Molecular characterization and epidemiology of Streptococcus pneumoniae serotype 8 in Denmark

Infections with Streptococcus pneumoniae affect all age groups, although predominantly children and the elderly. Invasive pneumococcal diseases (IPD) such as bacteremia, meningitis, and pneumonia cause high morbidity and mortality worldwide [1] and can be divided into serotypes based on their capsular polysaccharide with up to least 100 acknowledged serotypes [2, 3].

The introduction of the pneumococcal conjugate vaccine (PCV) – 7-valent (PCV7) in 2007 and 13-valent (PCV13) in 2010 – has changed the epidemiology of serotype-specific IPD in Denmark [4]. While the IPD incidence for the majority of serotypes included in the PCV13 vaccine has decreased, the incidence of non-PCV serotypes such as serotype 8 has increased post PCV vaccination [5]. The frequency of serotype 8 IPD has increased globally from 2013 to 2017 with 120% [6]. The observed replacement and increase of serotype 8 is similar to the emergence of the virulent and multidrug resistant serotype 19A observed in Massachusetts, USA, after introduction of PCV7, although an increase in multidrug resistance has not been observed [7].

In Europe, the current serotype 8 multi locus sequence type (MLST) is dominated by ST53 [8, 9], which constitutes up to 90.1% in Spain [9], and is related to the major clone Netherlands⁸–33 (https://​www.​pneumogen.​net/​pmen/​clone-collection.​html, accessed 14–04–2021) [10]. Alteration in the clonal epidemiology or other virulence related pneumococcal genes could be the explanation for the post PCV increase of serotype 8, as observed with serotype 19A ST320 post PCV7 in USA [7] or with serotype 19A ST199 and ST994 and the change in PCV strategies in Belgium [11]. In Denmark, we have observed a post PCV13 increase in serotype 24F ST162 [12]. However, even though the non-PCV serotype 8 for several years has been the dominant cause of IPD in Denmark, little is known about the serotype.

Serotype 8 is an example of successful serotype replacement due to PCV introduction in Denmark. It is, therefore, the intention of this study to investigate the mechanism behind the significant increase in serotype 8 IPD cases post PCV, by evaluating epidemiological data, the clonal epidemiology, and the presence/absence of virulence related pneumococcal genes. Whole-genome sequencing (WGS) analysis was performed on a representative number of serotype 8 isolates to investigate potential changes in the clonal distribution, molecularly susceptible related genes, and species-specific virulence genes pre- and post- PCV vaccination.

Introduction of the PCV vaccines has reduced IPD in children and other age groups. However, the introduction of the PCV vaccines has also contributed to an increase in the proportion of the non-vaccine serotypes such as serotype 8 in countries like Denmark, the United Kingdom, Spain, and other European countries [6], showing a post-vaccination serotype replacement [5, 6, 33, 34].

The serotype 8 IPD incidence in Denmark increased after the PCV13 introduction, predominantly affecting the age groups above 65 years, and a significant increase was observed for the age group 85+ after the introduction of PCV7; the serotype 8 IPD incidence increased in 2008–2009 and then decreased to pre-PCV7 levels in 2010 (Fig. 2, Table 3). The large increase of serotype 8 in Denmark (Fig. 2, Tables 1 and 3) after the introduction of PCV13 was not foreseen in any Danish published IPD and pneumococcal carriage data up to December 2013 [4, 13, 35]. It was observed that around 80% of IPD cases in 2012–2013 were caused by non–vaccine serotypes (8, 10A/B, 12F, 15B/C, 20, 22F, 33F, 38, 23B, 24F), with no clear predominance of any specific serotype [4]. In 2014, Danish IPD data on non-vaccine serotypes indicated the dominance of serotype 8, although at that time it was not clear that serotype 8 would continue to be the leading cause of IPD in Denmark (Table 1) [5]. Neither did Danish carriage studies in children below 5 years of age in 2000 [35] and below 2 years of age in 2014 to 2016 [36] show any indication of high carriage of serotype 8, which could explain the transmission to the elderly. Similar carriage data on serotype 8 in children below 5 years of age showing limited carriage have been observed in other countries [37]. It has furthermore been found that there is a limitation in using carriage data from children to forecast changes in general IPD epidemiology, and that serotype 8 is a possible example of a serotype transmitted directly among older age groups [38]. This observation is supported by studies from the UK performed on other age groups than children, in which they observed serotype 8 carriage [37, 39]. In Denmark, no carriage studies on other age groups than children have been performed, which suggests a direct transmission among other age groups [36].

The current Danish pneumococcal data are not able to provide an explanation or warning of the present dominance of serotype 8 [4, 5, 13, 35, 36]. Moreover, the epidemiological data does not provide an explanation for the dominance of serotype 8 IPD cases observed in Denmark.

At present only the pneumococcal polysaccharide vaccine (PPV23) includes serotype 8, which has shown a significant vaccine efficacy against serotype 8, although the protection is of limited duration [40]. The duration of protection can explain the limited effect of PPV23 in England against serotype 8 IPD despite a national PPV23 immunization program for the age group of 65+ since 2003 [40, 41]. The serotype 8 IPD in Denmark predominantly affects the age groups above 65 years (Fig. 2, Tables 1 and 3), and it will be important to monitor the serotype 8 IPD incidence with the introduction of PPV23 into a vaccination program for risk groups and the elderly 65+ [42].

Serotype 8 is often observed to be susceptible to antimicrobial drugs [8]. Spain has, however, seen an emergence and spread of S. pneumoniae serotype 8 ST63, a multidrug resistant clone resistant to erythromycin, clindamycin, tetracycline, and ciprofloxacin [8].

In Denmark we have not observed any occurrence of non-susceptible serotype 8 isolates (DANMAP, https://​www.​danmap.​org/​, accessed 10–03-2021), and the post PCV13 increase in serotype 8 incidence has not shown any changes in the susceptibility of serotype 8 isolates. The PBP profiles of the sequence isolates in this study corresponded well with the predicted PBP profile and the phenotypic susceptibility testing (Fig. 1) [12, 32].

The S. pneumoniae serotype 8 MLST type is ST53 belonging to cluster GPSC3 [43‐46], constituting 80% of the sequenced isolates in this study. The ST53 clone was found to be dominant both before and after the introduction of PCV7 and PCV13 (Table 2). The increase in serotype 8 can, therefore, not be related to changes in serotype 8 clones. Other serotype 8 MLST types observed in this study, such as ST404 and ST1480, have been reported in other European countries, Brazil, and The UK [45, 47‐51], while MLST types ST3714 and ST2234 have only been observed in Denmark, Sweden, Turkey, Belarus, the UK, Saudi Arabia, and Kenya (PubMLST DataBase, https://​pubmlst.​org/​spneumoniae/​, accessed 10–03-2021). The historical isolate 8-4-1962 (ST7203) was related to clone ST404 and was in the same GPSC98 cluster. An unknown ST type was detected in isolate 243–2010, which had six of seven identical allelic variants with ST53 and was in the same GPSC3 cluster (Fig. 1). Overall, all MLST types in this study were known as susceptible clones, although three isolates showed the presence of the tet(M) gene (Table 2).

The SNP phylogenetic tree showed that it was not possible to see any clades of isolates segregated by the year before and after the PCV introduction, indicating that it might not be a gene mutation causing the serotype 8 increase (Fig. 1). The tree illustrates two clades of twelve and three ST53 isolates, respectively, that were separated from the majority of ST53 isolates. The differentiation of the clades could, however, not be linked to the year of isolation. We do not know the basis of the difference for the twelve isolates based on the genes selected in this study, and further gene analysis needs to be performed to reveal which genes were responsible for the discrepancy. The clade of three isolates showed molecular tetracycline resistance, differentiating them from the majority of the ST53 isolates.

Evaluation of species-specific genes described in various studies [12, 20, 30, 52] did not show a clear presence/absence of genes defined by the PCV introduction (Table 2). The generally used lytA gene and other genes suggested for species identification of S. pneumoniae were detected in all our isolates (Table 2) similar to our previous observations [12]. Some genes were not observed in all our isolates; SP2020 [30] was not found in two of our isolates (Table 2). The zmpC gene was present in all ST53 isolates and in the clonally related isolate, while it was absent in all other ST types, which is consistent with observations from previous studies [22]. However, interestingly the zmpC gene has been described to suppress S. pneumoniae virulence in experimental models of pneumococcal meningitis [21]. In this study, specific meningitis data are too limited to evaluate the effect on the number of meningitis cases; however, the zmpC gene was found in isolates from cerebrospinal fluid and did not seem to be linked to reduced invasiveness of serotype 8 (Table 2). The genes piaA/piaB/piaC were present in all isolates before the introduction of PCV13. However, they were lacking in 12.5% of the isolates (8 isolates) after the introduction. Although the absence of the genes piaA/piaB/piaC first appeared after the PCV13 introduction, it does not explain the increase in serotype 8, as only a limited number of isolates lacked the genes (Table 2).

Interestingly, the SP2020 or piaB gene in combination with the lytA gene has been suggested for the detection of pneumococcal pure cultures or swab samples [21, 30]. However, when analyzing the 96 isolates in this study, we observed isolates which did not include the SP2020 or piaB gene (Table 2). It is therefore questionable how favorable these genes are compared to the use of the ply gene for detection of Danish pneumococcal isolates. All isolates in this study (Table 2) and the study by Kavalari et al [12] showed the presence of the ply gene. It has furthermore been described that the piaB gene only lacks in non-typeable pneumococci [21]. In this study, however, the piaB gene was not found to be unique for the invasive capsulated isolates, as 7 isolates lacked the gene (Table 2).

A limitation of the study includes that not all serotype 8 isolates were sequenced with the caveat that we might not detect possible mini-outbreaks of specific clones as a possible explanation for the observed serotype 8 replacement in Denmark. However, the number of isolates was sufficient enough to find interesting sequence results (e.g. isolates not possessing the SP2020 or piaB genes (Table 2).