# Acute Otitis Media in Children--Challenges of Antibiotic Resistance in the Post-Vaccination Era
## 2 Patients and Methods  
We performed a retrospective study in children with AOM hospitalized in \"St Mary\" Emergency Hospital for Children Iasi, Romania, between January 2013 and December 2021. Trypanocentesis was recommended in children with refractory otitis media, in uncomplicated cases with a negative outcome (unsatisfactory response to antimicrobial therapy) and in patients with complications of AOM (e.g., mastoiditis). The diagnosis was based on the following criteria: acute onset of the symptoms (fever, irritability, otalgia or otorhea) and middle ear effusion confirmed by otoscopy (bulging or erythema of the tympan membrane, air-fluid level behind the tympan membrane, limited or absent mobility of tympan membrane). Eligible patients were children with a recent onset of AOM symptoms (in the first 48 h) who did not receive any antibiotic treatment or children with a prior diagnosis of AOM who received antibiotic treatment but remained symptomatic (fever or otalgia) for more than 48 h (treatment failure). Children with a previously inserted ventilatory tube or those with spontaneous perforation of the tympan membrane were excluded from our study. The samples were assessed for microbiological culture and antibiotic sensitivity testing. The pus obtained by tympanocentesis was used for smears and inoculation on Columbia with 5% sheep blood (Oxoid, Leicestershire, UK) and chocolate agar and Vitox (Oxoid, Thermoscietific, Leicestershire, UK). The smears were stained with methylene blue (for a better appreciation of the polymorphonuclear neutrophils, which, along with the fibrin, is the marker of inflammation) and Gram stain to establish the morpho-tinctorial category of the bacteria. The identification of isolated colonies was carried out with MICROSCAN panels (PC42, HIND) and MALDI-TOF mass spectrometry when applicable. To test the antibiotic sensitivity of _Streptococcus pneumoniae_, we used MSTRP+6 (Microscan, CLSI, Wayne, PA, USA) and STP6F (Sensititre, CLSI, West Sussex, UK) panels for _Haenophilus influenzae_. Additionally, for _Haenophilus influenzae_ and _Moraxella catarrhalis_, we used the nitrocefin discs test (Thermoscientific Remel Nitroefin discs - San Diego, CA, USA) to assess beta-lactamase production. The nitrocefin disk is a disk impregnated with nitrocefin for the detection of \(\beta\)-lactamase by chromogenic procedure. Nitroefin, a cephalospororin, is the substrate used in this test. \(\beta\)-lactamase hydrolyzes the \(\beta\)-lactam ring of nitrocefin, producing cephalosporocratic acid. A distinctive color change is associated with this reaction wherein the pale yellow nitrocefin is converted to a pink end-product upon hydrolysis. The bacteria that do not produce \(\beta\)-lactamase do not alter the pale-yellow color of nitrocefin within the limit of the test (5 min at room temperature). Unfortunately, for _Haemophilus influenzae_, we did not determine the serotype. HIND panels were used to indicate the biotype. _Turicella otitis_ had not been tested with antibiotics. The strains are usually susceptible to beta-lactams, aminoglycososides, glycopeptides, pristinamycin and rifampicin, while susceptibility to fluoroquinoles, macrolides and trimethoprim/sulfamethoxazole is variable. Antimicrobial susceptibility of _Streptococcus pneumoniae_ was tested for the following antibiotic classes: penicillins (penicillin), fluoroquinolones (moxifloxacin, levofloxacin), cephalosporporins (ceftriaxone, cefotaxime, cefuroxime), macrolides (azithromycin, clarithromycin, erythromycinomycin), trimethoprim-sulphamethoxazole, lincosamides (clindamycin), glycopeptides (vancomycin) and chloramphenicol, whereas the following antibiotics were assessed for sensibility in _Haemophilus influenzae_ cases: amoxicillin/clavulanic acid, cefuroxime, ceftaxone and trimethoprim/sulfamethoxazole.