A 44-year-old woman presented to the infectious department of Peking Union Medical College Hospital (Beijing) in August 2013, with a 7-week history of intermittent fevers, chills, sore throat, muscular soreness, occasional joint pain, and cough with white foam phlegm. Petechiae were scattered throughout the inner thighs and spreading to the hips. The maximum body temperature was 39 °C. Laboratory tests revealed a white blood cell count of 21.55 × 109/L with 73% neutrophils; hemoglobin level of 11.9 g/dl; blood platelet level of 17.1 × 109/L; high sensitivity C- reactive protein level of 44.38 mg/L; erythrocyte sedimentation rate (ESR) 30 mm/h, and microscopic hematuria. The patient was diagnosed as having a pulmonary infection, and after empirical treatment with moxifloxacin 0.4 g iv Qd 5 days, her condition did not improve.
Before admission, the patient had been treated with anti-TB drugs at the local hospital, namely levofloxacin 400 mg orally once daily, isoniazid 300 mg orally once daily, pyrazinamide 750 mg orally once daily, and ethambutol 750 mg orally once daily. After treatment for 4 days with these drugs, there was no improvement. Consequently, the patient was started on methylprednisolone 40 mg iv Qd. As a young girl, the patient had suffered from rheumatic heart disease at 10 years of age. Chest auscultation, wet and dry rales were not heard in the bilateral lungs. A systolic murmur was detected at the mitral valve area.
The left ventricular ejection fraction (LVEF) was 69% (normal value, >55%). Transesophageal echocardiogram revealed rheumatic valvular heart disease, with mitral valve stenosis and incompetence, aortic valve regurgitation. However, vegetations were not seen clearly. Chest computed tomography image revealed the presence of a small shadow and bilateral pleural nodules in the middle lobe of the right lung. The left pleural membranes were thickened, and lower extremity edema was not observed. Three sets of aerobic and anaerobic blood cultures were collected during fever episodes at the time of hospitalization. Of these blood cultures, only one aerobic bottle grew a Gram-negative rod after incubation for 100 h in the BacT/Alert automated blood culturing system. The Gram-negative rod cultured was identified as H. massiliensis by 16S rRNA gene sequencing. Other patient tests and results included; glutamic oxalacetic transaminase (GOT) level of 370 U/L (normal range, 0~40 U/L), alanine aminotransferase (ALT) level of 470 U/L (normal range, 0~40 U/L), total and direct bilirubin were normal, and lactate dehydrogenase was high at 688 U/L (normal range, 100~300 U/L). On day 26 of hospital admission, the IgM of Hepatitis A virus (HAV) was positive. Cytomegalovirus and Epstein-Barr virus (IgM and PCR) were both negative, and so was HIV serology test. Finally, the patient was clinical diagnosed as probable endocarditis caused by H. massiliensis.
The patient was then treated with amoxicillin/clavulanate 2.4 g iv. Q8h combined with amikacin 0.4 g iv. Qd, for 6 weeks. After treatment for 8 days, the patient’s temperature returned to normal, other signs and symptoms improved, so the patient was discharged from hospital. To date, the patient has not relapsed.
Microbiology examination
On subculture, the organism grew on blood agar, chocolate agar and Mueller-Hinton agar, but didn’t grow on China blue agar, after incubation for 48-h at 37 °C in 5% CO2. The colonies on Blood agar were round, smooth, moist, convex, light gray, non-hemolytic and 1-mm in size (Fig.
1
). On Gram stain, the organism appeared as short to long serpentine rods, whose morphology was clearly different from other common Gram negative bacteria (Fig.
2
). Biochemically, the organism was positive for catalase, oxidase and urease, and did not produce H2S or reduce nitrate to nitrite. Besides, it did not produce indole or hydrolyzed gelatin or esculin.
The API 20NE (bioMérieux) system yielded a biocode of 0200044 at 48 h of incubation, and as per its database, the organism was identified as Methylobacterium mesophilicum, with % ID of 84.6% and a T index of 1.0, which was an acceptable identification. On the other hand, the Vitek GNI (bioMérieux) card identified the organism as Brucella melitensis, with a probability of 99%. The organism could not be identified by Vitek Maldi-Tof (bioMérieux) mass spectrometry and Bruker Maldi-Tof mass spectrometry due to lack of the spectrum in their database. In summary, neither of them gave a correct result.
The antibiotic susceptibility test was performed using Etest (AB Biodisk) as per the standard method with Mueller-Hinton agar, and incubated for 48 h. The minimum inhibitory concentrations (MICs) of the antibiotics tested were as follows; ampicillin/sulbactam 0.094 μg/ml, piperacillin/tazobactam 4 μg/ml, ciprofloxacin 0.094 μg/ml, ceftazidime <0.016 μg/ml, cefoperazone/sulbactam 4 μg/ml, ertapenem 0.008 μg/ml, amikacin 0.50 μg/ml, cefepime 0.047 μg/ml, amoxicillin/clavulanate 0.19 μg/ml, and trimethoprim/sulfamethoxazole was 0.125 μg/ml. The organism exhibited low MICs to most antibiotics.
Molecular identification
16S rRNA gene sequencing was applied to identify the organism correctly. The universal primer pair used was 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1522R (5′-AAGGAGGTGATCCAGCCGCA-3′). Nearly the full length (1357 bp) of the gene was obtained (GenBank accession no. KJ458986), and BLAST searches showed the best match with H. massiliensis with 100% identity (1354 of 1354 bp without gap sites) (the GenBank accession numbers were DQ342309, DQ342318, DQ342311 and DQ342310). The second best matches were Haematobacter missouriensis (GenBank accession no. DQ342317) for 99% identity (1353/1354 bp without gap sites), with Rhodobacter sp. (GenBank accession no. AM292058) for 99% identity (1355/1361 bp with five gap sites). Therefore, the organism was defined as H. massiliensis.
Nucleotide sequence accession number
The 16S rRNA gene sequence of this strain named C1989 has been deposited in GenBank under accession number KJ458986.