Multilocus sequence types of invasive pneumococcal isolates from Danish infants (0–90 days) 2003–2013

Background A pneumococcal conjugate vaccine (PCV) has been part of the Danish childhood immunization programme since October 2007. It is administered at the ages of 3, 5 and 12 months and healthy infants younger than 90 days are consequently not vaccinated. Initially the PCV-7 vaccine was used but this was replaced by the PCV-13 in April 2010. Vaccination coverage in Denmark is approximately 90 %.The aim of this study was to present multilocus sequence typing (MLST) profiles of Streptococcus pneumoniae isolates from Danish infants (0–90 days) with invasive pneumococcal disease (IPD) in the period 2003–2013. Findings 32 IPD isolates were investigated for their MLST profiles. The identified sequence types (STs) had previously been observed in other European countries. Among the clones were ST 306 (serotype 1), ST 180 (serotype 3) and ST 191 (serotype 7F). Conclusions The ST profile distribution in this study is similar to that observed in other European studies and show a variety of STs. Our data show that the majority of STs found in Denmark is also observed in other European countries, indicating that the IPD isolates were from clones generally circulating in Europe.


Findings
Invasive pneumococcal disease (IPD) is one of the most frequent types of bacteraemia and meningitis in infants in Denmark as well as globally [1,2]. With the introduction and use of the pneumococcal conjugate vaccines (PCV) in children, an effective protection has been provided against IPD caused by the serotypes included in the vaccines [3,4]. The PCV-7 vaccine was introduced into the Danish childhood immunisation programme in October 2007, and in April 2010, it was replaced by a PCV-13 vaccine [5,6]. Several studies have shown the effect of PCVs on the reduction of vaccine serotypes causing IPD [2,5,7].
The vaccination of children in Denmark starts at the age of 3 months; thus infants younger than 90 days are not vaccinated [8]. In a previous study [8], the serotype distribution for IPD cases from Danish infants (0-90 days) during the years 1943-2013 was described. Isolates from this study for the period 2003-2013 were tested for their clonal relationship using multi locus sequence typing (MLST). The present study aims at presenting a descriptive investigation on (1) whether the IPD isolates found in this group of children are dominated by a few clonal complexes or a variety of isolates consisting of singletons, and (2) if these IPD isolates belong to clones that circulate in Denmark only or are generally found in Europe.
Eligible for inclusion in the study were 44 IPD cases from the period 2003 to 2013. Detailed background information, e.g. inclusion criteria and pneumococcal identification details including phenotypic serotyping has previously been published [8]. Of the 44 cases, viable biological material was non-existent for 12 of them, all from before 2007. Thus, only 32 isolates could be analysed for their MLST profiles. The study is based on isolates from Open Access *Correspondence: hcs@ssi.dk Neisseria and Streptococcus Reference Laboratory, Department of Microbiology and Infection Control, Statens Serum Institut, Artillerivej 5, 2300 Copenhagen, Denmark the national laboratory surveillance data on IPD. Since data and samples from patients were collected routinely for diagnostic and national surveillance purposes, no ethical approval or informed consents from parents or guardians were required. The study was approved by the Danish Data Protection Agency (record number 2007-41-0229).
The MLST analysis was performed at the Neisseria and Streptococcus Reference (NSR) laboratory at Statens Serum Institut (SSI) using a protocol previously described [9]. Briefly, the MLST primer pairs for aroE, gdh, gki, recP, spi, xpt, and ddl were used as described in the MLST online database (http://pubmlst.org/). All sequences were analysed using the BioNumerics software (Applied Maths, Sint-Martens-Latem, Belgium). Analysis and annotation of sequences for MLST were performed by the online tool at http://pubmlst.org/. From the allelic distance, groups of closely related isolates were identified. In this study, a clonal complex was defined as isolates sharing six of the seven loci that define the allelic profile, as defined by Feil et al. [10] and as used in recent studies [11,12]. Clonal relationships based on the sharing of five out of the seven loci was also investigated. BioNumerics and eBURSTV3 software (http://pubmlst.org/) were used to establish the relationship among the isolates. Table 1 presents the serotypes and MLST profiles of the 32 viable isolates. A variety of sequence types (STs) were found, and for some serotypes the same ST was observed over several years. eBURST analysis based on similarities in 6 out of 7 loci revealed that the STs were dominated by 16 singletons and 3 groups of related STs and that there were no predicted founder. In the study by Lambertsen et al. [9] invasive penicillin non-susceptible S. pneumoniae from Denmark in the period from 2001 to 2005 were described. The study presented the MLST profiles of 43 isolates, showing that there were dominating clonal complexes, but also that the isolates showed many singletons. To the authors' knowledge, this is the only other study describing the MLST distribution of invasive pneumococcal isolates in Denmark. It has to be considered that Lambertsen et al. [9] presented selected data (only penicillin nonsusceptible isolates from before the PCV introduction in Denmark) while the present study presents data on isolates obtained after the PCV introduction and moreover only from infants. Hence, the two studies cannot be compared. The MLST profiles from the two studies are also very different, and only a few STs were either identical or clonally related between both studies, while the majority were different STs. 37 of the 43 tested isolates in the study by Lambertsen et al. [9] belonged to serotypes included in the PCV-7 and two belonged to the PCV-13 serotypes. Today, the circulation of serotypes included in the PCV-7 has been extensively reduced due to the impact of the vaccine. Moreover, since penicillin nonsusceptibility is often associated with certain serotypes, the current study and the Lambertsen study would be expected to be very different with respect to serotypes as well as STs. The effect of the PCV-7 introduction in 2007 in Denmark [5] might be the reason why the PCV-7 serotypes found in the Lambertsen et al. study [9] are very seldomly observed today in Denmark [5].
The MLST profiles found in this study belong to wellknown STs also found in other European countries [13][14][15]. Among these are ST 306 (serotype 1), ST 180 (serotype 3) and ST 191 (serotype 7F) ( Table 1) (Table 1). This occurrence of STs over several years has also been described by Ardanuy et al. [13], who found ST 306 (serotype 1) and ST 180 (serotype 3) in 1999-2000 and again in 2007-2009. The observation that the STs in this study are generally seen in other European countries [13][14][15]  In conclusion, the MLST profiles of IPD isolates from Danish unvaccinated infants younger than 90 days in this study are similar to the profiles observed in Europe. The isolates show a variety of STs that exceeds the number of different serotypes. There are, however, STs that reappear over the years from 2003 to 2013. The serotypes appearing after the PCV introduction are in general clones observed in other European countries, suggesting that future serotypes appearing in Denmark may well be clones already circulating in Europe, although domestic pneumococcal clones such as ST 9760 (serotype 27) also appear.

Availability of supporting data
The dataset supporting the results of this article is included within the article.