In Vitro Development of Resistance to Erythromycin, Other Macrolide Antibiotics, and Lincomycin in Mycoplasma pneumoniae

The molecular mechanisms of resistance to fluoroquinolones, tetracyclines, an aminocyclitol, macrolides, a lincosamide, a phenicol, and pleuromutilins were investigated in Mycoplasma bovis. For the identification of mutations responsible for the high MICs of certain antibiotics, whole-genome sequencing of 35 M. bovis field isolates and 36 laboratory-derived antibiotic-resistant mutants was performed. In vitro resistant mutants were selected by serial passages of M. bovis in broth medium containing subinhibitory concentrations of the antibiotics. Mutations associated with high fluoroquinolones MICs were found at positions 244 to 260 and at positions 232 to 250 (according to Escherichia coli numbering) of the quinolone resistance-determining regions of the gyrA and parC genes, respectively. Alterations related to elevated tetracycline MICs were described at positions 962 to 967, 1058, 1195, 1196, and 1199 of genes encoding the 16S rRNA and forming the primary tetracycline binding site. Single transversion at position 1192 of the rrs1 gene resulted in a spectinomycin MIC of 256 g/ml. Mutations responsible for high macrolide, lincomycin, florfenicol, and pleuromutilin antibiotic MICs were identified in genes encoding 23S rRNA. Understanding antibiotic resistance mechanisms is an important tool for future developments of genetic-based diagnostic assays for the rapid detection of resistant M. bovis strains. KEYWORDS antibiotic resistance, cattle, Mycoplasma bovisA ntibiotics are among the most important therapeutic tools in the veterinary and human medicine, but their use is limited since resistance tends to evolve in pathogenic bacteria. The microorganisms are exposed to selective pressure by the use of antimicrobials in medicine and agriculture, favoring the development, survival, and spread of resistant clones (1).Mycoplasma spp. are members of the class Mollicutes and comprise the simplest life form that can replicate independently from the host (2). Mycoplasma spp. have no cell wall and they have a limited number of metabolic pathways. The greatly reduced genome size and coding capacity of Mycoplasma spp. makes them a good model for genetic studies. Mycoplasma spp. are fast-evolving bacteria with several human and animal pathogens; however, their importance is often underestimated (2). Mycoplasma bovis is a major cause of calf pneumonia, mastitis and arthritis, and it is responsible for significant economic losses (3). Adequate housing and appropriate antibiotic treatment

Section: Discussionmentioning

confidence: 48%

Mutations Associated with Decreased Susceptibility to Seven Antimicrobial Families in Field and Laboratory-Derived Mycoplasma bovis Strains

Sulyok

Kreizinger

Wehmann

et al. 2017

“…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).…”

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confidence: 99%

Transition mutations in the 23S rRNA of erythromycin-resistant isolates of Mycoplasma pneumoniae

Lucier

Heitzman

Liu

et al. 1995

168

146

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 ...

“…Niitu et al reported the development of erythromycin resistance in M. pneumoniae that was accompanied by cross-resistance to other macrolide antibiotics in vitro; they also reported the isolation of M. pneumoniae that was resistant to erythromycin and other antibiotics after erythromycin therapy (14,15). New quinolones might exhibit antimycoplasmal activity against macrolide-and tetracycline-resistant strains of M. pneumoniae because of their different mechanisms of action.…”

Section: Discussionmentioning

confidence: 99%

“…Macrolide and tetracycline antibiotics have been widely used in chemotherapy against M. pneumoniae infections because of the susceptibility of the infectious agent to these antibiotics (1,4,13,17,23). At the same time, the reported incidences of mutants resistant to erythromycin from patients treated with or without the antibiotics have increased (14,15).Recently, the efficacy of new quinolones against various respiratory pathogens, including M. pneumoniae, has been demonstrated (4,16,22). We have also reported that temafloxacin, ofloxacin, and ciprofloxacin, among several new quinolones, possess more mycoplasmacidal activity against M. pneumoniae than macrolide and tetracycline antibiotics.…”

mentioning

confidence: 98%

See 1 more Smart Citation

Effects of new quinolones on Mycoplasma pneumoniae-infected hamsters

Arai

Gohara

Akashi

et al. 1993

The efficacies of the new quinolones temafloxacin, ofloxacin, and ciprofloxacin were investigated against Mycoplasma pneumoniae in an experimental hamster pneumonia model. Hamsters were infected intratracheally with M. pneumoniae and sacrificed 18 h after the final medication, and their lungs were aseptically removed, homogenized, and cultured quantitatively. The efficacies of these drugs were determined by the CFU of M. pneumoniae in lungs. Temafloxacin and ofloxacin, but not ciprofloxacin, were active when the oral administration of200 mg/kg ofbody weight per day (once per day) for 5 days was initiated 24 h after infection. Although no effect on the elimination of M. pneumoniae was observed after the administration of these drugs at 200 mg/kg/day at 5 days after infection, the continuous administration for 15 days of temafloxacin, but not ofloxacin or ciprofloxacin, significantly reduced viable M. pneumoniae in the lungs. These results suggest that temafloxacin and ofloxacin are effective in the acute phase of infection and, moreover, that temafloxacin is effective in the late stage of infection during which progressive lung alterations and continuous increases in mycoplasmal growth occurred. The peak levels of temafloxacin in sera and lungs after oral administration were similar to those of ofloxacin and higher than those of ciprofloxacin. The areas under the curve of temafloxacin in the lung tissue, however, were higher than those of ofloxacin and ciprofloxacin. On the basis of these results, temafloxacin and ofloxacin might be promising antimicrobial agents for the treatment of mycoplasmal infection.Mycoplasma pneumoniae is the causative agent of upper respiratory tract infections and pneumonia in humans and the experimental pneumonia of hamsters (3,5,8,10). Macrolide and tetracycline antibiotics have been widely used in chemotherapy against M. pneumoniae infections because of the susceptibility of the infectious agent to these antibiotics (1,4,13,17,23). At the same time, the reported incidences of mutants resistant to erythromycin from patients treated with or without the antibiotics have increased (14,15).Recently, the efficacy of new quinolones against various respiratory pathogens, including M. pneumoniae, has been demonstrated (4,16,22). We have also reported that temafloxacin, ofloxacin, and ciprofloxacin, among several new quinolones, possess more mycoplasmacidal activity against M. pneumoniae than macrolide and tetracycline antibiotics. In this report, we evaluated the in vivo potency of new quinolones, administered orally against M. pneumoniae pneumonia in hamsters, which exhibited the potent mycoplasmacidal activity in vitro described previously (1). MATERIALS AND METHODSM. pneumoniae. M. pneumoniae 242, freshly isolated from the throat swab of a patient with mycoplasma pneumonia and passaged five to seven times in broth, was used as the challenge strain. Culture samples were kept at -80°C until used for inoculation. The MICs of temafloxacin, ofloxacin, and ciprofloxacin for the strain were 1.56,...