ABSTRACT
<p><b>OBJECTIVE</b>Mutations in 23S rRNA gene are known to be associated with macrolide resistance in Mycoplasma pneumoniae (M. pneumoniae). However, these mutations alone do not fully explain the high resistance rates in Asia. The aim of this study was to investigate other possible mutations involved in macrolide resistance in M. pneumoniae.</p><p><b>METHODS</b>The whole genomes of 10 clinical isolates of M. pneumoniae with macrolide resistance were sequenced by Illumina HiSeq2000 platform. The role of the macrolide-specific efflux transporter was assessed by efflux-pump inhibition assays with reserpine and carbonyl cyanide m-chlorophenyl-hydrazone (CCCP).</p><p><b>RESULTS</b>A total of 56 single nucleotide polymorphisms (SNPs) were identified in 10 clinical isolates in comparison to the reference strains M129 and FH. Strikingly, 4 of 30 SNPs causing non-synonymous mutations were clustered in macrolide-specific efflux system gene macB encoding macrolide-specific efflux pump protein of the ATP-binding cassette transporter family. In assays of the minimal inhibitory concentrations (MIC) of macrolide antibiotics in the presence of the efflux pump inhibitors caused a significant decrease of MICs, even under detectable levels in some strains.</p><p><b>CONCLUSION</b>Our study suggests that macrolide efflux pump may contribute to macrolide resistance in M. pneumoniae in addition to the common point mutations in 23S rRNA gene.</p>
Subject(s)
Anti-Bacterial Agents , Pharmacology , Drug Resistance, Bacterial , Genetics , Genome-Wide Association Study , Macrolides , Pharmacology , Microbial Sensitivity Tests , Mutation , Mycoplasma pneumoniae , GeneticsABSTRACT
The researches on chlamydia in recent years show that chlamydia bacteriophage may be a potential and effective means to solve the clinical infection of chlamydia trachomatis (Ct).We investigated the biological effect of chlamydiaphage phiCPG1 capsid protein Vp1 on Ct both in McCoy cells and genital tract of mice.Different concentrations of Vp1 were co-incubated with Ct E serotype strain in McCoy cells.Female BALB/c mice were used to establish Ct E strain-induced urogenital infection model.They were randomly divided into five groups and given different treatments on the fifth day after Ct inoculation.Animals in groups 1 and 2 were given 30 μL different concentrations of Vp1 in the genital tract respectively,those in group 3 were intramuscularly injected with 30 μL Vp1,those in the infected group did not receive any intervention,and those in the control group received 30 μL PBS in the genital tract.The vaginal discharge was collected to identify the live chlamydia by cell culture and gene fragment by real time PCR different days after infection.Inhibition rate of 100 μg/mL and 50 μg/mL Vpl proteins against Ct E strain in the McCoy cell cultures was 91％ and 79％ respectively,The number of intracellular Ct inclusion in the McCoy cells co-cultured with vaginal discharge of group 1 and group 2 was less than in the infected group,and that in group 1 was less than in group 2,on the 7th day after Ct inoculation.Real-time PCR showed that chlamydia concentration of the vaginal discharge in group 2 was lower than in the infected group,and that in group 1 was lower than in group 2 on the 10th day.It was suggested that Vp1 capsid proteins had inhibitory effect on the proliferation of Ct serovar E strain in cell culture and mouse genital tract.
ABSTRACT
<p><b>BACKGROUND</b>Mycoplasma pneumoniae (M. pneumoniae) is one of the common pathogens causing atypical pneumonia. In recent years, resistance to macrolides has become more common, especially in China. Previous studies have confirmed that the mutation at position 2063 in domain V of the 23S rRNA is the most prevalent, followed by the mutation at position 2064. Reported molecular detection methods for the identification of these mutations include direct sequencing, restriction fragment length polymorphism analysis, real-time polymerase chain reaction (PCR) with high-resolution melt analysis, and nested PCR-linked with capillary electrophoresis, etc. The most commonly used method for monitoring resistance-conferring mutations in M. pneumoniae is direct DNA sequencing of PCR or nested PCR products. However, these methods are time-consuming, labor-intensive or need expensive equipments. Therefore the development of rapid and sensitive methods is very important for monitoring the resistance globally.</p><p><b>METHODS</b>In this study, we reported a fast and cost-effective method for detecting 2063 and/or 2064 macrolide resistant mutations from specimens using a modified allele-specific PCR analysis, and all results were compared with the sequencing data. We also analyzed the clinical courses of these samples to confirm the modified allele-specific PCR results.</p><p><b>RESULTS</b>Among 97 M. pneumoniae specimens, 88 were found to possess mutations by this method, and all modified allele-specific PCR analysis results were consistent with the sequencing data. The data of the clinical courses of these 97 cases showed that they suffered from severe pneumonia. Erythromycin showed better efficacy on cases from which no macrolide resistance mutation was found on their specimens. However, in some cases from which mutations were detected, erythromycin monotherapy had poor efficacy, and on these patients severe symptoms improved only when azithromycin was added to the treatment.</p><p><b>CONCLUSIONS</b>The drug-resistant M. pneumoniae is very common in Beijing, China. Our modified allele-specific PCR analysis can identify erythromycin resistant mutations more rapidly from specimens than any other method currently available. Erythromycin is still effective for treating patients infected with the mutation negative M. pneumoniae, but this treatment fails to work on mutant organisms. This method can facilitate clinicians in selecting appropriate therapy within short timescales.</p>