Effect of tobramycin on protein synthesis in 2-deoxystreptamine aminoglycoside-resistant clinical isolates of Haemophilus influenzae

Abstract

Clinical isolates of Haemophilus influenzae resistant to a broad range of 2-deoxystreptamine aminoglycosides (2-DAM) were studied. The gene responsible for resistance could be mobilized by transformation into a 2-DAM susceptible laboratory strain of H. influenzae, enabling isogenic comparisons. The transformants had the same resistance phenotype as the parental strains. There was close linkage between 2-DAM resistance and streptomycin resistance, a chromosomal marker, but weak linkage between 2-DAM and erythromycin resistance. Resistant transformants exhibited a decreased accumulation of gentamicin due to the absence of the rapid, energy-dependent phase of uptake. Resistance was not through metabolic inactivation of the antibiotic; no aminoglycoside-acetylating, -adenylylating, or -phosphorylating activity was detected in the wild-type strains or in the 2-DAM-resistant transformants. Protein synthesis in 2-DAM-susceptible H. influenzae strains increased in the presence of low (1 ,ug/ml) and moderate (50 ,ug/ml) concentrations of tobramycin. With higher concentrations (100 and 500 ,ug/ml), protein synthesis was progressively inhibited. In contrast, protein synthesis in 2-DAM-resistant clinical isolates and transformants was inhibited by 1 ,ug of tobramycin per ml, and inhibition increased with higher drug concentrations. Since the stimulating effect of low concentrations of tobramycin in susceptible H. influenzae strains is probably due to misreading, these findings suggest that 2-DAM-resistant strains of H. influenzae have reduced sensitivity to misreading, indicating that altered ribosomes are responsible for the resistance.Although its pathogenic role has not been defined with certainty, nontypable Haemophilus influenzae can be cultured in high densities from the bronchial secretions of patients with cystic fibrosis (CF) or chronic bronchitis (21,26,33,38). With the use of selective culture media, this organism frequently can be recovered in the presence of other pathogenic organisms, such as members of the Enterobacteriaceae, Pseudomonas aeruginosa, Streptococcus pneumoniae, and Staphylococcus aureus (33,45). When patients with CF harbor P. aeruginosa and H. influenzae in similar densities in their bronchial secretions, the former organism is generally considered the main pathogen, and antimicrobial therapy that is effective against P. aeruginosa (i.e., an antipseudomonal penicillin and a 2-deoxystreptamine aminoglycoside [2-DAM]) is administered. The diffusion of these antibiotics into the bronchial secretions is poor, and their bioactivity in this environment is reduced; antipseudomonal penicillins are inactivated by P-lactamases (15), and aminoglycosides are bound to DNA from lysed cells (24,40) and antagonized by the low pH (3) and the ionic content of purulent bronchial secretions (24). Thus, high densities of H. influenzae in the bronchial secretions may be exposed for prolonged periods to subinhibitory concentrations of 2-DAM.We hypothesized that this situation could favor the emergence of resistance. ...

Correlation of tobramycin-induced inhibition of protein synthesis with postantibiotic effect in Escherichia coli

“…Our results confirm that during exposure of E. coli to tobramycin, global macromolecular synthesis was eventually inhibited, as has been shown by several investigators (21,22,32,36). Gentamicin has been shown to bind to DNA in purulent material and lysed neutrophils (34 The precise mechanism of action of aminoglycoside-induced PAE remains a subject of speculation.…”

Effect of N-Acetylcysteine in Combination with Antibiotics on the Biofilms of Three Cystic Fibrosis Pathogens of Emerging Importance

“…Our third choice, tobramycin, an aminoglycoside, binds to bacterial ribosomal subunits and inhibits bacterial protein synthesis [41]. Nebulised tobramycin is also commonly used in the treatment of P. aeruginosa in CF patients, however in recent years, there has been a pattern of emerging resistance of CF bacteria against aminoglycoside antibiotics [42].…”

“Affect of anaerobiosis on the antibiotic susceptibility of H. influenzae”

“…Tetracycline prevents the association of aminoacyl tRNA with the ribosome, which stops protein synthesis [13] and has no immediate effect on the cell membrane. We had previously shown that therapeutic tobramycin concentrations caused mistranslation in aminoglycoside susceptible H. influenzae [14]. …”