Macrolide resistance determination and molecular typing of Mycoplasma pneumoniae by pyrosequencing

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Abstract

The first choice antibiotics for treatment of Mycoplasma pneumoniae infections are macrolides. Several recent studies, however, have indicated that the prevalence of macrolide (ML)-resistance, which is determined by mutations in the bacterial 23S rRNA, is increasing among M. pneumoniae isolates. Consequently, it is imperative that ML-resistance in M. pneumoniae is rapidly detected to allow appropriate and timely treatment of patients. We therefore set out to determine the utility of pyrosequencing as a convenient technique to assess ML-resistance. In addition, we studied whether pyrosequencing could be useful for molecular typing of M. pneumoniae isolates. To this end, a total of four separate pyrosequencing assays were developed. These assays were designed such as to determine a short genomic sequence from four different sites, i.e. two locations within the 23S rRNA gene, one within the MPN141 (or P1) gene and one within the MPN528a gene. While the 23S rRNA regions were employed to determine ML-resistance, the latter two were used for molecular typing. The pyrosequencing assays were performed on a collection of 108 M. pneumoniae isolates. The ML-resistant isolates within the collection (n = 4) were readily identified by pyrosequencing. Moreover, each strain was correctly typed as either a subtype 1 or subtype 2 strain by both the MPN141 and MPN528a pyrosequencing test. Interestingly, two recent isolates from our collection, which were identified as subtype 2 strains by the pyrosequencing assays, were found to carry novel variants of the MPN141 gene, having rearrangements in each of the two repetitive elements (RepMP4 and RepMP2/3) within the gene. In conclusion, pyrosequencing is a convenient technique for ML-resistance determination as well as molecular typing of M. pneumoniae isolates.

Introduction

Mycoplasma pneumoniae is a common bacterial agent of upper and lower respiratory tract infections (RTIs), especially in children. About 20–40% of all community-acquired pneumonias is caused by this small, cell wall-less bacterium (Atkinson et al., 2008). Effective treatment of M. pneumoniae infections is usually achieved by the use of macrolides (MLs), which are generally considered as the first choice antibiotics in both children and adults for two reasons: (1) M. pneumoniae lacks a cell wall and is therefore intrinsically resistant to cell wall targeting drugs such as penicillin, and (2) MLs have little side effects and are easy to administer, which is of particular importance in children. However, the widespread empirical use of these antibiotics in treatment of RTIs may eventually lead to an increase in the prevalence of ML-resistance of M. pneumoniae. Indeed, a recent study (Morozumi et al., 2008) reported a dramatic increase of ML-resistance among M. pneumoniae isolates, from 0% in 2002 to over 40% in 2008, following the increased use of ML in Japan. In concurrence with this finding, ML-resistant M. pneumoniae isolates have also been reported to emerge in other countries (Dumke et al., 2010, Li et al., 2009, Peuchant et al., 2009). Given the apparent increase in the prevalence of ML-resistance, it is crucial that ML-resistant isolates are detected in a timely fashion to allow targeted treatment adjustments for M. pneumoniae-induced RTIs and to create more awareness of this upcoming problem in the general clinical community.

The ML-resistant phenotypes are defined by specific point mutations in the V domain of the single-copy 23S rRNA gene of M. pneumoniae. The mutations in this gene that induce a high-level of ML-resistance include an A to G transition at position 2063 and an A to C transversion at position 2064 (Morozumi et al., 2010). Low-level ML-resistance is induced by an A to G transition at position 2067 and a C to G or A transversion at position 2617 of the 23S rRNA gene (Okazaki et al., 2001, Pereyre et al., 2004). The presence of these mutations can be detected by PCR of the 23S rRNA gene followed by dideoxy chain termination sequencing. This procedure, however, can be laborious and time-consuming. Other methods, such as those based on real-time PCR followed by (high-resolution) melt analysis, have clear advantages over previous assays, in particular regarding the speed at which data can be obtained (Dumke et al., 2010, Wolff et al., 2008). Nevertheless, these methods often rely on the execution of multiple rounds of PCR and require ample experience in interpretation of the results. It is therefore important to seek novel, convenient molecular tests for the detection of ML-resistance.

Because molecular diagnostic tests have already proven their value in both detection and genotyping of M. pneumoniae, it would be useful to design assay platforms in which these tests can be combined with those aimed at the determination of ML-resistance. Currently, real-time PCR is the predominant technique used for the detection of M. pneumoniae in clinical microbiological laboratories. The methods and approaches used for genotyping of M. pneumoniae, however, are more diverse. The majority of these methods are aimed at the detection of variation within the MPN141 gene. This gene, which encodes the immunodominant and essential cytadhesin P1, contains two DNA elements, termed RepMP4 and RepMP2/3, which are variable among M. pneumoniae isolates (Dumke et al., 2006, Kenri et al., 1999, Pereyre et al., 2007, Spuesens et al., 2010, Spuesens et al., 2009). It is hypothesized that recombination between these elements and their homologs located at other positions in the bacterial genome is the cause of this variation. In total, the genome of M. pneumoniae strain M129 was found to include 8 variants of RepMP4 and 10 variants of RepMP2/3 (Himmelreich et al., 1996). In principle, the determination of the structure of the variable regions (RepMP4 and RepMP2/3) within the MPN141 gene can address two separate questions. First, it addresses whether sequences from another RepMP element within the genome have been introduced into the MPN141 gene (Spuesens et al., 2009). This information may be clinically and epidemiologically useful if the changes on the DNA level lead to an altered P1 amino acid sequence, which could have an influence on the virulence of M. pneumoniae isolates. Second, the sequences of the RepMP elements within the MPN141 gene indicate to which of the two major M. pneumoniae subtypes (subtypes 1 and 2) an isolate can be classified. However, the sequence differences between subtype 1 and 2 strains can be observed throughout the genome of M. pneumoniae, including all RepMP elements, and are not restricted to the MPN141 gene (Cousin-Allery et al., 2000, Dumke et al., 2003, Spuesens et al., 2009). The two subtypes of M. pneumoniae therefore appear to represent two separate evolutionary lineages (Spuesens et al., 2009). In this respect, it is important to discriminate between determination of the subtype of isolates on the one hand, and determination of the MPN141 genotype on the other. In principle, a ‘genotype switch’ of MPN141 can occur at any given moment in a single bacterium within a population due to a RepMP recombination event. This switch can occur irrespective of the subtype of the bacterium. However, a bacterium cannot switch from subtype 1 to subtype 2 (or the reverse). From an evolutionary point of view it is therefore primarily important to classify isolates on the basis of their subtype. With this in mind, we set out to evaluate whether the application of the pyrosequencing technique would be useful in the design of a simple and reliable molecular procedure aimed at: (i) the determination of the subtype of M. pneumoniae isolates, and (ii) the detection of 23S rRNA mutations responsible for ML-resistance. The pyrosequencing technique, which is a real-time sequencing-by-synthesis method, is of particular interest because it is highly suitable for the generation of relatively short DNA sequences.

In this study, we describe the design and evaluation of a set of four different pyrosequencing assays. Two of these were aimed at the determination of the subtype of M. pneumoniae strains by exploiting single-nucleotide polymorphisms (SNPs) between subtype 1 and subtype 2 strains. The other two assays were used for the detection of the aforementioned ML-resistant genotypes. We report that these assays are convenient and reliable, providing a 100% correlation with conventional molecular methods for typing and ML-resistance determination. In addition, we describe the identification of two M. pneumoniae isolates carrying novel MPN141 variants, having rearrangements in both the RepMP2/3 and RepMP4 element of the MPN141 gene.

Section snippets

M. pneumoniae strains, culturing and DNA isolation

A total of 108 M. pneumoniae isolates were used in this study. These isolates included the three reference strains M129 (ATCC 29342), MAC (ATCC 15492) and FH (ATCC 15531) and 100 clinical isolates, obtained between 1973 and 2005, (kindly provided by R. Dumke) (Dumke et al., 2003, Maquelin et al., 2009). Briefly, these included 69 subtype 1 strains (one of which carrying an MPN141 rearrangement) and 31 subtype 2 strains (four of which with an MPN141 rearrangement). Three of the isolates were

Identification of ML-resistant genotypes in M. pneumoniae by pyrosequencing

In the design of a pyrosequencing procedure that is suitable for the detection of all 23 S rRNA mutations known to be involved in ML-resistance, two different pyrosequencing reactions were developed. This was required because pyrosequencing produces DNA sequences with lengths up approximately 60 nucleotides, whereas the mutations involved in ML-resistance are situated approximately 550 bp apart, around positions 2065 and 2617 of the 23S rRNA. The first pyrosequencing reaction (termed 23S rRNA 1;

Discussion

In this study, we have demonstrated that pyrosequencing is highly useful for the genetic analysis of M. pneumoniae. Specifically, we showed that pyrosequencing can have two important diagnostic applications, i.e. (i) the detection of mutations within the 23 S rRNA gene that are known to be involved in ML -resistance of M. pneumoniae, and (ii) the molecular typing (or subtyping) of M. pneumoniae isolates. The main advantages of pyrosequencing as opposed to other sequencing protocols are that: (i)

Acknowledgments

AMCvR is supported by grants of the European Society for Pediatric Infectious Diseases, ZonMW, and the Erasmus MC. We are thankful to Dr. R. Dumke for making available his strain collection. D. Stumpel and Dr. R.W. Stam are thanked for their assistance and advice concerning the pyrosequencing procedure.

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