Catheter-associated bacteremia by Mycobacterium senegalense in Korea

Background Rapidly growing mycobacteria is recognized as one of the causative agents of catheter-related infections, especially in immunocompromised hosts. To date, however, Mycobacterium senegalense, which was known as the principal pathogen of bovine farcy, has not been reported in human infection. Case presentation We describe the first case of human infection by M. senegalense, which has caused catheter-related bloodstream infection in a cancer patient in Korea. The microorganism was identified by the 16S rRNA gene, rpoB, and 16S-23S rRNA gene internal transcribed spacer (ITS) sequence analyses. Conclusion Our first report of catheter-associated bacteremia caused by M. senegalense suggests the zoonotic nature of this species and indicates the expansion of mycobacterial species relating to human infection. M. senegalense should be considered as one of the causes of human infections in the clinical practice.


Background
Mycobacterium senegalense was originally described by Chamoiseau in 1973 as a subspecies of Mycobacterium farcinogenes [1]. However, it was later recognized as a distinct species closely related to M. fortuitum [2]. M. senegalense is known as the principal pathogen of bovine farcy, which is a chronic disease of skin and superficial lymphatics of cattle in East and Central Africa [3]. Unlike other rapidly growing mycobacteria, human infection by M. senegalense has not been reported to date. Here we report the first case of central venous catheter (CVC) infection caused by M. senegalense.

Case presentation
A 49-year-old woman with non-Hodgkin's lymphoma was admitted to the hospital because of fever for several hours. The patient had been treated for lymphoma since 3 months ago. Five days before admission, the patient was treated with the third cycle of CHOP plus rituximab (R-CHOP). She had no history of travel or contact with animals including cows or their products. Physical examination revealed high fever of 39.8°C. The patient had a subclavian cuffed-CVC(Hickman catheter) on the right side with no evidence of the inflammation at the exit site. Laboratory data and chest radiograph were within normal limits. Three sets of blood samples for cultures were drawn through CVC lines (2 sets) and a peripheral vein (1 set), respectively. The patient was treated empirically with vancomycin (1 g every 12 h intravenously). On the second hospital day, all 3 sets of blood cultures grew gram-positive, acid-fast bacilli. The cultures from the CVC became positive more than 2 hours earlier than that from a peripheral vein. Non-pigmented and pinpoint-shaped colonies were observed on blood or chocolate agar plate after 3 days of incubation at 37°C. It did not grow well on McConkey agar plate. As it grows, the color of the colonies becomes pale-yellow. Vancomycin was replaced by imipenem/cilastatin (500 mg every 6 h intravenously) and amikacin (375 mg every 12 h intravenously). On the sixth hospital day, the CVC was removed because of persistent fever. After removal of CVC, the patient became afebrile and the repeated blood cultures became negative. In vitro susceptibility test was performed by broth microdilution test as described by the National Committee for Clinical Laboratory Standards (NCCLS) guidelines [4]. The result of in vitro susceptibility test was shown in Table 1. The isolates were susceptible to most antimicrobial agents tested except vancomycin. The patient was further treated with oral ciprofloxacin (500 mg every 12 hours) and doxycycline (100 mg every 12 hours) for 4 weeks. She had been doing well with no evidence of recurrence for the next 3 months.

Molecular identification
Conventional automated methods in the clinical microbiology laboratory such as VITEK 2 system (bioMérieux, Hazelwood, Mo.) failed to identify this isolate to a given species. Thus, this isolate ("SMC-7485"), was subjected to the 16S rRNA gene, rpoB, and 16S-23S rRNA gene internal transcribe spacer (ITS) sequence analyses for bacterial identification. Genomic DNA was extracted by using the G-Spin Genomic DNA Extraction Kit (iNtRON, Seoul, Korea). DNA amplification of 16S rRNA gene, rpoB, and ITS were performed by using primer sets 16S-F3  [5][6][7], respectively. Template DNA (ca. 50 ng) and 20 pmol of each primer were added to a PCR mixture tube (AccuPower PCR PreMix; Bioneer, Daejeon, Korea) containing 1 unit of Taq DNA polymerase, each deoxynucleoside triphosphate at a concentration of 250 µM, 10 mM Tric-HCl (pH 8.3), 10 mM KCl, 1.5 mM MgCl 2 , and gel loading dye [8]. The reaction mixture was then subjected to 35 cycles for amplification. Each cycle consisted of 30 sec at 95°C for denaturation, 30 sec at 60°C, and 1 min at 72°C for extension, followed by final extension at 72°C for 5 min. Amplified PCR product was purified for sequencing using PCR purification kit (CoreOne, Seoul, Korea). The purified PCR product was sequenced directly using the same primers of PCR amplification at both directions. Sequence editing and analyses were performed with the EditSeq and MegAlign programs in DNASTAR (Windows version 3.12e; Madison, Wis.

Discussion
Rapidly growing mycobacteria such as the M. fortuitum group, the M. chelonae/abscessus group, and the M. smegmatis group are capable of thriving in even the most hostile environments [10]. Due to their ubiquitous capability, human infections by the rapidly growing mycobacteria have been identified with increasing frequency worldwide. Especially, rapidly growing mycobacteria are being recognized as one of the significant pathogens of catheterrelated infections in immunocompromised hosts. Among rapidly growing mycobacteria, the M. fortuitum group is the most common mycobacterial pathogen for this clinical condition [10,11].
Phylogenetic relationships of SMC-7485 and other Mycobacterium species based on ITS sequences, which were retrieved from GenBank database  [12]. Of these, M. senegalense was originally described by Chamoiseau in 1973 as a subspecies of M. farcinogenes. Although M. farcinogenes and M. senegalense have identical 16S rRNA gene sequences, M. senegalense could be identified as a different species based on differences in growth rate, chemical activity and DNA homology [1,6,13,14]. While most species of M. fortuitum group have been reported to be responsible for various human diseases, human infection by M. senegalense has not been described to date [3,10,12]. Instead, it causes the chronic infectious disease of zebu cattle known as bovine farcy, endemic to East and Central Africa [3,9]. Moreover, M. senegalense, which was originally found in Africa, has never been described elsewhere [3,10].
In this report, we have first documented the CVC infection caused by M. senegalense. Because conventional automated methods failed to identify it at the species level, we tried to sequence 16S rRNA gene, rpoB gene, and ITS region. By 16S rRNA gene, rpoB gene, and ITS sequence analyses, we concluded that an agent of CVC infection in our patient was M. senegalense. rpoB gene and ITS sequences could differentiate M. senegalense from M. farcinogenes clearly as in previous reports [6,14]. Moreover, ITS sequence analysis indicated that M. senegalense might consist of at least two heterogeneous groups (Fig. 1). There is possibility that M. senegalense isolate related to human infection has been misidentified as different species because it is difficult to identify nontuberculous mycobacteria (NTM) at the species level [9,16]. However, the strain SMC-7485 is the first described M. senegalense isolate, which is associated with human infection and is found outside Africa, to our knowledge.
In most cases with mycobacterial infection of CVC, the line should be removed for successful control of infection [17]. In this study, the patient failed to respond to an initial regimen of imipenem and amikacin, to which the isolate was susceptible. Persistent infection was controlled after removal of catheter, which emphasized the importance of catheter removal. Because of differences in susceptibilities among species and even within species, rapid identification and subsequent susceptibility testing are essential for selection of appropriate antibiotic agent(s) against rapidly growing mycobacteria [18]. In this study, the strain SMC-7485 was susceptible to amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, and imipenem. Because of the high frequency of relapse and resistance, combination therapy with multiple antibiotics is usually recommended for serious infections by rapidly growing mycobacteria. However, the optimal antibiotic regimen has yet to be defined for catheter-related infection by these mycobacteria. In our experience, oral antibiotic therapy subsequent to a short course of intravenous antibiotics seemed to be effective and safe. Although it was not possible to determine the optimal duration of antibiotic therapy in our case, this episode was successfully treated with short-term (approximately 5 weeks) antibiotic therapy. Further studies will be required to confirm these findings.

Conclusion
In this paper, we firstly reported catheter-associated bacteremia by M. senegalense. This case suggested that M. senegalense can cause human infections. This pathogen should be included in the list of nontuberculous mycobacteria causing human infections.