Abstract:?Objective?To analyze retrospectively the molecular and Mass spectrometer characteristics of levofloxacin-resistant?Streptococcus?agalactiae?isolated clinically in Suzhou area.?Methods?A total of 58 isolates of non-repetitive levofloxacin-resistant?Streptococcus?agalactiae?were collected from January 2018 to January 2019. PCR and Sanger gene sequencing methods were used to detect the gene mutations of?gyrA,?parC?and?parE, as well as the serotype-associated genes and multi-locus sequence typing. ClinPro Tools 3.0 software was used to screen and verify the levofloxacin-resistant specific peaks of mass spectrometry (MS) of proteomics in the?Streptococcus?agalactiae?isolates.?Results?The results of sequencing showed that levofloxacin-resistant?Streptococcus?agalactiae?strains mainly carried the mutations of gyrA_Ser81Leu+parC_Ser79Tyr+parE_His225Tyr, accounting for 44.8% (26/58). Serotype Ⅲ and ST19 were the main types of levofloxacin-resistant streptococcus agalactiae isolates accompanied by prevalent serotype Ⅰb and ST23. Mass spectrometry profile analysis for?streptococcus?agalactiae?showed that 6 894, 3 445, 5 076, 4 544 and 3 721 m/z were the dominant specific differential peaks of the levofloxacin-resistant?streptococcus?agalactiae?isolates.?Conclusion?The gene mutations of levofloxacin-resistant?Streptococcus?agalactiae?isolates in the local area were mainly gyrA_Ser81Leu+parC_Ser79Tyr+parE_His225Tyr with serotype Ⅲ and ST19, and 6 894 m/z was represented as the specific mass spectrometric peak.