This is a retrospective study about serotype distribution and antibiotic resistance of MEF S. pneumoniae and H. influenzae isolates collected from children before introduction of PCV10 and Hib conjugate vaccines nationwide. Microbiological diagnosis of AOM is not done on a routine basis in Bulgaria and this mild disease is underrepresented in our data set. Also, the tympanocentesis is not a common practice in our country. Thus, bacterial cultures studied here most likely represent cases of severe otitus media or patients with a complicated AOM.
The analysis included 128S. pneumoniae
isolates, of which 86% occurring in children aged
5 years. We found a relatively high rate of serotype 19F infections (20.3%), which is in accordance with the serotype distribution observed in the studies of AOM in German children [8
], and the Finland [7
]. Serotypes 6B and 19A were next most prominent, each comprising about 16% and 11%, respectively of the overall data set. The coverage calculations for the serotypes included in PCV7 without cross-protection was 63.6% in children with AOM aged
5 years, which is almost the same as was the serotype coverage previously reported for invasive pneumococcal disease (IPD) for the same age group in Bulgaria [23
]. The PCV10 increase the coverage by 2.8% of pneumococcal AOM (66.4%). The PCV13, which includes three serotypes in addition to the PCV10, would increase the coverage to 85.5%, mainly due to serotype 19A followed by serotypes 3 and 6A (Table ). However the cross-protection of serotype 6B towards serotype 6A was not included in calculations for our study. We found 14 serotype 19A isolates, 13 of which were multidrug resistant.
The results showed a high rate of reduced oral penicillin nonsusceptibility (68%) and a very high rate of ERSP isolates (47%) among MEF strains. These rates are more than twice as high as previously recorded with IPD isolates in Bulgaria studied during the same year period [23
]. These high resistance rates may be due to clonal spread of certain MDR pneumococcal serotypes such as 6B, 19 F and 19A. This resistance reflects the complexity of many of the severe otitis media episodes which included therapy failures. Notably, we observed that the percentages of macrolide, clindamycin, amoxicillin, cefuroxime sodium and ceftriaxone nonsusceptibility increased among S. pneumoniae
strains isolated during the last 6 years of the study period. The high antibiotic consumption for the treatment of AOM represents the major pressure for resistance selection [24
Overall, macrolide resistance in S. pneumoniae
now is mainly due to erm
(B) gene ( 71.7%), including 15% of isolates with a dual macrolide resistance mechanism (Table ). This pattern of macrolide resistance determinants in the last years replaced the efflux mechanism of macrolide resistance (mef
genotype), which was predominant among ERSP in Bulgaria before 2005 [25
]. Macrolide-resistant S. pneumoniae
strains were found to be co-resistant to oral penicillin (Table ), and to other compounds, such as tetracycline and trimethoprim-sulfamethoxazole (data not shown). Thus, the prevalence of MLSB
genotype may be due to clonal spread of certain MDR pneumococcal serotypes as 6B, 19 F and 19A. Interestingly, we observed multi-resistant serotype 19A in many years ago. For the first time, this MDR serotype was found in 1995 with L4 mutations as the mechanism of macrolide resistance [21
]. Since 2000 and after, erm
(B) genotype was found in serotype 19A isolates (nine strains). Dual macrolide resistance mechanisms were observed recently, between 2008 and 2010 in two serotype 19A strains (Table and Table ). Both the emergence of a new clone or capsular switching might be associated with these changes in our 19A serotype isolates. A considerable increase in the disease caused by serotype 19A has been recorded after the introduction of PCV7 in some countries [24
]. Of note, MDR serotype 19A isolates have also been identified in Israel and Finland before the large-scale use of conjugate vaccines as was the case in our study [27
Our findings of 97.5% nontypeable H. influenzae
strains isolated from AOM are in accordance with the literature [11
]. Nontypeable H. influenzae
are the major etiologic agents of respiratory tract infections in our country as was shown before [30
]. Ampicillin/amoxicillin nonsusceptible H. influenzae
were found to be a quarter of our MEF isolates. Among BLNAR and BLPACR most of the isolates (5/6) belonged to Dabernat group II, defined by the Asn526Lys substitution. A high incidence of BLNAR H. influenzae
strains has been reported in Japan, Spain and France [11
]. The emergence of BLNAR and BLPACR strains among MEF H. influenzae
isolates may have implications for the treatment of AOM infections, because amoxicillin and amoxicillin-clavulanate are the most common antibiotics used in the treatment of AOM in our country.
The main limitations of our study are: 1) it covers a long period of time, and the power is low; 2) isolates were sent by microbiology labs in hospitals, and we do not have clinical information on the type of AOM; 3) as isolates came from hospitals, they are not representative of S. pneumoniae and H. influenzae isolates causing otitis in children attending primary medical centers; 4) the numbers of S. pneumoniae and H. influenzae strains are small, because only a portion of children with AOM in Bulgaria are tested. Despite these limitations, we believe that they may represent a rough estimate of the serotype distribution and antibiotic resistance among a group of children with complicated AOM. This information will be invaluable when planning vaccination strategies to protect against otitis media.
In conclusion, the levels of antibiotic resistance among S. pneumoniae and H. influenzae causing severe otitis media in children are high in our settings. This has important therapeutic implications because such antibiotic-resistant isolates compromise the choice of macrolides and amoxicillin as treatment of AOM episodes in children. The existence of MDR S. pneumoniae serotype 19A before the active pneumococcal immunization is of particular concern. The rate of 15% BLNAR and BLPACR strains among H. influenzae MEF isolates may have implications for the treatment of AOM. Ongoing surveillance is needed for both pathogens in order to observe any changes in serotypes prevalence and antibiotic resistance after introduction of conjugate vaccines in our country.