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Molecular typing of mycoplasma pneumoniae: where do we stand?

Brown, Rebecca, Spiller, Owen Bradley and Chalker, Victoria J 2015. Molecular typing of mycoplasma pneumoniae: where do we stand? Future Microbiology 10 (11) , pp. 1793-1795. 10.2217/fmb.15.96

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Mycoplasma pneumoniae is a respiratory bacterial pathogen causing upper and lower respiratory disease in humans of all ages. It is considered a major cause of pneumonia, especially in children of school age and in some cases can result in serious extrapulmonary sequelae. A large increase in reported M. pneumoniae cases was documented in several European countries in 2011 [1]. In England and Wales, seasonal peaks of infection are detected from December to February each year with epidemics at approximately four yearly intervals, lasting 12–15 months [2]. Epidemics are not concurrent worldwide, however, differing countries also report cyclical patterns, as observed in England and Wales, such as Denmark, Sweden, Norway, Finland, Korea and Japan [3,4]. Additionally, in differing countries, seasonal peaks of infection have been observed in either summer or autumn and no definitive factor has been proven to account for seasonal variation or the formation of epidemic peaks. Traditionally, molecular typing was used to characterize epidemic outbreaks of M. pneumoniae, however, it has been postulated that molecular typing of M. pneumoniae is hampered by the genetically homologous nature of the species [5]. Despite this, molecular typing methods have been developed for this organism including: PCR-restriction length polymorphism (RFLP) of the major surface adhesin P1 [5], multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) [6], multilocus sequence typing (MLST) [7] and the recent SNaPshot™ minisequencing assay [8]. The mechanisms driving fluctuations in incidence of M. pneumoniae infections have not been defined. It has been postulated that shifts in proportion of individual strains with specific P1 type or concurrent increased incidence of several strains may result in epidemics or immunity. Additionally, it is believed that the genotype of M. pneumoniae may be changing, generating diverse genetic material in each epidemic with a recent study reporting the detection of polyclonal strains in a single epidemic [9]. The initial molecular typing procedure targeted the gene encoding of the major surface adhesin, P1, of M. pneumoniae. RFLP analysis of the p1 gene was the most common genotyping method, enabling separation of M. pneumoniae isolates into two types, type 1 and 2 [5,10]. Studies utilizing the repetitive regions, RepMp2/3 and RepM4 in the p1 gene resulted in the identification of an additional six variants [11–13]. Speculation that a shift in P1 adhesin type may be the cause of epidemics has been disputed with evidence indicating the presence of multiple P1 adhesin types in observed increases of infection [6,9,10]. It was hypothesized that a decline in immunity or an increase of the immunologically naive population may result in the 4-year cycle of epidemic periods [14]. In other geographical locations, it has also been observed that multiple P1 types can be detected during outbreaks, and it has been suggested that although immunological pressure may favor shifts of P1 type, a co-circulation of P1 types appears to be common [15]. MLVA has been increasingly used internationally for strain characterization and is based on variation in the copy number of tandem repeated sequences, called VNTRs, found at different loci across the genome. The variation of the copy number of these tandem repeats depends on the isolate tested. Initially, 265 strains were grouped into 26 MLVA types, based on five VNTR loci (Mpn1, Mpn13–16) and additional novel types have since been reported [6,16]. MLVA was documented to be more discriminatory for M. pneumoniae strains than P1 typing, providing an additional level of classification for transmission studies. However, reports of observed instability in the Mpn1 locus has called into question the reliability of the marker. Additionally, inconsistency in nomenclature and identification of repeat regions has led to international standardization of the MLVA and the removal of Mpn1 as a locus [17]. Analysis of the 2010/2011 epidemic in the UK revealed a total of 11 distinct MLVA types present using the original typing method [14], however, reanalysis using international guidelines reduces the MLVA types detected to five distinct types [Unpublished Data]. The discriminatory power of the MLVA method for characterization of M. pneumoniae strains has reduced with the removal of the Mpn1 locus, necessitating either the identification of new loci or alternative typing methods. Initial attempts at developing an MLST scheme for M. pneumoniae were unsuccessful due to low levels of polymorphisms found in the housekeeping genes examined, suggested to be because of the homogeneity of the M. pneumoniae species, and it was concluded that the use of an MLST scheme with housekeeping and structural genes was not useful for molecular typing. However, three housekeeping genes were examined for polymorphisms across 30 isolates of either P1 type 1, 2 or a variant strain and the other genes selected for analysis were examined against a single representative strain from each P1 type [18]. Recently, an MLST scheme was successfully developed to differentiate M. pneumoniae isolates based on sequence polymorphisms in eight housekeeping genes, which improved on existing typing methods for M. pneumoniae [7]. This MLST scheme discriminated between 57 M. pneumoniae isolates with a higher level than both MLVA (with the removal of Mpn1) and P1 typing and it may prove more optimal for epidemiological studies than other existing methods. Population modeling and phylogenetic analysis of concatenated MLST profiles revealed two distinct genetic clades of M. pneumoinae, showing similar topology to phylogenetic data and distinct genetic clustering obtained using genomic sequence analysis. The typing profiles obtained using the MLST method infers representation of the genetic phylogeny, reflecting that M. pneumoniae can be subdivided into two distinct genetic lineages [7]. Nevertheless, this MLST scheme has not yet been applied to localized outbreak or epidemic strain analysis or has not been demonstrated direct on clinical specimens. Recent development of a SNaPshot™ mini sequencing assay has resulted in identification of nine SNP types [8]. This method is rapid and appears to have greater discriminatory ability than MLVA and P1 typing. A direct comparison of MLST and SNaPshot™ minisequencing assay has not been undertaken and both methods may have similar discriminatory abilities. However, MLST resulted in a larger number of defined sequence types. These methods are all PCR-based and do not necessarily require the growth of bacteria, which can be a lengthy process for M. pneumoniae. P1 typing, MLVA and MLST do not limit investigation through the requirement of specialist methodology. However, MLST can be laborious and expensive, with the cost of genomic sequencing reducing and becoming a more attractive option for genetic analysis of strains. Genomic sequencing may allow the concurrent identification of P1 type, MLVA profile and MLST sequence type directly from the genomic sequence as well as providing additional information, such as the presence of antibiotic resistance and toxin markers. Improvements in sequencing technology and the development of methodologies to produce longer sequence reads enables the reliable determination of repeated DNA sequences [19]. This is of importance for species such as Mycoplasma, in which large tracts of repeated sections within AT-rich genomes are common. For genetically homologous species such as M. pneumoniae, the use of genomic sequencing to analyze phylogeny inferred from single nucleotide polymorphism analysis will improve the ability to accurately segregate this species into distinct lineages allowing in-depth epidemiology studies. Due to the fastidious nature of M. pneumoniae and other human Mollicutes, such as Mycoplasma amphoriforme, the use of metagenomic approaches to identify pathogens in studies of human infections [20] will no doubt improve detection of infections caused by Mollicutes, while obviating the need for expensive and laborious culture and typing methods, simultaneously providing additional data such as the detection of mutations known to confer resistance.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Medicine
Subjects: R Medicine > R Medicine (General)
Uncontrolled Keywords: CAP, MLST, MLVA, Mycoplasma pneumoniae, P1 typing, WGS
Publisher: Future Medicine
ISSN: 1746-0913
Last Modified: 11 Jun 2019 10:02

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