Carnobacterium species are heterofermentative lactic acid bacteria usually isolated from the environment or food such as fish, meat, and dairy products . They share resemblance to lactobacilli in several aspects such as morphology and absence of a cytochrome system . Lactobacilli are generally known to live in human intestines and female vagina but Carnobacteria are not known members of the human gastrointestinal microbial community. The first mention of such unusual lactobacilli was made in 1950, and referred this strain to isolates from poultry meat . Currently 11 species are known in the Carnobacterium genus , but among them, only Carnobacterium divergens and Carnobacterium maltaromaticum are frequently isolated in food or fish.T hese two species are known to inhibit growth oLf isteria monocytogenes in fish and meat products by production of antimicrobial peptides. Especially, C. maltaromaticum (or C. piscicola) is known to show antilisterial activity in-vitro and in cooked ham . Up to date, there are only four reports of isolation of such pathogen in human world-wide. No reports in Korea have been published until now. Herein, we report a case of C. divergens bacteremia in an adult central nervous system (CNS) lymphoma patient who was receiving chemotherapy in Dong-A University Hospital.
A 65-year old Chinese male patient diagnosed with CNS lymphoma on July 2019 was admitted to our hospital due to mild fever of 37.8°C and severe oral mucositis for 3 to 4 days. Laboratory findings showed pancytopenia with moderate neutropenia (white blood cell 2,240 /uL, segmented neutrophils 41.0%, hemoglobin 9.9 g/dL, platelet 70,000 /uL) and increased CRP (2.91 mg/dL). No other physical findings were observed. Due to his primary CNS lymphoma, he had had osteoplastic craniotomy with tumor removal on July 29 2019. After surgery, treatment was started with R-MPV (rituximab, methotrexate, procarbazine and vincristine) induction chemotherapy from August 10 for five days. On November 8, he was treated with etoposide for peripheral blood stem cell transplantation. Due to oral mucositis, total parenteral nutrition was applied to his central venous catheter on the 6 day of his admission. For antimicrobial therapy and treatment of fever, two pairs of blood cultures were drawn on the day of admission, 5, 6 and 8 day of admission. They were inoculated into two aerobic and two anaerobic blood culture bottles and incubated in BacT/ALERT 3D blood culture instrument (bioMérieux, Marcy-LEtoile, France). On the 8 day of admission, after 48 hours of incubation at 37°C, one of the anaerobic bottles showed positive results. White to gray colonies with 1-2 mm diameter were grown on blood agar plates (Fig. 1). On biochemical studies, the colonies were catalase and oxidase negative, and alpha-hemolytic. Microscopic examination revealed either Gram-positive rods or cocci (Fig. 2). Carnobacterium divergens was identified by using MALDI-ToF/MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; bioMérieux, Marcy-l'Étoile, France) with 99.9% validality. The sample for MALDIToF was taken from the colony, not directly from blood samples. For confirmation, 16S rRNA gene sequencing using the MiSeq Microbial Identification System (Macrogen, Seoul, Korea) was performed.
The sequencing confirmed C. divergens with homology of 99%. Antimicrobial susceptibility test was performed using VITEK 2 system (bioMérieux, Marcy-LEtoile, France). The isolate was resistant to oxacillin (minimum inhibitory concentration (MIC) of 4 mg/L) but susceptible to vancomycin (MIC of 0.5 mg/L) and linezolid (MIC of 2 mg/L). Antimicrobial therapy with piperacillin/tazobactam (Tabaxin) was empirically started on the date of his admission to cover both Gram-positive and negative bacteria, but after isolation of C. divergens, vancomycin was added due to resistance to oxacillin. Two days later, subsequent peripheral and central venous catheter blood cultures showed no growing bacteria. As his fever subsided and oral mucositis was improved, the patient was discharged.
Unlike the previously published cases of Carnobacterium species (Table 1), this case was identified by MALDI-ToF mass spectrometry, which is now applied widely in clinical microbial laboratories. In comparison to classical methods of bacterial identification such as API or VITEK, mass spectrometry uses proteomic approach that allows rapid and accurate identification of bacteria as well as yeast and fungi . In fact, the conventional bacterial detection method, VITEK 2 (bioMérieux), identified the same colony as Enterococcus gallinarum with VITEK 2 GP card (code 2420916203). This implies that the conventional method cannot accurately identify such Gram-positive rod. The most recent case introduced a 57-year old female patient from France who was diagnosed with acute necrotziing esophagitis . She began parenteral nutrition after surgery, and her four sets of blood culture bottles showed positive results. Unclear results were showed on API Coryne and API Listeria systems (bioMérieux) but the physicians presumed the isolate was L. monocytogenes. Following 16S rRNA sequencing confirmed the pathogen was C. divergens. The second case from Austria presented a 43-year male patient with history of contact with seafood . He was diagnosed with meningitis, and bacterial growth was detected in one of his culture bottles. They also suspected the isolate to be L. monocytogenes. However, gene sequencing discovered that the pathogen was C. divergens. The specimens of the other two cases of Carnobacteria were necrotic tissue or pus [3,9]. They both had history of either contact with aqueous environment, feeding with parenteral nutrition or both.
It is well known that the mass spectrometry has higher diagnostic sensitivity and specificity than the conventional microbial identification methods  but there are three other factors that also support the causative relationship of the patients bacteremia and such pathogen in our case report. First, the patients fever subsided after adding vancomycin which covers methicillin/oxacillin resistant Gram-positive bacteria. Subsequent culture from central catheter showed negative results. Second, although the patient history of contact with seafood or other special environment was not definitely confirmed, he was on parenteral nutrition, like the other two published reports. Third, his previous three sets of blood cultures showed no growth, but only after he started nutrition on feeding tube, bacterial growth was observed. However, since the bacteria were present in only one of the four bottles, the possibility of contamination cannot be completely ruled out. We assume the origin of infection was bacterial contamination of the parenteral nutrition bag or colonization of the feeding tube. To confirm the pathogen, culture of parenteral nutrition solution could have been implemented.
Similar to lactobacilli, which is used as biological preservatives in food, Carnobacteria can also rarely cause bacteremia [9,10]. In summary, we report a case of C. divergens bacteremia in an immunocompromised patient identified by MALDI-ToF MS and review of the literature. As more Grampositive bacteria and other atypical bacteria, which were previously neglected or undetected, are detected with such relatively new diagnostic tool, more attention should be paid.