Macrolide Resistance Determination and Molecular Typing of Mycoplasma pneumoniae in Respiratory Specimens Collected between 1997 and 2008 in The Netherlands

Abstract

dAn important role in the treatment regimens for Mycoplasma pneumoniae infections is played by macrolide (ML) antibiotics. In the past few years, however, a steady increase has been detected in the worldwide prevalence of ML-resistant (ML r ) M. pneumoniae strains. It is obvious that this increase necessitates a continuous monitoring of ML r and, when detected, modification of antibiotic treatment modalities. Previously, we developed a pyrosequencing-based assay system for the genetic determination of ML r as well as molecular typing of M. pneumoniae. In this study, the sensitivity of this system was improved by the inclusion of a nested-PCR protocol. The modified system was applied to 114 M. pneumoniae-positive specimens that were obtained from a collection of 4,390 samples from patients with acute respiratory tract infections. These samples were collected between 1997 and 2008 in The Netherlands. The pyrosequencing system produced reliable data in 86% of the specimens that contained >500 M. pneumoniae genome copies/ml of patient sample. Each of these samples contained DNA of the ML-sensitive genotype. While 43% of the samples were found to harbor the M. pneumoniae subtype 1 genotype, 57% contained the subtype 2 genotype. We conclude that the pyrosequencing-based assay system is a useful tool for ML r determination and molecular typing of M. pneumoniae in patient samples. ML r -associated M. pneumoniae genotypes, however, were not found in the current study population. Mycoplasma pneumoniae is a pathogen of the human respiratory tract and one of the most prevalent causes of community-acquired pneumonia. General treatment options for M. pneumoniae infections include macrolides (MLs), tetracyclines, and fluoroquinolones. In young children, however, tetracyclines and fluoroquinolones are contraindicated due to unfavorable effects, such as discoloration of teeth by tetracyclines and putative cartilage damage induced by fluoroquinolones. The antibiotics of choice for this patient group are therefore ML antibiotics, such as clarithromycin and azithromycin. In the past decade, however, increasing numbers of M. pneumoniae clinical isolates were found to be resistant against these antibiotics. ML resistance (ML r ) was first reported in Japan in 2002 (14), and since then it has also been detected in the United States (10, 26), Europe (3,7,17,18), eastern Asia (13), and the Middle East (1). The prevalence of ML r varies widely, from 1 to 2% in Denmark (18) to as high as 90% in China (11). Clearly, the emergence of ML r poses a significant threat to the use of MLs in the treatment of M. pneumoniae infections. It is therefore important that ML r among M. pneumoniae isolates is rapidly and efficiently monitored in order to allow effective antibiotic treatment.Although the sensitivity of M. pneumoniae to antibiotics can be determined unambiguously on cultured bacterial isolates, the culturing of M. pneumoniae is time-consuming and precludes the timely identification of antibiotic-resistant isolates. The monitoring of ML r in...

Regional Differences in Prevalence of Macrolide Resistance among Pediatric <i>Mycoplasma pneumoniae</i> Infections in Hokkaido, Japan

“…Macrolides are generally considered the drugs of choice for treatment of children with M. pneumoniae infection (2). Since approximately the year 2000, macrolide-resistant (MR) M. pneumoniae has been appearing in Asia, Europe, Canada, and the USA (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). The rate of macrolide resistance among M. pneumoniae infections ranges from 3 to 26z in Europe (9,15), from 63 to 97z in China (16)(17)(18)(19), and from 25 to 93z in Japan (20)(21)(22).…”

Evaluation of Five Real-Time PCR Assays for Detection of Mycoplasma pneumoniae

“…Despite differences among the methods regarding PCR efficiency, C T values, and analytical and clinical sensitivity, all systems detect at least 20 CFU/5 l of sample. With regard to the quantitative results of other studies based on real-time PCR (16,17,20,21), this can be considered an acceptable level of test sensitivity for most clinical questions. …”

Multilocus Variable-Number Tandem-Repeat Analysis of Mycoplasma pneumoniae Clinical Isolates from 1962 to the Present: a Retrospective Study

“…Methods such as PCR-restriction fragment length polymorphism (RFLP) analysis of the P1 gene (4,18) and real-time PCR followed by high-resolution melt analysis (HRM) targeting the region of M. pneumoniae repetitive elements 2 and 3 (RepMp2/3) of the P1 gene (19,20) were used to genotype M. pneumoniae into two subtypes (PCR-RFLP) and variants of subtypes 1 and 2 (PCR-HRM). Both sequencing (9) and pyrosequencing (6,21,22) techniques have also been developed to differentiate subtypes of M. pneumoniae. Recently, Degrange et al (6) developed a multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) method for M. pneumoniae based on whole-genome analysis that was able to differentiate 26 distinct VNTR types.…”