Introduction of the PCV vaccines has greatly 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 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 profound increase of serotype 8 in Denmark (Fig. 2, Tables 1 and 3) after the PCV13 introduction 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 of using carriage data from children to forecast changes in 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 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 the 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], Although the serotype 8 IPD in Denmark predominantly affects the age groups above 65 years (Fig. 2, Tables 1 and 3), it cannot be expected that a great decline in the serotype 8 IPD incidence will be observed with the introduction of PPV-23 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 01-10-2020), and the post PCV13 increase in serotype 8 incidence has not shown any change 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 dominating both before and after the introduction of PCV7 and PCV13 (Table 2). The serotype 8 increase can therefore not be related to changes in serotype 8 clones. Other serotype 8 MLST types such as ST404 and ST1480 observed in this study have been reported in other European countries, Brazil, and The United Kingdom [45, 47–51], while MLST types ST3714 and ST2234 have been observed in Denmark, Sweden, Turkey, Belarus, the United Kingdom, Saudi Arabia, and Kenya (PubMLST DataBase, https://pubmlst.org/spneumoniae/, accessed 20-10-2019). The historical isolates 8-4-1962 (ST7203) were 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 were 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 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 show which genes were responsible for the discrepancy. The clade of three isolates showed a molecular tetracycline resistance, differentiating them from the majority of the ST53 isolates.
Comparing the SNP tree with a tree based on the 53 rMLST genes from PubMLST species identification showed nearly identical branches, although the SNP tree showed more details in the branches, as the clade with the three isolates containing the tet(M) gene was not present in the rMLST tree (data not shown). In general, the authors found the species ID identification using PubMLST rMLST was very easy to use; it did, however, not provide any additional information to the cause for the increase in serotype 8.
Evaluation of species-specific genes described in different 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; the 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 seemed not to be link to reduced invasiveness of serotype 8 on the contrary (Table 2). The genes piaA/piaB/piaC were present in all isolates before PCV13 introduction. 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 have 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 were lacking the gene (Table 2).