Erythromycin is the drug of choice for treatment of Mycoplasma pneumoniae infections due to its susceptibility to low levels of this antibiotic. After exposure of susceptible strains to erythromycin in vitro and in vivo, mutants resistant to erythromycin and other macrolides were isolated. Their phenotypes have been characterized, but the genetic basis for resistance has never been determined. We isolated two resistant mutants (M129-ER1 and M129-ER2) by growing M. pneumoniae M129 on agar containing different amounts of erythromycin. In broth dilution tests both strains displayed resistance to high levels of several macrolide-lincosamide-streptogramin B (MLS) antibiotics. In binding studies, ribosomes isolated from the resistant strains exhibited significantly lower affinity for Mycoplasma pneumoniae is a common cause of respiratory tract infections particularly in school-aged children and young adults. All levels of the respiratory tract are involved, and while the most characteristic manifestations are acute bronchitis and pneumonia (8, 11), serious complications can occur (5). The sensitivity of M. pneumoniae to erythromycin and many other macrolide antibiotics makes them the drugs of choice for chemotherapy (3, 33). Treatment with erythromycin results in relatively rapid alleviation of symptoms; however, viable M. pneumoniae can frequently be isolated from infected individuals for a prolonged period of time following therapy (6,41,45). Isolation of resistant strains from patients following treatment is not uncommon (26,28,(43)(44)(45), and erythromycinresistant mutants are readily derived by selection in vitro (27,43,44). Phenotypic studies have demonstrated that most of these strains simultaneously developed resistance to macrolide, lincosamide, and group B streptogramin (MLS) antibiotics (48). It was suspected that development of resistance to erythromycin contributed to the prolonged colonization of the respiratory tract following chemotherapy.As part of our efforts to understand what role, if any, development of antibiotic resistance may play in the course of M. pneumoniae infection, we are attempting to determine the molecular mechanisms by which resistance to erythromycin can arise. While the genetic basis for MLS resistance has been extensively studied for other bacteria, except for a rudimentary study of Ureaplasma urealyticum (30) it has not been explored in mycoplasmas. We show that MLS-type resistance in two isolates derived in vitro is correlated with A-to-G transitions at two conserved sites in the central loop in domain V of the 23S rRNA which are known to result in similar patterns of resistance in other organisms. The possible clinical implications of these mechanisms are discussed. MATERIALS AND METHODSMedia and growth conditions. M. pneumoniae M129-B16 (ATCC 33530) was grown in glass culture bottles or 96-well microtiter plates in modified Hayflick's medium (19) without penicillin. One-percent Bacto-Agar (Difco, Detroit, Mich.) was added for growth on solid medium. Antibiotics were obtained ...

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Molecular cloning and sequencing showed that Mycoplasma gallisepticum, like Mycoplasma capricolum, contains both tRNAUCA and tRNACCA genes, while Mycoplasma pneumoniae and Mycoplasma genitalium each appear to have only a tRNAUCA gene. Therefore, these mycoplasma species contain a tRNA with the anticodon UCA that can translate both UGA and UGG codons.Although UGA is a stop (opal) (Fig. 1A) were employed as probes, and they gave identical results (not shown). The hybridizing DNA fragments from M. genitalium (AluI, DraI, and EcoRI fragments of 390 bp, 622 bp, and 5 kbp, respectively) and M. gallisepticum (410-and 850-bp AluI and 5.45-and 5.75-kbp EcoRI fragments) were cloned in E. coli. These regions were mapped, and relevant portions of both strands were sequenced.The cloned M. genitalium DNA was found to contain a tRNA gene with the anticodon UCA (Fig. 2B), while the M. gallisepticum clones contained both a tRNA gene with the anticodon UCA (Fig. 2C) and one with the anticodon CCA 504 on June 7, 2019 by guest

Employment of recombinant viruses as gene transfer vectors is limited by constraints on the size and functional design of the genetic material to be transferred as well as potential safety hazards deriving from obligatory co-transfer of viral genetic elements. As an alternative strategy that capitalizes on the efficient cellular entry mechanisms of viruses, we have derived adenovirus-polylysine-DNA complexes whereby foreign DNA is transferred bound to the exterior of the virion. This linkage was accomplished utilizing an antibody bridge in which a monoclonal antibody was rendered competent to carry DNA by the attachment of a polylysine residue. Attachment of the antibody-polylysine to the virus was by virtue of the antibody's specificity for the virion. The resulting vector system mediates high-efficiency gene transfer to target cells in vitro. In addition, this vector design allows greatly enhanced flexibility in terms of the size and design of heterologous sequences that can be transferred. Since this strategy selectively exploits viral entry functions, which are independent of viral gene expression, the potential exists to derive vectors that avoid the hazards deriving from transfer of parent virus genome.

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