title: Microbiological characterization of Streptococcus pneumoniae and non-typeable Haemophilus influenzae isolates as primary causes of acute otitis media in Bulgarian children before the introduction of conjugate vaccines.
abstract: BACKGROUND
  Pneumococcal and Haemophilus influenzae type b (Hib) vaccines were introduced in our national immunisation program in April 2010. The aims of this retrospective, laboratory-based study were to determine the serotypes and antibiotic resistance of Streptococcus pneumoniae and H. influenzae isolates from middle ear fluid (MEF) collected before the introduction of immunization.
  
  
  METHODS
  S. pneumoniae (n = 128) and H. influenzae (n = 40) strains isolated from MEF of children with AOM between 1994 and 2011 were studied. MICs were determined by a microdilution assay. Serotyping of S. pneumoniae was done by Quellung method and PCR capsular typing was used for H. influenzae. Macrolide resistance genes were detected by PCR for erythromycin resistant S. pneumoniae (ERSP). DNA sequencing of ftsI gene was performed for ampicillin nonsusceptible H. influenzae.
  
  
  RESULTS
  The most common serotypes found among children with pneumococcal AOM were 19 F (20.3%), 6B (15.6%), and 19A (10.9%). The potential coverage rates by the PCV7, PCV10 and PCV13 of children aged < 5 years were 63.6%, 66.4% and 85.5%, respectively. Reduced susceptibility to oral penicillin was seen in 68.1%; resistance to erythromycin was 46.9%. We found erm(B) gene in 56.7% of the ERSP, mef(E) gene in 25%; 15% harbored both genes erm(B) + mef(E) and 3.3% had mutations of L4 ribosomal protein. Of the 40 H. influenzae isolates 97.5% were nontypeable. Nonsusceptibility to ampicillin occurred in 25%. Ampicillin resistance groups were: β-lactamase-positive ampicillin resistant (BLPAR) strains (10%), β-lactamase-negative ampicillin resistant (BLNAR) strains (12.5%) and β-lactamase-positive amoxicillin-clavulanate resistant (BLPACR) strains (2.5%). Among BLNAR and BLPACR most of the isolates (5/6) belonged to group II, defined by the Asn526Lys substitution.
  
  
  CONCLUSIONS
  The levels of antibiotic resistance among S. pneumoniae and H. influenzae causing severe AOM in children are high in our settings. The existence of multidrug-resistant S. pneumoniae serotype 19A is of particular concern. The rate of BLNAR and BLPACR strains among H. influenzae isolates was 15%.
authors.affiliation: Department of Medical Microbiology, Medical University of Sofia, Faculty of Medicine, Sofia, 1431, BULGARIA. lenasetchanova@hotmail.com
authors.forename: Lena P
authors.initials: LP
authors.lastname: Setchanova
authors.forename: Tomislav
authors.initials: T
authors.lastname: Kostyanev
authors.forename: Alexandra B
authors.initials: AB
authors.lastname: Alexandrova
authors.forename: Ivan G
authors.initials: IG
authors.lastname: Mitov
authors.forename: Dimitar
authors.initials: D
authors.lastname: Nashev
authors.forename: Todor
authors.initials: T
authors.lastname: Kantardjiev
mesh_terms: D000293:Adolescent; D019943:Amino Acid Substitution; D002031:Bulgaria; D002648:Child; D002675:Child, Preschool; D024881:Drug Resistance, Bacterial; D024901:Drug Resistance, Multiple, Bacterial; D006192:Haemophilus Infections / Q000382:microbiology; D006193:Haemophilus influenzae / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D006801:Humans; D017589:Immunization Programs; D007223:Infant; D008826:Microbial Sensitivity Tests; D010033:Otitis Media / Q000382:microbiology*; D046915:Penicillin-Binding Proteins / Q000235:genetics; D011008:Pneumococcal Infections / Q000382:microbiology; D012189:Retrospective Studies; D013296:Streptococcus pneumoniae / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D018074:Vaccines, Conjugate / Q000627:therapeutic use; D001618:beta-Lactamases / Q000378:metabolism
chemical_list: D046915:Penicillin-Binding Proteins; D018074:Vaccines, Conjugate; D001618:beta-Lactamases