Mutations determining generalized resistance to aminoglycoside antibiotics in Escherichia coli.

Summary Mutations conferring resistance to low levels of kanamycin in Escherichia coli have been mapped at 3 locations: the unc locus (min. 83), a locus we have designated, kanA (min. 72), close to strA (rpsL), and a locus at min. 86.5 previously discovered by Plate (1976) that we have designated ecfB. The unc and ecfB mutations are associated with defects in energy metabolism, while mutations at kanA may be in the gene coding for ribosomal protein S12 (rpsL). The three types of mutations cause cross resistance to a number of different aminoglycoside antibiotics and the effects of the mutations are cumulative in combination.     

Inheritance of low-level resistance to penicillin, tetracycline, and chloramphenicol in Neisseria gonorrhoeae.

The genetics of low-level resistance to penicillin and other antibiotics in a clinical isolate and a multistep laboratory mutant of Neisseria gonorrhoea was studied by transformation. Mutations at three loci affected sensitivity to penicillin. Mutation at penA resulted in an eightfold increase in resistance to penicillin without affecting response to other antimicrobial agents. Mutation at ery resulted in a two- to fourfold increase in resistance to penicillin and similar increases in resistance to many other antibiotics, dyes, and detergents. Mutation at penB resulted in a fourfold increase in resistance to penicillin and tetracycline, the phenotypic expression of which was dependent on the presence of mutation at ery. The cumulative effect of mutations at penA, ery, and penB was an approximate 128-fold increase in penicillin resistance, to a minimum inhibitory concentration of 1.0 mug/ml. Low-level resistance to tetracycline or chloramphenicol was due to similar additive effects between mutations at the nonspecific ery and penB loci and a locus specific for resistance to each drug (tet and chl, respectively). No evidence was found for penicillinases or other drug-inactivating enzymes.     

Gene in the major cotransduction gap of the Escherichia coli K-12 linkage map required for the expression of chromosomal resistance to tetracycline and other antibiotics

In Escherichia coli K-12, amplifiable resistance to tetracycline, chloramphenicol, and other unrelated antibiotics was mediated by at least four spatially separated loci. Tetracycline-sensitive mutants were isolated by Tn5 insertional inactivation of an amplified multiply resistant strain. One of these, studied in detail, showed coordinate loss of expression of all other resistance phenotypes. The Tn5 element in this mutant mapped to 34 min on the E. coli K-12 linkage map. We have designated the locus marA (multiple antibiotic resistance). Tetracycline-sensitive mutants containing marA::Tn5 regained all resistance phenotypes at frequencies of 10(-8) to 10(-7) upon precise excision of Tn5. Moreover, a newly described tetracycline efflux system (A. M. George and S. B. Levy, J. Bacteriol. 155:531-540, 1983) was inactivated in tetracycline-sensitive mutants, but recovered in tetracycline-resistant revertants. In merodiploids, F-prime marA+ expressed partial or complete dominance over corresponding mutant chromosomal alleles. Dominance tests also established that a previously amplified host and a mutant marA allele were preconditions for the expression of phenotypic resistances.     

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.     

Selection for loss of tetracycline resistance by Escherichia coli

An improved medium for the direct, positive selection of tetracycline-sensitive clones from a population of tetracycline-resistant strains of Escherichia coli is described.     

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.     

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.     

Expression in Escherichia coli of a staphylococcal gene for resistance to macrolide, lincosamide, and streptogramin type B antibiotics.

Plasmid pBD9, which comprises two plasmids from Staphylococcus aureus, pE194 and pUB110, was joined to plasmid pBR322 by in vitro recombination to form plasmid pKH80. The ermC gene of plasmid pE194 confers inducible resistance to macrolide, lincosamide, and streptogramin type B antibiotics. When pKH80 was transferred to Escherichia coli K-12, the bacteria became resistant to several of these antibodies.