Background: Rheumatic fever and rheumatic heart disease remain significant causes ofrncardiovascular diseases in Ethiopian children.rnObjectives: This study was conducted to determine the throat carriage rate and antibioticrnsusceptibility pattern of beta-hemolytic Streptococci and gram negative oral commensal bacteria,rnsuch as Neisseria species in order to have a better understanding of their composition andrndynamics in children having rheumatic heart disease who received on-going monthly PenicillinrnG prophylaxis.rnMethods: Throat swab from 234 children who had rheumatic heart disease and received ongoingrnsecondary prophylaxis was collected and inoculated onto Sheep blood and ModifiedrnThayer-Martin agar plates. The bacterial strains were analyzed by conventional methods as wellrnas advanced mass spectrometry and molecular methods. Antimicrobial susceptibility testing ofrnstreptococci was performed by Kirby Bauer disc diffusion method. ASO titer determination wasrnalso done for infection or colonization differentiation.rnResults: Out of 234 participants, 38.03% were referred for possible surgical intervention butrnonly three participants received this treatment. Throat carriage rate of beta-hemolyticrnstreptococci was 23.93 % (56/234). Of these, 4 were S. pyogenes. Streptococcus dysgalactaiernsubsp. equisimilis possessing Lancefield group A, C and G were isolated. Six different emm generntypes with one newly discovered subtype (stGrobn.1) were identified. The Streptococcalrnglycosyltransferase in strain emm 68.2 (multidrug resistant strain) had N-linked glycosylationrncarrying a unique HexNAc-deoxyhexose; a novel post-translational modification not previouslyrnrecognized or studied which indicates it is not only M protein responsible for rheumatic feverrndisease. All beta- hemolytic Streptococci were susceptible to penicillin except S. agalactaie.rnErythromycin and tetracycline resistant S. pyogenes were isolated. Among 234 participants,rn29.49% (69) was positive to ASO. Of the positives, 66.67 % (46 / 69) showed increased ASOrntiters (ASO > 200 units/Î¼L) and the rest 33.3% (23/69) was ASO =200 units/ Î¼L. Children whornreceived antibiotic prophylaxis within two weeks showed significantly lower beta- hemolyticrnstreptococcal throat carriage than 4 weeks of injection (p=0. 004). The isolation rate of gramrnnegative oropharyngeal commensals was 55.1% (129/234). Throat carrier rate of N. meningitidis,rnN. lactamica, M. catarrhalis, K. denitrificans and K. kingae were 4.27%, 4.27%, 19.66 %,rn15.81% and 2.56 % respectively.rnIXrnConclusion: Characterization of carrier strains and increased ASO titer in rheumatic heartrndisease is likely to elucidate the significance and mechanisms for carriage and drug resistancernduring on-going penicillin G monthly prophylaxis. The throat carriage rate of gram negativerncommensals seems affected by on-going penicillin G prophylaxis.rnRecommendation: The current schedule benzathine penicillin prophylaxis injection should bernrevised (changing schedule of benzathine penicillin G prophylaxis from four to two weeks).rnTreatment intervention has to be established to provide services for those who need repair orrnreplacement of their damaged heart valves. Penicillin failure to clear pathogenic streptococcirnneeds further investigations.rnKey words: Group A streptococcus, beta-hemolytic streptococci, rheumatic heart disease,rnrheumatic fever, antibiotic prophylaxis, susceptibility pattern, emm type, proteinrnprofile, gram negative oral commensal, Ethiopia