ArticleEUROPEAN JOURNAL OF MEDICAL RESEARCH. 13(3): 133-135 (2008)journal articl

Hidaka,  E

Honda,  T

Kasuga,  E

Kawakami,  Y

Matsubara,  M

Matsumoto,  T

Oana,  K

Ota,  H

Shigemura,  T

Suzuki,  T

Yamauchi,  K

English

Shinshu University Institutional Repository

EU RO PE AN JOUR NAL OF MED I CAL RE SEARCH 133AbstractBacteremia due to Capnocytophaga sputigena occurredin a 4-year and 9-month-old Japanese girl patient withacute erythroblastic leukemia in Shinshu UniversityHospital, Japan. On her admission to the hospital, shehad a temperature of  38.2 °C with canker sore. Priorto the commencement of  chemotherapy, peripheralblood culture was carried out with the BacT/Alert 3DSystem ver. 4.00D (bioMérieux Japan Ltd., Tokyo,Japan) using both the PF and the SN bottles. At 48 hrsof  incubation, the System showed the positive signonly in the anaerobic SN bottle for bacterial growth.The strain isolated from the SN bottle was morpho-logically, biochemically, and genetically characterized,and finally identified as Capnocytophaga sputigena. Thecausative Capnocytophaga sputigena isolate was foundto be a β-lactamase-producer demonstrating to pos-sess cfxA3 gene. The gene responsible for the produc-tion of  CfxA3-β-lactamase was proved to be chromo-some-encoded, by means of  southern hybridizationanalysis. This was the first case of  bacteremia causedby chromosome-encoded CfxA3-β-lactamase-produc-ing Capnocytophaga sputigena.Key words: Bacteremia, Capnocytophaga sputigena,CfxA3-β-lactamaseINTRODUCTIONCapnocytophaga species are capnophilic, gram-negativefusiform rods with gliding motility, and are known tobe common inhabitants in oral cavity. In immunocom-promised granulocytopenic patients, a number of  com-plications, including bacteremia, endocarditis, and peri -pheric lesions, have been reported to date [2, 3, 11]. Anisolation of  TEM-type- β-lactamase-producing Capno-cytophaga ochracea from blood has already been report-ed [14]. Moreover, some isolation cases of  CfxA3-type-β-lactamase-producing Capnocytophaga species, such asC. granulose, C. ochracea, C. gingivalis, together withC. sputigena, had been reported [6, 8]. Indeed, CfxA3-type-β-lactamase-producing strains have already beendescribed and are well known as gene-encoded β-lacta-mase. However, to the best of  our knowledge, no casereport of  bacteremia associated with CfxA3-type-β-lactamase-producing Capnocytophaga species has beendocumented. This was the first isolation of  CfxA3-type-β-lactamase-producing Capnocytophaga sputigenafrom a blood stream infection.A CLINICAL CASE OF CAPNOCYTOPHAGABACTEREMIAPatient: The patient was a 4-year and 9-month-old girl,transferred from other private hospital to our ShinshuUniversity Hospital on 31 January in 2007, with the di-agnosis of  suspected Behçet's syndrome. She was hos-pitalized in a pediatric ward and finally diagnosed aserythroblastic leukemia in our hospital. On the veryday of  her hospitalization in our hospital, she had atemperature of  38.2 °C with canker sore, clinically sus-pected of  having contracted bacteremia.Isolation from the Blood of  the Patient: On initiatingan empirical antimicrobial dual chemotherapy withboth ceftazidime (CAZ) and piperacillin (PIPC), oneset of  peripheral blood culture sample, that is, a PFbottle (bioMérieux Japan Ltd., Tokyo, Japan) for aer-obes and an SN (bioMérieux Japan Ltd.) for anaer-obes, was collected for bacteriological examination bymeans of  the BacT/Alert 3D System ver. 4.00D (bio-Mérieux Japan Ltd., Tokyo, Japan).At the 48 hrs of  incubation, only the SN (bio-Mérieux Japan Ltd.) bottle in the BacT/Alert 3D Sys-tem (bioMérieux Japan Ltd.) showed the positive sig-nal for bacterial growth, demonstrating gram-negative,long, fusiform, straight to slightly curved rod-shapedmorphology, by means of  Gram stain. Hence, analiquot (approximately 100µl) from the positive SN(bioMérieux Japan Ltd.) bottle was inoculated ontomodified Drigalski (Nippon Becton Dickinson, Tokyo,Japan), Sheep Blood (Nippon Becton Dickinson,Tokyo, Japan), Chocolate agar plates (Nippon BectonDickinson), and Anaero Columbia agar plates (NipponBecton Dickinson), and incubated at 35 °C. After incu-bation for 48 hrs, yellowish colored colonies withMarch 31, 2008Eur J Med Res (2008) 13: 133-135                                                                                                                       © I. Holzapfel Publishers 2008FIRST CASE OF BACTEREMIA DUE TO CHROMOSOME-ENCODEDCfxA3-β-LACTAMASE-PRODUCING CAPNOCYTOPHAGA SPUTIGENAIN A PEDIATRIC PATIENT WITH ACUTE ERYTHROBLASTIC LEUKEMIAT. Matsumoto1, M. Matsubara2, K. Oana2, E. Kasuga1, T. Suzuki1, E. Hidaka1, T. Shigemura3, K. Yamauchi1, T. Honda1, H. Ota2, Y. Kawakami21Department of Laboratory Medicine, Shinshu University Hospital, 2Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University School of Medicine, 3Department of Pediatrics, Shinshu University Hospital, Nagano, Matsumoto, Japanslightly gliding were grown both on Chocolate agarplates (Nippon Becton Dickinson) and Sheep Bloodagar plates (Nippon Becton Dickinson) in 5% CO2 in-cubator, and on Anaero Columbia agar plates in ananaerobic chamber. The isolate was revealed to be fac-ultatively anaerobic Gram-negative fusiform, andcapnophilic rod shaped morphology reminiscent ofCapnocytophaga species, at a glance.Identification of  the Isolate: Biochemical characteriza-tion of  the isolate was investigated with ID-Test HN20(Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) kit sys-tem. Inoculated ID-Test HN20 (Nissui PharmaceuticalCo., Ltd.) kit system was kept at 35 °C in the atmos-phere for 4 hrs, and final readings were carried out asstated by the instructions of  the manufactures. Theisolate was shown to be oxidase- and catalase- negative,and identified as Capnocytophaga sputigena accordingto the profile number 7077133 obtained. Moreover, inorder to confirm the identification to the species level,16S rRNA gene of  the isolate was amplified by PCR,as previously described [15], and the resulting 1,300-nucleotides product was sequenced with an ABIPRISM 3,100 genetic analyzer (Applied BiosystemsJapan, Tokyo, Japan). The sequence data obtained werecompared with those of  the published sequences in theGenBank database of  the National Center for Biotech-nology Information with the BLAST N algorithm. Theclosest match of  the 16S rRNA sequence with Capno-cytophaga sputigena was obtained with 99% identity(GenBank accession no. X67609), thus confirming theidentification result of  C. sputigena based on biochem-ical and morphological properties.Susceptibility Tests of  the Isolate: Antimicrobial sus-ceptibility tests were carried out with the Epsilometertest (E-test, Aska Diagnostics, Inc., Tokyo, Japan)strips impregnated with gradient of  different concen-trations of  each antimicrobial, on Mueller-Hinton agarsupplemented with 5% sheep blood (Nippon BectonDickinson) plates, as previously described [9].The isolate was proved to be positive for the pro-duction of  β-lactamase by means of  Cefinase diskprocedure (Nippon Becton Dickinson, Tokyo, Japan).Moreover, the isolate was demonstrated to be resistantto many β-lactam antibiotics including amoxicillin,ceftazidime (CAZ) and cefotaxime (CTX), but suscep-tible to imipenem, amoxicillin-clavulanic acid, CAZ-clavulanic acid, CTX-clavulanic acid and cefoperazon-sulbactam, as shown in Table 1.Treatment was altered to intravenous administrationof  meropenem (500 mg, every 8 hrs. for 9 days) andclindamycin (200 mg, every 8 hrs. for 4 days). The pa-tient’s clinical state became in good condition and im-proved satisfactorily after intravenous administrationfor 7 days.Mechanism of  Resistance to β -Lactams: For the pur-pose of  clarifying the resistance mechanism against ß-lactams, the experiment for detecting β-lactamase genewas carried out by PCR with cfxA -specific primers aspreviously described [6], and successfully found cfxAgene in the isolate. DNA sequencing of  the PCRproducts was demonstrated to be cfxA gene. Carefulanalysis of  the nucleotide sequence verified that thecfxA-β-lactamase gene coding for the isolate was dif-ferent from that coding for Bacteroides vulgatus [12] bythe substitution of  two amino acids, that is, K272Eand Y239D. Therefore, the β-lactamase gene of  theisolate was finally confirmed to be cfxA3. The cfxA3gene was chromosome-encoded, as demonstrated bySouthern hybridization study.Although several investigators reported that TEM-and CfxA-type-β-lactamases from Capnocytophagaspecies were inhibited its activities in the presence ofclavulanic acid [6, 8, 14], the C. sputigena isolate fromblood in this case was not exceptional. That is, our iso-late was resistant to penicillins and cephems, but sus-ceptible to those antimicrobials in conbination withclavulanic acid, a kind of  β-lactamase inhibitor.DISCUSSIONThe species in the genus Capnocytophaga are made upof  slow-growing, capnophilic, fusiform, and filamen-tous gram-negative bacilli. They were a member of  thenormal gingival flora and known to cause gingivitisand periodontitis. For that reason, it has been suggest-ed that bloodstream infections develop when Capnocy-tophaga break through the normal mucosal barrier inpatients who have severe oropharyngeal mucositis orperiodontal disease [2, 11].The susceptibility of  Capnocytophaga species to ex-tended-spectrum cephalosporins has been reported tobe variable [1, 7]. Hence, bacteremias in neutropenicpatients are often empirically treated with extended-spectrum cephalosporins prior to the detection ofCapnocytophaga species because of  its slow-growingproperty [11]. As a matter of  fact, the patient of  thiscase was demonstrated to develope an oral ulcer prob-ably due to the diagnosis of  suspected Behçet syn-drome. The site of  oral ulcer might possibly be theportal of  entry of  C. sputigena in this case.EUROPEAN JOURNAL OF MEDICAL RESEARCH134 March 31, 2008Table 1. Antimicrobial susceptibility of CfxA3-β-lactamaseproducing Capnocytophaga sputigena isolate as determinedwith Epsilometer test strips.Antimicrobial agent                          MIC (µg/ml)Amoxicillin                                      >256Amoxicillin + clavulanic acid                0.25Aztreonam                                             0.19Cefepime                                               3Cefoperazone + sulbactam                    0.19Cefotaxime                                        >16Cefotaxime + clavulanic acid              <0.016Cefoxitin                                                0.25Cefpirome                                             2Ceftazidime                                         48Ceftazidime + clavulanic acid            <0.064Imipenem                                              0.064Recently, plasmid-encoded extended-spectrum TEM- 17 β-lactamase, chromosome- or plasmid-encodedCfxA β-lactamase have been identified for Capnocyto -phaga species [6, 8, 15]. It is thus becoming essential todetermine which antibiotics are effective against suchclinical isolates. A Medline search of  the literature dis-closed a few clinical reports in bacteremia caused byCapnocytophaga species [4, 5, 10, 11, 13], however, nota single CfxA β-lactamase-producing case has beendocumented.We describe here the first case of  bacteremiacaused by a strain of  C. sputigena possessing a chro-mosome-encoded cfxA3-β-lactamase gene. The iso-late was recovered from only an SN bottle usually in-applicable for pediatric patients. Thus indicating thatthis case would be difficult to be observed unless ap-plied an SN bottle for blood culture.In conclusion, the carbapenem or beta-lactam drugin combination of  a β-lactamase inhibitor is favorableto be administered in cases of  bacteremia due toCfxA3-β-lactamase producing Capnocytophaga species.REFERENCES1.   Arlet G, Sanson-Le Pors MJ, Casin IM, Ortenberg M,Perol Y. In vitro susceptibility of 96 Capnocytophagastrains, including a beta-lactamase producer, to new beta-lactam antibiotics and six quinolones. Antimicrob AgentsChemother. 1987;31(8): 1283-1284.2.   Bilgrami S, Bergstrom SK, Peterson DE, Hill DR, Daini-ak N, Quinn JJ, Ascensao JL. Capnocytophaga bacteremiain a patient with Hodgkin's disease following bone mar-row transplantation: case report and review.  Clin InfectDis. 1992; 14(5): 1045-1049.3.   Buu-Hoi AY, Joundy S, Acar JF. Endocarditis caused byCapnocytophaga ochracea. J Clin Microbiol. 1988; 26(5):1061-1062.4.   Funada H, Machi T, Yoneyama H, Matsuda T, Miura H,Ezaki T, Yokota Y. Capnocytophaga sputigena bacte remiaassociated with acute leukemia. Kansenshogaku Zasshi.1993; 67(7): 622-628.5.   Gomez-Garces JL, Alos JI, Sanchez J, Cogollos R.  Bac-teremia by multidrug-resistant Capnocytophaga sputigena.J Clin Microbiol. 1994; 32(4): 1067-1069.6.   Handal T, Giraud-Morin C, Caugant DA, Madinier I,Olsen I, Fosse T. Chromosome- and plasmid-encodedbeta-lactamases in Capnocytophaga spp. Antimicrob.Agents Chemother. 2005; 49(9): 3940-3943.7.   Jolivet-Gougeon A, Buffet A, Dupuy C, Sixou JL, Bon-naure-Mallet M, David S, Cormier M.  In vitro suscepti-bilities of Capnocytophaga isolates to beta-lactam antibi-otics and beta-lactamase inhibitors. Antimicrob. AgentsChemother. 2000; 44(11): 3186-3188.8.   Jolivet-Gougeon A, Tamanai-Shacoori Z, Desbordes L,Burggraeve N, Cormier M, Bonnaure-Mallet M. Geneticanalysis of an ambler class A extended-spectrum beta-lac-tamase from Capnocytophaga ochracea. J Clin Microbiol.2004; 42(2): 888-890.9.   Kawakami S, Ono Y, Kato J, Miyazawa Y. Evaluation ofa new method for susceptibility testing against fastidiousand unusual bacteria. Kansenshogaku Zasshi. 1998; 72(10): 1017-1026.10. Martino R, Ramila E, Capdevila JA, Planes A, Rovira M,Ortega MD, Plume G, Gomez L, Sierra J.  Bacteremiacaused by Capnocytophaga species in patients with neu-tropenia and cancer: results of a multicenter study. ClinInfect Dis. 2001; 33(4): E20-22.11. Maury S, Leblanc T, Rousselot P, Legrand P, Arlet G,Cordonnier C.  Bacteremia due to Capnocytophaga speciesin patients with neutropenia: high frequency of beta-lacta-mase-producing strains. Clin Infect Dis. 1999; 28(5):1172-1174.12. Parker AC, Smith CJ. Genetic and biochemical analysis ofa novel Ambler class A beta-lactamase responsible for ce-foxitin resistance in Bacteroides species. AntimicrobAgents Chemother. 1993; 37(5): 1028-1036.13. Ramos JM, Pacho E, Bernacer M, Fernandez-Roblas R,Soto C, Soriano F. Capnocytophaga spp. bacteremia incompromised patients. Enferm Infect Microbiol Clin.1993; 11(4): 206-209.14. Rosenau A, Cattier B, Gousset N, Harriau P, Philippon,A, Quentin R.  Capnocytophaga ochracea: characterizationof a plasmid-encoded extended-spectrum TEM-17 beta-lactamase in the phylum Flavobacter-bacteroides.  An-timicrob Agents Chemother. 2000; 44(3): 760-762.15. Weisburg WG, Barns SM, Pelletier DA, Lane DJ.  16S ri-bosomal DNA amplification for phylogenetic study. JBacteriol. 1991; 173(2): 697-703.Received: October 26, 2007 / Accepted: January 9, 2008Address for correspondence: Yoshiyuki Kawakami, Ph.D., ProfessorDivision of Clinical Microbiology,Department of Biomedical Laboratory Sciences,School of Health Sciences,Shinshu University School of Medicine,Matsumoto, 390-8621, JapanPhone:    +81-263-37-2381Fax:         +81-263-37-2370E-mail:    yk23724@gipac.shinshu-u.ac.jpEUROPEAN JOURNAL OF MEDICAL RESEARCHMarch 31, 2008 135

First case of bacteremia due to chromosome-encoded CfxA3-beta-lactamase-producing Capnocytophaga sputigena in a pediatric patient with acute erythroblastic leukemia

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First case of bacteremia due to chromosome-encoded CfxA3-beta-lactamase-producing Capnocytophaga sputigena in a pediatric patient with acute erythroblastic leukemia

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