BACKGROUND
streptococcus pneumoniae is a leading pathogen in bacterial pneumonia, sepsis and meningitis in humans worldwide  <cit> .
in many european countries the rate of resistance of s. pneumoniae to macrolides has exceeded that of penicillin  <cit> .
concerning penicillin, it has been described that treatment of patients with nonmeningeal invasive pneumococcal infections with nonsusceptible isolates was not associated with higher mortality rates  <cit> .
in  <dig> new penicillin breakpoints for s. pneumoniae were published by the clsi  <cit> , differentiating meningitis and non-meningitis cases of invasive pneumococcal disease .
their impact on susceptibility categorisation in germany was described previously by our group  <cit> .
however, for macrolides an increased risk of macrolide failure has been reported for pneumococcal isolates nonsusceptible in vitro  <cit> .
the aim of this study was to evaluate macrolide susceptibility of all isolates of s. pneumoniae with ipd that were sent to the german national reference center for streptococci  between  <dig> and  <dig> and to evaluate potential trends in nonsusceptibility over time.
the description of serotype specific resistance, was a major aim of the study.
the study was undertaken against the background of the recent observation of declining macrolide resistance rates especially among german children.
methods
study design
the nrcs has conducted surveillance for invasive pneumococcal disease in germany since  <dig>  a population- and laboratory-based approach was used to collect data on invasive pneumococcal disease among children <  <dig> years and adults ≥  <dig> years in germany.
isolates were sent to the nrcs by diagnostic microbiological laboratories throughout germany on a voluntary basis.
cases from january  <dig>   <dig> to december  <dig>   <dig> were included in this study.
a case of ipd was defined by the isolation of s. pneumoniae from a normally sterile site.
microbiological investigations
isolates were identified by standard procedures including bile solubility and optochin sensitivity.
minimal inhibitory concentrations  testing was performed using the broth microdilution method as recommended by the clinical and laboratory standards institute   <cit> .
macrolide resistance was investigated using erythromycin or clarithromycin, in which testing with erythromycin was replaced by clarithromycin over the years.
<dig> isolates were tested both for erythromycin and clarithromycin.
the susceptible, intermediate, and resistant breakpoints  were ≤  <dig> ,  <dig> , and ≥ <dig> μg/ml, both for erythromycin and clarithromycin  <cit> .
streptococcus pneumoniae atcc  <dig> was used as a control strain.
statistical analysis
all categorical data were expressed as frequencies.
to analyse a severe increase or decrease over time the cochran-armitage test was used.
the overall significance level was adjusted using the bonferroni correction to account for the problem of multiple testing.
due to  <dig> tests p-values ≤  <dig>  were considered as statistically significant test results.
all statistical analyses were conducted using sas version  <dig> .
<dig> .
RESULTS
in total,  <dig>  isolates from invasive pneumococcal disease were collected between january  <dig>   <dig> and december  <dig>   <dig>  the number of cases for each year vary between  <dig> and  <dig>  .
data on macrolide susceptibility were available for  <dig>  isolates, whereas  <dig>  isolates  originated from adults,  <dig>  isolates  were from children.
the overall nonsusceptibility rate of all isolates was  <dig> % .
higher resistance rates were observed among children  than among adults  .
sero
i%, intermediate isolates in percent; r%, resistant isolates in percent; s%, susceptible isolates in percent; n, number of isolates tested.
total † represents the number of isolates collected from both children and adults.
total ‡ represents the serotype distribution in percent in relation to the number of  <dig> ipd isolates serotyped.
others* includes the serotypes : 15f , 18b , 7c , 10f , 11b , 35a ,  <dig> ,  <dig> , 24a , 28f , 35c ,  <dig> , 22a ,  <dig> , 7a , 24b , 25f ,  <dig> ,  <dig> , 7b , 17a ,  <dig> ,  <dig> ,  <dig> ,  <dig> .
the sampling source related nonsusceptibility is shown in table  <dig>  highest nonsusceptibility rates were observed for pharyngeal isolates , pericardium  and mastoid .
nonsusceptibility rates for csf and blood were  <dig> %  and  <dig> % , respectively.
the serotype distribution broken down to the sampling source is shown in table  <dig>  for blood, csf and bal serotype  <dig> is most prevalent, whereas for pleural fluid serotypes  <dig> and  <dig> are most often found.
sero
only sampling sites with ≥  <dig> isolates were included in this table.
nt: nontypeable; n: number of isolates tested.
others* includes the serotypes : rough , 9l , 23b ,  <dig> , 18f , 12a ,  <dig> , 28a , 35b , 10b , 15f , 18b , 12b , 33b , 7c , 10f , 11b , 11f , 35a , 7a , 19c ,  <dig> ,  <dig> , 24a , 28f , 35c ,  <dig> , 19b , 22a ,  <dig> ,  <dig> , 7b , 24b , 25f ,  <dig> ,  <dig> ,  <dig>  17a ,  <dig> ,  <dig> ,  <dig> .
as for the childhood ipd isolates in the first year of this study ,  <dig> % were intermediate and  <dig> % resistant to macrolides.
maximum nonsusceptibility rates during the period under study were observed in  <dig> , while in  <dig>   <dig> % of isolates were intermediate and  <dig> % resistant.
ipd isolates obtained from adults were intermediate in  <dig> % and resistant in  <dig> % in  <dig>  maximum nonsusceptibility rates were observed in  <dig> as well .
nonsusceptibility rates in  <dig> were  <dig> %  and  <dig> % .
the increase in macrolide nonsusceptibility from  <dig> to  <dig> was statistically significant for children  and adults , as well as the decrease from  <dig> to  <dig> .
concerning the intermediate resistant isolates no significant trends were observed , adults ; 2005-2008: children , adults ).
detailed results of the macrolide susceptibility testing are shown in figure  <dig>  the mics of all invasive isolates are illustrated in figure  <dig>
overall, the leading serotypes were serotypes  <dig> ,  <dig> , 7f ,  <dig>  and 23f .
a ranking of serotype specific macrolide nonsusceptibility of ipd isolates is shown in table  <dig>  serotype  <dig>  was by far the most resistant serotype, followed by serotypes rough, 19b,  <dig> , 6b , 15a , 19f , and 19a .
however, absolute numbers for rough, 19b and  <dig> were very low.
serotypes contributing considerably to pneumococcal macrolide nonsusceptibility by combination of frequency among invasive isolates and relatively high macrolide nonsusceptibility are especially serotypes  <dig>  6b, 19f, 19a, 9v and 23f.
the development of nonsusceptibility of these serotypes over the years is shown in figure  <dig>  the nonsusceptibility among serotype  <dig> isolates increases considerably over the years up to around 80% .
for serotype 19f a significant increase  in nonsusceptibility was observed as well.
no significant trends were found for serotypes 6b , 9v , 19a  and 23f .
the peak in nonsusceptibility among 7-, 10- and 13-valent serotypes in adults from  <dig> to  <dig>  correlates to an increased incidence of serotype  <dig> during that time  <cit> .
generally, the rate of resistance is higher among the vaccine serotypes   than among the non vaccine serotypes  .
the proportion of nonsusceptible 7-valent vaccine serotypes remained largely constant from  <dig> to  <dig> among children .
among the non pcv <dig> serotypes the rate of nonsusceptibility is lower .
concerning adults, an increase of isolates sent to the nrcs can be noticed .
the fraction of nonsusceptible isolates has declined during the last years among 7-valent vaccine serotypes after a notable increase from  <dig> to  <dig> .
discussion and
CONCLUSIONS
this paper presents the results of  <dig> years of surveillance for macrolide susceptibility of invasive pneumococcal disease in germany.
the prevalence of antibiotic-resistant s. pneumoniae continues to increase worldwide but varies widely between countries  <cit> .
in europe, high resistance rates for macrolides have been reported from france, spain, italy and belgium  <cit> .
pneumococcal macrolide resistance rates reported from germany were low  <cit> .
nevertheless, a continuous and statistically significant increase of macrolide nonsusceptibility could be observed after publication of these studies, reaching maximum values in  <dig> .
the relatively high rate of variation in resistance among childhood isolates during the first years of the study is presumably due to the low number of cases, and a suspected bias for resistant isolates among the centers sending the isolates.
since  <dig>  a considerable and statistically significant decrease especially for childhood nonsusceptibility has been noticed.
these data are similar to those recently reported from spain, where a reduction of erythromycin nonsusceptibility from  <dig> % in  <dig> to  <dig> % in  <dig> has been described  <cit> .
an increased awareness of ipd among adults has been observed since  <dig>  this correlates to the general recommendation of pneumococcal conjugate vaccination for children <  <dig> years in germany at the end of july  <dig> and an increased interest in serotype information of ipd.
furthermore, in january  <dig> an internet based laboratory sentinel system  was established in germany, which enables participating laboratories to transfer anonymised basic patient information on a voluntary basis.
compared to children, only a minor reduction of nonsusceptibility has been observed among adults from  <dig>  to  <dig> , although this reduction was also statistically significant.
possible reasons for the decrease in macrolide nonsusceptibility include a reduced macrolide consumption due to the rising resistance rates, as well as the general recommendation of pneumococcal conjugate vaccination for children <  <dig> years in germany at the end of july  <dig>  since the introduction of the vaccine a considerable decrease of serotypes included in the 7-valent pneumococcal conjugate vaccine has been observed among german children, but also  among adults  <cit> , which is partly due to the association of serotypes with age  <cit> .
the antibiotic prescribing practices, which are thought to be among the most significant drivers for the spread of pneumococcal resistance, differ vastly between european countries  <cit> .
a decrease in the use of macrolides has been reported for instance in spain  <cit> , portugal  <cit> , belgium  <cit> , slovenia  <cit>  and taiwan  <cit> .
the influence of a decreased macrolide consumption on macrolide susceptibility is discussed controversially.
in spain a relation between the decrease in macrolide consumption and the decrease in erythromycin non-susceptibility among children could be shown, while this effect was absent among the adult population, probably due to the increase in non-vaccine serotypes such as 19a   <cit> .
reports from other countries showed no decrease in macrolide nonsusceptibility following a reduced macrolide consumption  <cit> .
besides the total macrolide consumption, the influence of long lasting macrolides, which may increase even in times of decreasing total macrolide consumption  <cit> , is discussed to be a cause of the macrolide nonsusceptibility  <cit> .
besides antibiotics, pneumococcal conjugate vaccination is another important factor associated with changes in macrolide susceptibility  <cit> .
in our study, high rates of serotype specific resistance among the more frequent serotypes were observed among the serotypes  <dig>  6b, 19f and 23f, in particular.
these results are in line with results from germany published previously by the nrcs for invasive  <cit>  and respiratory tract isolates  <cit>  and comparable to results reported from several european countries  <cit> .
notably, a statistically significant trend in increasing macrolide resistance was seen for serotypes  <dig> and 19f.
however, since both serotypes are included in the pneumococcal conjugate vaccines, a future reduction of these serotypes can be expected.
the low rate of macrolide nonsusceptibility among isolates not serotyped corresponds to the fact, that high resistance levels were a main trigger for initiation of serotyping during the early years of this study, when consistent serotyping of all isolates was not conducted due to excessive costs.
in spite of all these observations, because the impact of preventive and therapeutic strategies on pneumococcal evolution not only depends on, but also influences the serotype distribution, when normal temporal  <cit>  and regional  <cit>  variations of serotype distribution are taken into consideration, future developments remain difficult to predict  <cit> .
ongoing nationwide surveillance is necessary to observe further developments of pneumococcal macrolide resistance in germany.
authors' contributions
mi performed the analysis and drafted the manuscript.
cm performed the statistical analysis.
mi, rrr and ml participated in the laboratory analyses.
mi, rrr and ml conceived the study.
all authors read and approved the final manuscript.

 streptococcus pneumoniae is a leading pathogen in bacterial pneumonia, sepsis and meningitis in humans worldwide. in many european countries the rate of resistance of s. pneumoniae to macrolides has exceeded that of penicillin. concerning penicillin, it has been described that treatment of patients with nonmeningeal invasive pneumococcal infections with nonsusceptible isolates was not associated with higher mortality rates. in new penicillin breakpoints for s. pneumoniae were published by the clsi, differentiating meningitis and non-meningitis cases of invasive pneumococcal disease. their impact on susceptibility categorisation in germany was described previously by our group. however, for macrolides an increased risk of macrolide failure has been reported for pneumococcal isolates nonsusceptible in vitro. the aim of this study was to evaluate macrolide susceptibility of all isolates of s. pneumoniae with ipd that were sent to the german national reference center for streptococci between and and to evaluate potential trends in nonsusceptibility over time. the description of serotype specific resistance, was a major aim of the study. the study was undertaken against the background of the recent observation of declining macrolide resistance rates especially among german children. methods study design the nrcs has conducted surveillance for invasive pneumococcal disease in germany since a population- and laboratory-based approach was used to collect data on invasive pneumococcal disease among children < years and adults ≥ years in germany. isolates were sent to the nrcs by diagnostic microbiological laboratories throughout germany on a voluntary basis. cases from january to december were included in this study. a case of ipd was defined by the isolation of s. pneumoniae from a normally sterile site. microbiological investigations isolates were identified by standard procedures including bile solubility and optochin sensitivity. minimal inhibitory concentrations testing was performed using the broth microdilution method as recommended by the clinical and laboratory standards institute. macrolide resistance was investigated using erythromycin or clarithromycin, in which testing with erythromycin was replaced by clarithromycin over the years. isolates were tested both for erythromycin and clarithromycin. the susceptible, intermediate, and resistant breakpoints were ≤,, and ≥ μg/ml, both for erythromycin and clarithromycin. streptococcus pneumoniae atcc was used as a control strain. statistical analysis all categorical data were expressed as frequencies. to analyse a severe increase or decrease over time the cochran-armitage test was used. the overall significance level was adjusted using the bonferroni correction to account for the problem of multiple testing. due to tests p-values ≤ were considered as statistically significant test results. all statistical analyses were conducted using sas version..  in total, isolates from invasive pneumococcal disease were collected between january and december the number of cases for each year vary between and. data on macrolide susceptibility were available for isolates, whereas isolates originated from adults, isolates were from children. the overall nonsusceptibility rate of all isolates was %. higher resistance rates were observed among children than among adults. sero i%, intermediate isolates in percent; r%, resistant isolates in percent; s%, susceptible isolates in percent; n, number of isolates tested. total † represents the number of isolates collected from both children and adults. total ‡ represents the serotype distribution in percent in relation to the number of ipd isolates serotyped. others* includes the serotypes: 15f, 18b, 7c, 10f, 11b, 35a,,, 24a, 28f, 35c,, 22a,, 7a, 24b, 25f,,, 7b, 17a,,,,. the sampling source related nonsusceptibility is shown in table highest nonsusceptibility rates were observed for pharyngeal isolates, pericardium and mastoid. nonsusceptibility rates for csf and blood were % and %, respectively. the serotype distribution broken down to the sampling source is shown in table for blood, csf and bal serotype is most prevalent, whereas for pleural fluid serotypes and are most often found. sero only sampling sites with ≥ isolates were included in this table. nt: nontypeable; n: number of isolates tested. others* includes the serotypes: rough, 9l, 23b,, 18f, 12a,, 28a, 35b, 10b, 15f, 18b, 12b, 33b, 7c, 10f, 11b, 11f, 35a, 7a, 19c,,, 24a, 28f, 35c,, 19b, 22a,,, 7b, 24b, 25f,,, 17a,,,. as for the childhood ipd isolates in the first year of this study, % were intermediate and % resistant to macrolides. maximum nonsusceptibility rates during the period under study were observed in, while in % of isolates were intermediate and % resistant. ipd isolates obtained from adults were intermediate in % and resistant in % in maximum nonsusceptibility rates were observed in as well. nonsusceptibility rates in were % and %. the increase in macrolide nonsusceptibility from to was statistically significant for children and adults, as well as the decrease from to. concerning the intermediate resistant isolates no significant trends were observed, adults; 2005-2008: children, adults ). detailed results of the macrolide susceptibility testing are shown in figure the mics of all invasive isolates are illustrated in figure overall, the leading serotypes were serotypes,, 7f, and 23f. a ranking of serotype specific macrolide nonsusceptibility of ipd isolates is shown in table serotype was by far the most resistant serotype, followed by serotypes rough, 19b,, 6b, 15a, 19f, and 19a. however, absolute numbers for rough, 19b and were very low. serotypes contributing considerably to pneumococcal macrolide nonsusceptibility by combination of frequency among invasive isolates and relatively high macrolide nonsusceptibility are especially serotypes 6b, 19f, 19a, 9v and 23f. the development of nonsusceptibility of these serotypes over the years is shown in figure the nonsusceptibility among serotype isolates increases considerably over the years up to around 80%. for serotype 19f a significant increase in nonsusceptibility was observed as well. no significant trends were found for serotypes 6b, 9v, 19a and 23f. the peak in nonsusceptibility among 7-, 10- and 13-valent serotypes in adults from to correlates to an increased incidence of serotype during that time. generally, the rate of resistance is higher among the vaccine serotypes than among the non vaccine serotypes. the proportion of nonsusceptible 7-valent vaccine serotypes remained largely constant from to among children. among the non pcv serotypes the rate of nonsusceptibility is lower. concerning adults, an increase of isolates sent to the nrcs can be noticed. the fraction of nonsusceptible isolates has declined during the last years among 7-valent vaccine serotypes after a notable increase from to. discussion and  this paper presents the results of years of surveillance for macrolide susceptibility of invasive pneumococcal disease in germany. the prevalence of antibiotic-resistant s. pneumoniae continues to increase worldwide but varies widely between countries. in europe, high resistance rates for macrolides have been reported from france, spain, italy and belgium. pneumococcal macrolide resistance rates reported from germany were low. nevertheless, a continuous and statistically significant increase of macrolide nonsusceptibility could be observed after publication of these studies, reaching maximum values in. the relatively high rate of variation in resistance among childhood isolates during the first years of the study is presumably due to the low number of cases, and a suspected bias for resistant isolates among the centers sending the isolates. since a considerable and statistically significant decrease especially for childhood nonsusceptibility has been noticed. these data are similar to those recently reported from spain, where a reduction of erythromycin nonsusceptibility from % in to % in has been described. an increased awareness of ipd among adults has been observed since this correlates to the general recommendation of pneumococcal conjugate vaccination for children < years in germany at the end of july and an increased interest in serotype information of ipd. furthermore, in january an internet based laboratory sentinel system was established in germany, which enables participating laboratories to transfer anonymised basic patient information on a voluntary basis. compared to children, only a minor reduction of nonsusceptibility has been observed among adults from to, although this reduction was also statistically significant. possible reasons for the decrease in macrolide nonsusceptibility include a reduced macrolide consumption due to the rising resistance rates, as well as the general recommendation of pneumococcal conjugate vaccination for children < years in germany at the end of july since the introduction of the vaccine a considerable decrease of serotypes included in the 7-valent pneumococcal conjugate vaccine has been observed among german children, but also among adults, which is partly due to the association of serotypes with age. the antibiotic prescribing practices, which are thought to be among the most significant drivers for the spread of pneumococcal resistance, differ vastly between european countries. a decrease in the use of macrolides has been reported for instance in spain, portugal, belgium, slovenia and taiwan. the influence of a decreased macrolide consumption on macrolide susceptibility is discussed controversially. in spain a relation between the decrease in macrolide consumption and the decrease in erythromycin non-susceptibility among children could be shown, while this effect was absent among the adult population, probably due to the increase in non-vaccine serotypes such as 19a. reports from other countries showed no decrease in macrolide nonsusceptibility following a reduced macrolide consumption. besides the total macrolide consumption, the influence of long lasting macrolides, which may increase even in times of decreasing total macrolide consumption, is discussed to be a cause of the macrolide nonsusceptibility. besides antibiotics, pneumococcal conjugate vaccination is another important factor associated with changes in macrolide susceptibility. in our study, high rates of serotype specific resistance among the more frequent serotypes were observed among the serotypes 6b, 19f and 23f, in particular. these results are in line with results from germany published previously by the nrcs for invasive and respiratory tract isolates and comparable to results reported from several european countries. notably, a statistically significant trend in increasing macrolide resistance was seen for serotypes and 19f. however, since both serotypes are included in the pneumococcal conjugate vaccines, a future reduction of these serotypes can be expected. the low rate of macrolide nonsusceptibility among isolates not serotyped corresponds to the fact, that high resistance levels were a main trigger for initiation of serotyping during the early years of this study, when consistent serotyping of all isolates was not conducted due to excessive costs. in spite of all these observations, because the impact of preventive and therapeutic strategies on pneumococcal evolution not only depends on, but also influences the serotype distribution, when normal temporal and regional variations of serotype distribution are taken into consideration, future developments remain difficult to predict. ongoing nationwide surveillance is necessary to observe further developments of pneumococcal macrolide resistance in germany. authors' contributions mi performed the analysis and drafted the manuscript. cm performed the statistical analysis. mi, rrr and ml participated in the laboratory analyses. mi, rrr and ml conceived the study. all authors read and approved the final manuscript. 
the current study was undertaken against the background of the recent observation of declining macrolide resistance rates especially among german children. nationwide surveillance of invasive pneumococcal disease has been conducted in germany since a population- and laboratory-based approach was used to collect data on invasive pneumococcal disease, and isolates sent to the national reference center for streptococci by diagnostic microbiological laboratories from to were included in this study. higher resistance rates were observed among children than among adults. maximum nonsusceptibility rates during the period under study were observed in, while nonsusceptibility rates in were considerably lower, especially for children. the rate of resistance was higher among the vaccine serotypes than among the non vaccine serotypes. there has been a considerable and statistically significant decrease in macrolide nonsusceptibility in germany since especially among children. serotype proved to be the most resistant serotype. the overall nonsusceptibility rate of all isolates was %. from to data on macrolide susceptibility were available for invasive isolates.
 macrolide resistant streptococcus pneumoniae has been on a gradual increase in germany for over a decade. the current study was undertaken against the background of the recent observation of declining macrolide resistance rates especially among german children. nationwide surveillance of invasive pneumococcal disease has been conducted in germany since a population- and laboratory-based approach was used to collect data on invasive pneumococcal disease, and isolates sent to the national reference center for streptococci by diagnostic microbiological laboratories from to were included in this study.  from to data on macrolide susceptibility were available for invasive isolates. isolates were from adults, and isolates from children. the overall nonsusceptibility rate of all isolates was %. higher resistance rates were observed among children than among adults. maximum nonsusceptibility rates during the period under study were observed in, while nonsusceptibility rates in were considerably lower, especially for children. the rate of resistance was higher among the vaccine serotypes than among the non vaccine serotypes. serotype proved to be the most resistant serotype.  there has been a considerable and statistically significant decrease in macrolide nonsusceptibility in germany since especially among children. 