Increased macrolide resistance of Mycoplasma pneumoniae in France directly detected in clinical specimens by real-time PCR and melting curve analysis. J Antimicrob Chemother

Laboratoire de Bactériologie EA 3671, Université Victor Segalen Bordeaux 2 and CHU de Bordeaux, Bordeaux cedex, France.
Journal of Antimicrobial Chemotherapy (Impact Factor: 5.31). 06/2009; 64(1):52-8. DOI: 10.1093/jac/dkp160
Source: PubMed


Mycoplasma pneumoniae is a common aetiological agent of community-acquired respiratory tract infections for which macrolides are the treatment of choice. In France, only two macrolide-resistant isolates were reported in 1999. In contrast, several recent data reported that macrolide-resistant M. pneumoniae isolates have been spreading since 2000 in Japan. Mutations A2058G (Escherichia coli numbering), A2058C, A2059G, A2062G, C2611A and C2611G in domain V of the 23S rRNA gene were associated in vivo or in vitro with this resistance. The aim of this study was to determine whether macrolide resistance of M. pneumoniae is emerging in France.
We developed a duplex real-time PCR for the detection of the six 23S rRNA mutations associated with macrolide resistance in M. pneumoniae and a simplex real-time PCR for the identification of the A2058G mutation, the most common one. Both methods rely on fluorescence resonance energy transfer coupled to melting curve analysis and are directly applicable to clinical samples. The duplex real-time PCR assay, first validated on 40 genetically characterized M. pneumoniae strains, was then applied directly on 248 French respiratory tract clinical samples.
Among M. pneumoniae-positive specimens collected before 2005, no macrolide-resistant M. pneumoniae isolate was detected. In contrast, among 51 samples collected between 2005 and 2007, five (9.8%) yielded a resistant genotype, suggesting a recent increase in macrolide-resistant M. pneumoniae isolates in France.
The epidemiological monitoring of macrolide resistance in this species has become necessary in France and Europe, and will be made easier by using these PCR assays.

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    • "Previous studies have confirmed that a single base mutation at position 2063 in domain V of the 23S rRNA gene of M. pneumoniae is the most prevalent mutation, followed by a mutation at position 2064, both conferring high-level macrolide resistance. Current molecular techniques for identifying these mutations include direct sequencing, restriction fragment length polymorphism analysis, real-time PCR with high-resolution melting curve analysis, PCR with pyrosequencing, and allele-specific PCR (Bebear et al., 2011; Li et al., 2012; Matsuoka et al., 2004; Peuchant et al., 2009; Spuesens et al., 2010, 2012; Wolff et al., 2008). These methods all have various advantages and limitations (Li et al., 2012). "
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    ABSTRACT: The aim of this study was to develop a single-nucleotide polymorphism (SNP) PCR assay to be performed directly on respiratory samples for the simultaneous detection of Mycoplasma pneumoniae and its 23S rRNA gene mutations, which are responsible for macrolide resistance. For multiplex SNP PCR, two outer primers for amplification of the 23S rRNA gene and two mutant-specific primers for the discrimination of single base changes were designed. A total of 73M. pneumoniae-positive samples and 100M. pneumoniae-negative samples were analyzed using this assay. By SNP PCR, we detected two mutations conferring high-level macrolide resistance in 22 samples (A2063G from 20 and A2064G from 2 samples); these results are identical to those produced by the 23S rRNA gene sequencing of M. pneumoniae-positive samples. Thus, this assay can be used as a practical method for the simultaneous detection of M. pneumoniae and mutations associated with macrolide resistance directly from respiratory samples.
    Full-text · Article · Apr 2014 · Journal of microbiological methods
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    • "Similarly, since our team firstly reported the appearance of resistant strains of M. pneumoniae in 2005 [13], the frequency of macrolide-resistant M. pneumoniae remained high in China, ranging from 84.4% to 100% (not published) in our lab as well as in other Chinese researchers [9, 14]. In France, Pereyre et al. reported the emergence of M. pneumoniae drug resistant strains; only 2 of 155 showed resistance to macrolide isolated between 1994 and 2006 [11] but risen to 10% between 2005 and 2007 reported by Peuchant et al. [15]. In the other countries, the results were as follows: In USA, the frequency of macrolide-resistant M. pneumoniae was 8.2% during 2007 and 2010 [16], and in Italy that was 11 out of 43 in 2010 [17]; in Germany, 2 of 167 throat swabs were macrolide resistant collected between 2003 and 2008, reported by Dumke et al. in 2010, while during 1991 and 2009, only 3 of 99 isolation showed resistance [6], and so on. "
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    ABSTRACT: Throat swabs from children with suspected Mycoplasma pneumoniae (M. pneumoniae) infection were cultured for the presence of M. pneumoniae and its species specificity using the 16S rRNA gene. Seventy-six M. pneumoniae strains isolated from 580 swabs showed that 70 were erythromycin resistant with minimum inhibitory concentrations (MIC) around 32-512 mg/L. Fifty M. pneumoniae strains (46 resistant, 4 sensitive) were tested for sensitivity to tetracycline, ciprofloxacin, and gentamicin. Tetracycline and ciprofloxacin had some effect, and gentamicin had an effect on the majority of M. pneumoniae strains. Domains II and V of the 23S rRNA gene and the ribosomal protein L4 and L22 genes, both of which are considered to be associated with macrolide resistance, were sequenced and the sequences were compared with the corresponding sequences in M129 registered with NCBI and the FH strain. The 70 resistant strains all showed a 2063 or 2064 site mutation in domain V of the 23S rRNA but no mutations in domain II. Site mutations of L4 or L22 can be observed in either resistant or sensitive strains, although it is not known whether this is associated with drug resistance.
    Full-text · Article · Jan 2014
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    • "Macrolide-resistant M. pneumoniae is also emerging in other several countries especially in children [5,22-26]. A recent study in China has shown macrolide resistance in 69% of M. pneumoniae isolates from adolescent and adult patients [26]. "
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    ABSTRACT: Background Although the prevalence of macrolide-resistant Mycoplasma pneumoniae isolates in Japanese pediatric patients has increased rapidly, there have been no reports concerning macrolide-resistant M. pneumoniae infection in adolescents aged 16 to 19 years old. The purpose of this study was to clarify the prevalence and clinical characteristics of macrolide-resistant M. pneumoniae in adolescent patients with community-acquired pneumonia. Methods A total of 99 cases with M. pneumoniae pneumonia confirmed by polymerase chain reaction (PCR) and culture were analyzed. Forty-five cases were pediatric patients less than 16 years old, 26 cases were 16 to 19-year-old adolescent patients and 28 cases were adult patients. Primers for domain V of 23S rRNA were used and DNA sequences of the PCR products were compared with the sequence of an M. pneumoniae reference strain. Results Thirty of 45 pediatric patients (66%), 12 of 26 adolescent patients (46%) and seven of 28 adult patients (25%) with M. pneumoniae pneumonia were found to be infected with macrolide-resistant M. pneumoniae (MR patients). Although the prevalence of resistant strains was similar in pediatric patients between 2008 and 2011, an increase in the prevalence of resistant strains was observed in adolescent patients. Among 30 pediatric MR patients, 26 had an A-to-G transition at position 2063 (A2063G) and four had an A-to-G transition at position 2064 (A2064G). In 12 adolescent MR patients, 10 showed an A2063G transition and two showed an A2064G transition, and in seven adult MR patients, six showed an A2063G transition and one showed an A2064G transition. Conclusions The prevalence of macrolide-resistant M. pneumoniae is high among adolescent patients as well as pediatric patients less than 16-years old. To prevent outbreaks of M. pneumoniae infection, especially macrolide-resistant M. pneumoniae, in closed populations including among families, in schools and in university students, physicians should pay close attention to macrolide-resistant M. pneumoniae.
    Full-text · Article · May 2012 · BMC Infectious Diseases
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