(1) Background: Culture-negative endocarditis is challenging to diagnose. Here, we retrospectively identified 23 cases of Coxiella burnetii and Bartonella endocarditis by metagenomic next-generation sequencing. (2) Methods: Twenty-three patients with culture-negative endocarditis were retrospectively enrolled from Guangdong Provincial People's Hospital (n = 23) between April 2019 and December 2021. Metagenomic next-generation sequencing was performed on blood (n = 22) and excised cardiac valvular tissue samples (n = 22) for etiological identification, and Sanger sequencing was performed for pathogenic diagnostic verification. The demographic and clinical data of the 23 patients were obtained from hospital electronic health records. (3) Results: A total of 23 male patients (median age, 56 years (interquartile range, 16)) with culture-negative endocarditis were diagnosed with Coxiella burnetii (n = 21) or Bartonella (n = 2) species infection by metagenomic next-generation sequencing. All patients underwent cardiac surgery. The resected tissue exhibited both a significantly higher number of unique suspected pathogen read-pairs and more unique pathogen read-pairs than the blood specimens. The results of Sanger sequencing tests on all remaining tissue and blood specimens were positive. Oral doxycycline was added to the antibiotic regimen for at least 1.5 years according to etiology. A total of 21 patients (91%) were discharged, and 20 patients were healthy at the 21-month (interquartile range, 15) follow-up visit. One patient exhibited endocarditis relapse with the same pathogen from inadequate antibiotic administration. The last 2 patients (9%) developed septic shock and multiple organ dysfunction syndrome postoperatively and died shortly after discharge. (4) Conclusions: CNE caused by C. burnetii and Bartonella species is challenging to diagnose and exhibits poor outcome due to delayed treatment. In response, mNGS, characterized by high sensitivity and rapid results, is an effective alternative for the etiological identification of C. burnetii and Bartonella endocarditis.

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