3 phage in strain ML3-CMrI, which had been trained to grow in the presence of that CM concentration, despite the fact that cells of this strain were not phage-resistant per se. The drug had no direct virucidal action and did not prevent adsorption to or penetration of phage into the bacterium. Lysogenization did not occur. It is concluded that the block in phage development probably involves inhibition of synthesis of phage components, either involving deoxyribonucleic acid at an early stage or the phage coat protein at a later one.
IntroductionBiological Oxidation–Reduction Processes and Oxidative PhosphorylationBiochemistry and Pharmacology of Naturally Occurring Compounds Containing the Nitro or Nitroso GroupBiochemistry and Pharmacology of Synthetic Compounds Containing the Nitro or Nitroso GroupThe Role of Nitro and Nitroso Compounds in the Formation of MethemoglobinAddendum and Final RemarksReferences
Low concentrations of hydroxyurea stimulated the growth of the blue-green alga Anabaena variabilis that had been pretreated with sublethal concentrations of chloramphenicol or of certain nucleic acid base analogues. When supplemented to the culture medium, hydroxyurea also counteracted the growth inhibitory effect of chloramphenicol on this organism. In contrast, when A. variabilis cells grown in the presence of hydroxyurea were subsequently treated with chloramphenicol, they were found to have become highly susceptible to the growth inhibitory effects of chloramphenicol. The growth of hydroxyurea pretreated cells in basal medium was attended by a lag that was shorter than that of untreated controls; on the other hand, when hydroxyurea pretreated cells were inoculated into chloramphenicol-supplemented medium, they exhibited a longer lag than that shown by untreated cells in chloramphenicol.
The results obtained are discussed in terms of the probable effects of hydroxyurea and chloramphenicol on certain enzyme systems.
Resistance of Staphylococcus aureus 111 to tetracycline was due to an extrachromosomal genetic unit (plasmid) that could be eliminated by growth at 44°. The susceptible (eliminated) strain actively concentrated tetracycline from the nutrient medium by an energy-dependent transport system. The resistant culture accumulated the drug to a much lesser degree than the susceptible culture, both according to the E 380 of the bacterial extract and to its radioactivity after incubation with tritiated tetracycline. Accumulation of tetracycline was low and independent of the external concentration until this reached a level corresponding approximately to the minimal inhibitory concentration.
Pre-incubation with tetracycline at low concentrations decreased ability to accumulate the drug. This pre-incubation effect was not prevented by nalidixic acid but was by actinomycin D.
Antibiotics are found predominantly in the culture medium and not within the cells. This suggests that excretion is built into the biosynthetic process and, on present evidences, it is predicted that a mechanism to discourage reentry of the metabolite is an essential component in a majority of protective systems. Aurodox production by Streptomyces goldiniensis is self-limiting. The feedback effect is immediate and reversible. It is unrelated to the activity of aurodox as an inhibitor of prokaryotic protein synthesis since it is not reproduced by chloramphenicol or puromycin. However, structural analogs of aurodox, including some without antibiotic activity, mimic the effect. Mutants selected for tolerance to high levels of aurodox and able to produce greatly increased amounts of the antibiotic—are also released from the self-inhibition. In the chloramphenicol fermentation, addition of excess antibiotic inhibits further synthesis of the key enzyme, arylamine synthetase.
Germ-free swine were artificially contaminated with tetracycline (TC) sensitive strains of Escherichia coli and Klebsiella pneumoniae. One of these strains, E. coli 3306, was infected with a plasmid carrying kanamycin (KM) resistance, i.e., T-kan factor. Another strain, E. coli P-5, carried a conjugally transferable Col B factor. Among the nine strains used, only E. coli P-38 became TC-resistant after TC administration. Three types of TC-resistance E. coli P-38 strains were found; (a) one strain carried nontransferable TC resistance and could not produce colicin, (b) one strain carried TC resistance with a high transmission frequency which could not produce colicin, and (c) one strain carried TC resistance with a low transmission frequency that could produce colicin B. Genetic studies disclosed that the transmissible TC resistance factors, i.e., Rms105 (group b) and Rms104 (group c), were formed by recombination between Col B factor and nontransmissible TC-resistance (tet) determinant which appeared in E. coli P-38 mutants.
When staphylococci, resistant to 4 μg. tetracycline/ml., were grown in nutrient media at subinhibitory levels of the drug, phenotypical resistance increased until the cocci grew with 160 μg. tetracycline/ml. Resistance increased most rapidly at the highest concentration of tetracycline which did not significantly inhibit growth. Increase in resistance was also obtained by pre-incubation with β-apo-5-oxy-tetracycline. Increase in resistance could be prevented by chloramphenicol and actinomycin D, but not by nalidixic acid. When a highly resistant culture was transferred to tetra-cycline-free medium, phenotypical resistance decreased gradually; after four transfers on nutrient agar it returned entirely to the original level.
This chapter discusses the role of chloramphenicol. Chloramphenicol was the first broad-spectrum antibiotic introduced into the medicinal use to inhibit the growth of bacteria, rickettsiae, and organisms of the psittacosis-lymphograniiloma group. Several strains of chloramphenicol-producing Streptomyces have been described in the chapter. In a complex medium, growth and chloramphenicol production are dissociated, but in a defined medium, antibiotic production and growth are closely linked, and in most of the growth phase, cells are exposed to concentrations much higher than the 10 μg/ml, which inhibits the growth of many bacteria. Chloramphenicol is predominantly a bacteriostatic agent, inhibiting the growth of blue-green algae, bacteria, and related prokaryotic organisms. The chapter discusses the derived rules for predicting the effect of structure on biological activity in the chloramphenicol series. It describes the biosynthesis of chloramphenicol, and effect of chloramphenicol on the producing organism. One of the most important aspects of chloramphenicol biosynthesis is the ability of the producing organism to become resistant to concentrations that were originally inhibitory. In vivo, as well as in vitro experiments, shows that chloramphenicol inhibits protein synthesis in Streptomyces 13S.
Four R factors conferring chloramphenicol (CM) resistance were isolated from Escherichia coli strains of clinical origin. Strains carrying the factors were found to be incapable of inactivating the drug in the presence of acetyl coenzyme A. E. coli W3630 carrying R(70), one of these factors, became sensitive to CM after treatment with glycine, indicating that the spheroplasts of W3630 R(70) (+) were sensitive to the drug and suggesting that the cell membrane is important for CM resistance. The observation that cell-free protein synthesis in W3630 R(70) (+) was inhibited by CM is also compatible with a decrease in permeability. CM resistance in W3630 R(70) (+) appeared to be inducible, because (i) preincubation with subinhibitory concentrations of CM prevented the prolonged lag noted for growth in the presence of 25 mug of CM per ml, and (ii) the preincubation effect was lost after overnight growth in CM-free medium. By contrast, E. coli W3630 cml(+), in which the resistance determinant is integrated into the chromosome, was capable of rapid inactivation of CM. E. coli W3630 cml(+) R(70) (+), which contains the proposed permeability determinant (episomal) as well as levels of the inactivating enzyme (chromosomal) that are comparable with W3630 cml(+), was capable of brief inactivation of CM when inoculated into drug-containing medium. The absence of continued inactivation on more prolonged incubation favors the hypothesis that the R(70) factor inhibited further penetration of CM and that this property possesses the characteristics of induction.
Accumulation of (3)H-tetracycline in nonproliferating cells of susceptible and resistant strains of Escherichia coli and Staphylococcus aureus in tris(hydroxymethyl)aminomethane (Tris) buffer (10 mm, pH 7.5) was significantly decreased in the presence of 5 to 40 mm MgCl(2) and increased in the presence of 5 to 10 mm MnCl(2). When the bacteria first accumulated (3)H-tetracycline in plain Tris.HCl, and the metal salts were thereafter added, a prompt decrease or increase in radioactivity of the cells was observed after the addition of Mg(2+) or Mn(2+), respectively. In phosphate buffer (10 mm, pH 7.5), the effect of Mg(2+) was delayed. Three minutes after addition of (3)H-tetracycline, uptake was as in the control cell suspension, but thereafter it dropped rapidly. When (3)H-tetracycline was incubated with Mg(2+) before addition to the bacterial suspension, uptake was scarcely measurable. The addition of Mg(2+) to growing cultures of S. aureus and E. coli caused a marked decrease in susceptibility; in contrast, no increase in susceptibility could be demonstrated when Mn(2+) was added. It was also demonstrated that Mg(2+) and Mn(2+) had distinct influences on the absorption spectrum, the optical rotatory dispersion, the circular dichroism, and the lipid solubility of tetracycline.
The resistance factor R1 may exist in either of two stable physical states in Proteus mirabilis PM-1. In one case, the R1 deoxyribonucleic acid (DNA) has a buoyant density of 1.711 g/cm(3) and replicates under stringent control. Cells harboring R1 in this form may transfer drug resistance by conjugation. In the other case, R1 DNA shows two buoyant density classes at 1.707 and 1.714 g/cm(3). The 1.714 g/cm(3) component is replicated under a degree of relaxed control, and strains carrying this form generally cannot transfer drug resistance by conjugation. Intracellular amounts of the R factor-coded enzyme, chloramphenicol acetyltransferase, did not correspond to amounts of plasmid DNA in Proteus, and the enzyme was present in lower amounts than in Escherichia coli. It is proposed that the two states of R1 in Proteus may represent stable associated and dissociated forms of the plasmid.
This chapter discusses the biochemical mechanisms of transferable drug resistance. The biochemical mechanisms of extrachromosomal antibiotic resistance might have seemed premature only a few years ago, the rapidity with which information has accumulated recently has made a preliminary appraisal necessary. Since the advent of antimicrobial therapy, the “emergence” of drug-resistant bacteria has been a problem of interest to clinicians, microbial geneticists, and pharmacologists. An unanticipated benefit from an initially adverse development has been the insights which certain types of resistance have provided for a better understanding of the mechanisms of action of agents such as streptomycin and related inhibitors of ribosome function. There is now little doubt that the conventional view of the spread of drug-resistant bacteria by the mechanism of mutation and selection must be revised to include a consideration of those resistance genes that reside on extrachromosomal elements which, in the most general sense, are best described as “plasmids” to connote their autonomous existence and replication apart from the bacterial chromosome.
Chloramphenicol (CM)-resistant mutants of Streptococcus lactis strain ML3 were obtained either as a consequence of continuous transfer of the bacteria in broth containing increasing amounts of CM or by selecting for high-level resistant derivatives after mutagenic treatment of the bacteria. Some CM-resistant cells obtained by the first method were also resistant to the homologous bacteriophage. Cells trained to grow in the presence of CM developed resistance to some heterologous attacking phages but not to phage ml(3). Mutants selected for phage resistance were not resistant to CM. There appear to be two different loci for CM resistance on the bacterial chromosome: the one for high-level resistance is associated with the phage-resistance locus and the other is independent of it. A concentration of CM (280 mug/ml) that was bacteriostatic for ML3 inhibited the intracellular growth of ml(3) phage in strain ML3-CM(r)I, which had been trained to grow in the presence of that CM concentration, despite the fact that cells of this strain were not phage-resistant per se. The drug had no direct virucidal action and did not prevent adsorption to or penetration of phage into the bacterium. Lysogenization did not occur. It is concluded that the block in phage development probably involves inhibition of synthesis of phage components, either involving deoxyribonucleic acid at an early stage or the phage coat protein at a later one.
Naturally occurring chloramphenicol resistance in bacteria is normally due to the presence of the antibiotic inactivating enzyme chloramphenicol acetyltransferase (CAT) which catalyzes the acetyl-S-CoA-dependent acetylation of chloramphenicol at the 3-hydroxyl group. The product 3-acetoxy chloramphenicol does not bind to bacterial ribosomes and is not an inhibitor of peptidyltransferase. The synthesis of CAT is constitutive in E. coli and other Gram-negative bacteria which harbor plasmids bearing the structural gene for the enzyme, whereas Gram-positive bacteria such as staphylococci and streptococci synthesize CAT only in the presence of chloramphenicol and related compounds, especially those with the same stereochemistry of the parent compound and which lack antibiotic activity and a site of acetylation (3-deoxychloramphenicol). Studies of the primary structures of CAT variants suggest a marked degree of heterogeneity but conservation of amino acid sequence at and near the putative active site. All CAT variants are tetramers composed in each case of identical polypeptide subunits consisting of approximately 220 amino acids. The catalytic mechanism does not appear to involve an acyl-enzyme intermediate although one or more cysteine residues are protected from thiol reeagents by substrates. A highly reactive histidine residue has been implicated in the catalytic mechanism.
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.
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Chloramphenicol resistance to Salmonella typhi in 1989 was 16% in Hinduja Hospital. From Jan to June 1990, this resistance has increased to 81%. 74 strains of blood culture isolated of S typhi obtained from Jan to June were subjected to antibiotic sensitivity testing by disc diffusion technique of Kirby and Bauer 60 strains were found to show a block resistance tochloramphenicol, Ampicillin, Co-trimoxazole, Streptomycin, Tetracycline and Carbenicillin. All strains were sensitive to quinolones. Resistance was record and MIC done in 50 resistant strains by modified National Committee for Clinical Laboratory Standards [NCCLS] according to recommended break points and resistance to Chloramphenicol was confirmed. Of the 47 strains isolated, 34 had received treatment with either chlorampheniicol, Ampicillin or Cotrimoxazole in adequate dosages. In 39 of these 47 patients, Salmonella typhi was isolated after 14 days duration of fever. Plasmid analysis revealed presence of 100 megadalton plasmid in majority of cases. Most patients responded to fluoroquinolones but amongst the admitted patients complications such as Myoglobinuria in 2 cases, perforation in 4 cases, acute Renal failure in 2 cases and typhoid spine in 1 case were seen. Phage typing results of 19 strains were E-8, 0-6.
SUMMARY The protein-synthesizing activity of cell-free preparations of Escherichia coli was estimated by adding radioactive amino acids according to the system of Matthei & Nirenberg (1961). Cell-free systems were prepared from antibiotic-sensitive strains of E. coli and from resistant variants, and the sensitivity of amino acid incorporation to inhibition by chloramphenicol and tetracycline determined. The same sensitivity was shown for the sensitive as for the resistant strains.
Virtually all (94.5 per cent of 110) chloramphenicol-resistant strains of Sbigella, Escherichia, and Staphylococcus isolated from clinical cases caused significant inactivation of the antibiotic, but none of the 29 resistant pseudomonads did so. None of the 235 clinically isolated sensitive strains, representing five genera, inactivated the drug. Resistant organisms derived in vitro from initially sensitive ones by repeated subculture in increasing concentrations of chloramphenicol had only limited ability to inactivate the antibiotic, but those produced via transformation inactivated it almost completely. Such transferred resistance was lost following treatment with acriflavine. Simultaneously, ability to inactivate chloramphenicol was lost.
A multi-step Chloramphenicol (CM)-resistant derivative of an RC-stringent strain of Escherichia coli auxotrophic for threonine and leucine was resistant also to Aureomycin (AM) and Puromycin (PM). All three antibiotics released the repression of RNA synthesis due to amino acid starvation in the CM-sensitive parent strain, their relative activities being about 1:10:100 for AM: CM: PM. High doses of AM and CM failed to induce RNA synthesis. The CM-resistant strain required greater concentrations of each antibiotic than the sensitive strain to induce the same level of RNA synthesis, and appeared to be about one hundred times, ten times and five times more resistant to CM, AM and PM, respectively, than the sensitive strain.
IN an investigation of the biochemical mechanism of chloramphenicol (CM) resistance in Pseudomonas fluorescence, Kuschner1 reported that oxidation of succinate by intact cells of a CM-resistant strain was not affected by CM, although this reaction by the wild strain was moderately inhibited. On the other hand, the same reaction in cell-free systems of both strains was found to be equally sensitive to CM inhibition. From this result, he proposed that CM resistance in the resistant strain was due to decreased permeability against CM. Ramsey2 investigated the action of CM on the formation of inducible enzymes by ultra-socially disrupted cell preparation of Staphylococcus aureus, and observed that the preparation from the CM-resistant strain still retained the resistance of intact cells so that changes in permeability was not the main source of resistance in this organism.
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