From July 1997 until June 2011, a total of 3328 isolates from invasive pneumococcal disease were sent to the German National Reference Center for Streptococci. Isolate numbers varied from a minimum of 152 in 1998/99 to a maximum of 295 in 2006/07 (Table ). The average number of isolates per year was 238. Isolates were sent in from all over Germany, with no geographical bias. A total of 159 infections were caused by serotype 19A. The percentage of serotype 19A isolates remained below 5% until 2006, and then increased constantly over time, with a significantly higher proportion (8.1%) during the post-PCV-vaccination period when compared to the pre-PCV-vaccination period (2.8%) (Table ). Interestingly, the stepwise introduction of higher-valent vaccines in 2009 only slightly reduced the increase in serotype 19A.
Isolates from IPD in children from Germany from July 1997 to June 2011, proportion of serotype 19A and antibiotic resistance among serotype 19A isolates
Forty-four 19A isolates (27.7%) were from patients with meningitis. Among these, 15 isolates (34.1%) were resistant to penicillin, according to the new (parenteral) CLSI breakpoints (MIC ≥0.12 μg/ml) [17
]. Penicillin resistance rates post-vaccination were considerably higher (40.0%) than pre-vaccination (26.3%), though the difference was not statistically significant (p=0.52). Among the 115 non-meningitis isolates, only intermediate penicillin G resistance (MIC=4 μg/ml) was found (4 cases, 3.5%).
As described in the materials and methods section, the ‘oral’ penicillin breakpoints (which correspond to the old CLSI breakpoints [20
]), were used to assess the development of penicillin non-susceptibility among the 19A isolates, since they give better insight into resistance development over time. Applying these breakpoints, 54 isolates (9 resistant, 45 intermediately resistant) were classified as penicillin non-susceptible (NS), and the proportion of penicillin non-susceptible isolates increased significantly (p=0.002) between the pre- and the post-PCV-vaccination period (Table ). Rates of resistance to macrolides, clindamycin, and tetracycline were relatively high, but did not change significantly over time (Table ). Seventy-five isolates were resistant to at least one, 59 to at least two and 35 to three or more classes of antibiotics (using oral penicillin breakpoints). The latter isolates were classified as multidrug-resistant (MDR) (Table ). The proportion of isolates with resistance to at least one drug and the proportion of MDR isolates also increased moderately between the pre- and post-PCV-vaccination period, but the differences were statistically not significant (≥1 drug: p=0.7418, MDR: p=0.1655).
Multilocus sequence typing was performed for 158 of the 159 isolates included in this study (one isolate could not be regrown). The most common sequence types found were ST199 (10.7%), ST416 (9.4%) and ST994 (8.8%) (Figure ). When categorized according to clonal complex (CC) CC199 was the most prevalent (30.4%), followed by CC230 with 18.4%.
eBURST snapshot of 19A isolates from IPD in children in Germany before (black) and after (gray) the introduction of pneumococcal conjugate vaccination. Circle size is proportional to the amount of isolates.
When the clonal composition of the serotype 19A isolates before and after the start of the pneumococcal conjugate vaccine recommendation in July 2006 was compared, several changes in clonal composition could be observed. Eight CCs or groups accounted for 77.2% and 65.3% of all isolates before and after vaccination, respectively. Three CCs and 7 other STs were not detected after vaccine introduction. Clonal complexes 81, 320, 733 and 994 and STs 482, 5204 and 8031 were detected only after vaccine introduction. A significant prevalence increase after the introduction of vaccination was only detected for CC994 (p=0.0006) and CC320 (p= 0.0496) (Table ).
ST distribution among serotype 19A isolates from IPD in German children, before and after introduction of pneumococcal conjugate vaccine
The increased proportion of MDR isolates was mainly associated with increased proportions of CC230 and CC320. Of note, in the period before vaccination, only three MDR isolates with intermediate penicillin resistance were found, all three belonging to CC230 (ST230). After vaccination, 9 MDR isolates with high resistance to penicillin were found, belonging to CC230 (ST276, n=2), CC320 (ST320, n=5, ST2432, n=1) and CC81 (ST81, n=1) (Table ). Three of the ST320 isolates could be traced back to children that had recently come to Germany. On the clinical data sheets sent in with the isolates, it was stated that one child had entered from the US shortly before being infected, one from the UK, and a third had recently spent a longer holiday in India. In fact these were the first, second and third ST320, serotype 19A isolates ever detected in Germany (Nov. 2008, Mar. 2009 and Dec. 2009, respectively). The only earlier detected ST320 isolate was a serotype 19F, found in June 2006. The other two ST320 serotype 19A isolates (Jan. and May 2010), as well as the ST2432 isolate (Apr. 2009), had no data which indicated a foreign origin.
MDR serotype 19A isolates from IPD in German children, before and after introduction of pneumococcal conjugate vaccine
Data on antibiotic use in Germany were available for 1997–2011. Significant increases in cephalosporin and azithromycin use were noted when comparing the pre- and post-vaccination periods (p=0.00001 and p=0.0013 respectively). However, with 23.4% of all macrolide use in 2011, the proportional use of azithromycin remains relatively low in Germany. Penicillin, macrolides (without azithromycin) + clindamycin, and tetracycline use increased incrementally, but did not show significant change when comparing the pre- and post- vaccination periods (Figure ).
Figure 2 Yearly antibiotic prescription rates (DDD per 1,000 population and day, left axis), marketed doses of PCV per 10 children under 2 years of age (squares, right axis), and percentage of IPD cases caused by serotype 19A in Germany (triangles, right axis). (more ...)
The number of marketed doses of PCV7 (since 2001) and PCV13 (since Dec. 2009) in Germany was around 200,000 per year before the general vaccination recommendation. After the recommendation was issued in July 2006, this amount rose to an average of 2 million doses per year (Figure ). The birth cohorts in Germany have been an average 736,000 births/year from 1997–2006 and an average 675,000 births/year from 2007–2011.