In order to determine the possible asymptomatic carriage of Mycoplasma pneumoniae in the upper respiratory tracts of children, Emiel Spuesens and colleagues investigate the prevalence of M. pneumoniae in symptomatic and asymptomatic children at a hospital in The Netherlands. Please see later in the article for the Editors' Summary

The gene encoding major adhesin protein P1 of Mycoplasma pneumoniae, MPN141, contains two DNA sequence stretches, designated RepMP2/3 and RepMP4, which display variation among strains. This variation allows strains to be differentiated into two major P1 genotypes (1 and 2) and several variants. Interestingly, multiple versions of the RepMP2/3 and RepMP4 elements exist at other sites within the bacterial genome. Because these versions are closely related in sequence, but not identical, it has been hypothesized that they have the capacity to recombine with their counterparts within MPN141, and thereby serve as a source of sequence variation of the P1 protein. In order to determine the variation within the RepMP2/3 and RepMP4 elements, both within the bacterial genome and among strains, we analysed the DNA sequences of all RepMP2/3 and RepMP4 elements within the genomes of 23 M. pneumoniae strains. Our data demonstrate that: (i) recombination is likely to have occurred between two RepMP2/3 elements in four of the strains, and (ii) all previously described P1 genotypes can be explained by inter-RepMP recombination events. Moreover, the difference between the two major P1 genotypes was reflected in all RepMP elements, such that subtype 1 and 2 strains can be differentiated on the basis of sequence variation in each RepMP element. This implies that subtype 1 and subtype 2 strains represent evolutionarily diverged strain lineages. Finally, a classification scheme is proposed in which the P1 genotype of M. pneumoniae isolates can be described in a sequence-based, universal fashion.

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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...

The P1, P40, and P90 proteins of Mycoplasma pneumoniae and the MgPa and P110 proteins of Mycoplasma genitalium are immunogenic adhesion proteins that display sequence variation. Consequently, these proteins are thought to play eminent roles in immune evasive strategies. For each of the five proteins, a similar underlying molecular mechanism for sequence variation was hypothesized, i.e., modification of the DNA sequences of their respective genes. This modification is thought to result from homologous recombination of parts of these genes with repeat elements (RepMp and MgPar elements in M. pneumoniae and M. genitalium, respectively) that are dispersed throughout the bacterial genome. Proteins that are potentially involved in homologous DNA recombination have been suggested to be implicated in recombination between these repeat elements and thereby in antigenic variation. To investigate this notion, we set out to study the function of the RecA homologs that are encoded by the M. pneumoniae MPN490 and M. genitalium MG339 genes. Both proteins, which are 79% identical on the amino acid level, were found to promote recombination between homologous DNA substrates in an ATP-dependent fashion. The recombinational activities of both proteins were Mg 2؉ and pH dependent and were strongly supported by the presence of single-stranded DNA binding protein, either from M. pneumoniae or from Escherichia coli. We conclude that the MPN490-and MG339-encoded proteins are RecA homologs that have the capacity to recombine homologous DNA substrates. Thus, they may play a central role in recombination between repetitive elements in both M. pneumoniae and M. genitalium.Mycoplasma pneumoniae is a human pathogen that causes a range of respiratory infections, including atypical pneumonia. This bacterium causes up to 40% of all community-acquired pneumonias and approximately 18% of cases requiring hospitalization for children (for reviews, see references 1 and 35). The closest known relative of M. pneumoniae on the basis of sequence similarity is Mycoplasma genitalium, which is an etiological agent of various diseases of the human reproductive tract, such as urethritis (see reference 28 for a review).Although M. pneumoniae and M. genitalium represent the smallest self-replicating species regarding both cellular dimensions and genome size, it is interesting to note that a significant part of their genomes consists of repeated DNA elements. In M. pneumoniae strain M129 (6, 12), approximately 8% of the 816-kb genome is composed of variants of four different types of repetitive DNA elements (RepMP1, RepMP2/3, RepMP4, and RepMP5) (29, 34, 36), while in M. genitalium strain G-37 T , 4% of the 580-kb genome consists of MgPa repeats (or MgPar sequences) (10,26,27). Common features of the two types of repetitive elements are that (i) their representatives are similar but not identical in sequence and (ii) they are also contained in open reading frames (ORFs) encoding surface-exposed, antigenic proteins. Among these proteins is the M. pneumoniae P1 p...

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