TEM-producing Capnocytophaga sputigena primary bactaeremia in a breast cancer patient

Case presentation: C. sputigena was isolated from blood in a breast cancer patient who suffered from oral mucosal barrier breakage for several years. The bacterium was initially identified in the blood culture of the patient by conventional techniques and confirmed by mass spectrometry and sequencing of the 16S rRNA gene. Antibiotics susceptible testing revealed the bacterium was resistant to penicillins, first-, secondand third-generation cephalosporins and monobactam. PCR was used to detect common b-lactamase genes; the TEM gene was detected and confirmed by sequencing.


Introduction
Capnocytophaga spp.are recognized as part of the normal human oral and buccal cavity.Blood infection caused by Capnocytophaga spp. is extremely uncommon, due to its relatively low pyrogenicity and very low toxicity, as shown using a sonic extract of the organism (Stevens et al., 1980).It usually appears in immunocompromised patients (Gomez-Garces et al., 1994), especially in neutropenic patients, because neutrophils play an important role in impairing Capnocytophaga (Miyasaki, 1990).Capnocytophaga spp.are important pathological bacteria for oral infections, but opportunistic pathogens in various extra-oral infections (Atmani et al., 2008;Chan et al., 2010;Douvier et al., 1999;Garcia-Cia et al., 2004;Rysselaere & Van Cauwenberge, 1983;Torrus et al., 1995).Reports of bloodstream infections caused by C. sputigena are relatively rare (Matsumoto et al., 2008;Vandemeulebrouck et al., 2008).Here, we reported a case of bacteraemia with C. sputigena in a breast cancer patient.

Case report
A 52-year-old Chinese woman was admitted to our hospital on 6 October 2013 due to invasive breast carcinoma with an oral ulcer (Fig. 1).After the third phase of chemotherapy on 18 November 2013, the patient got a fever of 38.5 u C. The white blood cell count of the patient was decreased to 0.6610 9 ml 21 (reference range is 4610 9 -10610 9 ml 21 ).Blood cultures were performed immediately under aerobic and anaerobic conditions.The anaerobic blood culture bottle became positive on day 3 of incubation, and a direct Gram stain of the aspirate revealed Gram-negative bacilli.The aspirate was plated onto 5 % sheep blood agar, chocolate blood agar and China blue agar (a substitute for MacConkey agar) in 5 % CO 2 at 35 u C.After 3 days of incubation the 5 % sheep blood agar grew tiny, brown, irregular colonies of Gram-negative slender fusiform bacilli.The bacterium was oxidase and catalase negative and nitrate reduction test positive.There was no colony growth on the chocolate and China blue agars after 7 days of incubation.The bacterium was identified as Capnocytophaga spp.using an NH identification system (VITEK2 Compact: bioMe ´rieux) with 99 % confidence levels.The bacterium was then subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Bruker Daltonik), using the Bruker Biotyper system and database with a score of 2.035 for C. sputigena, which was higher than the determined cutoff of 1.8 used for accurate identification of the bacterial species and genus.Sequencing of the 16S rRNA gene was performed to further identify the bacterium.PCR of 16S rRNA gene gave a product about 1500 bp using the following primers: F, 59-CAGAGTTTGATCCTGGCT-39, and R, 59-AGGAGGTGATCCAGCCGCA-39. The sequencing result showed 100 % nucleotide identity with the C. sputigena sequence in GenBank (accession no.AF133536) by BLAST analysis (http://blast.ncbi.nlm.nih.gov/).
Antimicrobial susceptibility was tested using automatic instrument methods (GNS-09, VITEK2 Compact; bioMe ´rieux) and K-B disk diffusion method (Oxoid) on blood agar plates incubated for 48 h at 35 uC in 5 % CO 2 , and the results were interpreted using Clinical and Laboratory Standards Institute breakpoints for HACEK group infections.The bacterium showed resistance (with bacteriostatic diameter ,10 mm or MICs .256mg l 21 ) to penicillin, ampicillin, cefuroxime, cefazolin, cefotaxime, ceftazidime, aztreonam, erythromycin, clindamycin, levofloxacin and tetracycline but susceptibility (with bacteriostatic diameter .27mm or MICs ,2 mg l 21 ) to imipenem.b-Lactamase was positive using Cefinase (BD), and b-lactamase genes were detected by PCR, using a standard PCR protocol (denaturation for 4 min at 94 u C; 30 cycles of 45 s at 94 u C, 45 s at 55 u C and 1 min at 72 u C; and a final extension of 10 min at 72 u C).The primer sequences are given in Table 1.The PCR products showed that the organism was positive for the TEM gene (Fig. 2); it was confirmed as TEM (GenBank accession no.FJ223605) by DNA sequencing and a BLAST search.

Discussion
The growth of C. sputigena is very slow and needs fastidious conditions, strictly requiring CO 2 .Thus, it is easy to escape examination in a clinical laboratory.The timely detection of C. sputigena plays an important role in the diagnosis of bloodstream infections, especially in patients with a damaged oral mucosa or who are immunocompromised.It is possible that the bacteraemia in this patient was caused by a lesion of the oral cavity, which allowed C. sputigena to pass into the bloodstream as a result of damage to the oral mucous membrane and induce infection of the blood.
C. sputigena was first reported and named in 1979, and was separated from other Capnocytophaga spp. on the basis of morphological and physiological characteristics (Leadbetter et al., 1979).Conventional methods can identify Capnocytophaga spp.only to the genus level.MALDI-TOF MS has been shown to provide rapid identification of bacteria and thus theoretically allow earlier intervention; it can identify Capnocytophaga to the species level quickly and easily, making it a powerful tool for bacterial identification.
Reports of C. sputigena carrying the TEM gene are rare.TEMproducing strains make treatment more difficult; the patient in this report was successfully cured by imipenem.To the best of our knowledge, this study is the first report of bacteraemia in a breast cancer patient caused by TEM-producing C. sputigena.It is important to focus on bloodstream infections caused by conditional pathogenic bacteria.

Table 1 .
PCR primers used in this study