Multicenter Study To Evaluate Bloodstream Infection by Helicobacter cinaedi in Japan

Surveillance.We performed prospective laboratory-based multicenter surveillance in 13 hospitals located in Tokyo, Japan, from 1 October 2003 to 31 March 2004. All hospitals had automated blood culture systems (BACTEC; BD Diagnostic Systems, Tokyo, Japan), and all provide medical care to adults and children in different medical specialties. The research protocol was approved by the local institutional review board of each site.

Blood culture microbial identification.Blood was collected in BACTEC culture bottles and incubated in a BACTEC 9050 blood culture system for at least 4 days (Becton Dickinson, BD Biosciences, Tokyo, Japan). Positive blood culture samples were further investigated for the microbial identification. To identify the bacterial isolates, phenotypic tests commonly used to characterize helicobacters were performed. Growth was examined under aerobic, microaerobic, and anaerobic conditions at 35°C. Bacteria were Gram stained and tested for urease activity by using a selective rapid urea test. Dihydrogen sulfide production, γ-glutamyltransferase activity, hippurate hydrolysis, and nitrate reduction were determined by using the Campy identification system (bioMerieux Vitek, Tokyo, Japan). Some currently available biochemical tests have been reported to be unable to conclusively identify or distinguish H. cinaedi from other fastidious Campylobacter species or Helicobacter species (13). Therefore, we decided to examine both suspected Campylobacter isolates and suspected Helicobacter species for further identification.

Patients.Patients with H. cinaedi bacteremia were reviewed to determine the clinical background, the source of infection, and the outcome. The case report form contained the following information: age, gender, date of admission, ward, date of bacteremia, underlying medical conditions, exposure to invasive medical procedures, use of antibiotics or corticosteroids, management of bacteremia (antimicrobial treatment, catheter removal), and outcome.

Amplification of 16S rRNA.Bacteria were grown on blood agar plates for 48 h, and chromosomal DNA was prepared using hexadecyltrimethyl ammonium bromide as described previously (17). PCR of the 16S rRNA gene was performed with the primers 8F (5′-AGA GTT TGA TCM TGG CTC AG-3′) and 15R (5′-AAG GAG GTG ATC CAR CCG CA-3′), which were designed based on the Escherichia coli 16S rRNA numbering system. The PCR was performed in a DNA thermal cycler (PE Applied Biosystems Division, Foster City, CA) in reaction mixtures of 25 μl containing 20 ng of genomic DNA, 2.5 μl of 10-fold-concentrated reaction buffer (QIAGEN, Inc., Tokyo, Japan), 160 nM (each) primer, 0.625 U of Taq DNA polymerase (QIAGEN), and 250 μM (each) deoxynucleotide (Amersham-Pharmacia Biotech, Tokyo, Japan). Samples were incubated at 94°C for 2 min to denature the target DNA. They were then cycled 30 times at 94°C for 1 min and annealed at 55°C for 1 min and 72°C for 2 min, with a final incubation at 72°C for 10 min to complete the extension.

16S rRNA data analysis.The fragment for sequencing was amplified by PCR, and the product was purified with a QIAquick PCR purification kit (QIAGEN). The nucleotide sequence was determined directly from the PCR fragment with a PCR-based reaction using the ABI PRISM BigDye Terminator cycle sequencing ready reaction kit (PE Applied Biosystems Division) and analyzed using the PE Applied Biosystems 310 DNA sequencer (PE Applied Biosystems Division) at the Miyazaki University Gene Research Center. To determine the central region of the 16S rRNA fragment, the primers MF (5′-AAT ATT GCG CAA TGG GGGAA-3′) and MR (5′-GGC CAT GAT GAC TTG ACG TC-3′) were used for sequencing (9). Computer analyses of the DNA sequences were performed using Genetics Computer Group programs; database similarity searches were performed through the National Center for Biotechnology Information using the BLASTX algorithm.

Similarity matrices were constructed from the aligned sequence, and a phylogenetic tree was constructed based on the 16S rRNA gene sequences using the unweighted pair-group method with arithmetic averages with Genetyx-win software (version 5.0.4).

Positivity rate of blood cultures.A total of 16,743 blood culture samples were obtained during the study period, and 2,718 samples (17.7%) yielded positive culture results with various organisms containing coagulase-negative staphylococci. The positivity rate of blood culture including all types of bacteria in each institution ranged from 10.6 to 32.1% (average, 17.7%) (Table 1). Among these positive blood cultures, nine isolates were suspected to be Helicobacter and other related organisms.

Bacterial identification according to biochemical characteristics.All nine Helicobacter and other related isolates were found to be gram-negative spiral rods, and urease was negative. However, three of nine isolates were suspected to be Campylobacter spp. according to the positive growth at 25°C and 42°C and on charcoal cefoperazone deoxycholate agar (Table 2). Six other isolates were biochemically more consistent with Helicobacter than with Campylobacter based on a failure to hydrolyze hippurate, to reduce nitrate, or to grow at 42°C; we were unable to identify the species of these isolates based on these biochemical characteristics.

Identification of organisms based on 16S rRNA sequences.Identification results for isolates based on the 16S rRNA sequences are shown in Table 3. The 16S rRNA gene sequences of each suspected H. cinaedi strain, strains 5, 9, 11, 12, 14, and 16, and H. cinaedi reference strain ADN0413 revealed a high level of similarity (98 to 99%). Furthermore, the 16S rRNA gene sequences of each suspected Campylobacter strain, no. 4, 13, 15, and 17, and of Campylobacter fetus subsp. fetus reference strains also revealed a high level of similarity (98 to 99%).

Sequence homology among H. cinaedi isolates.A phylogenetic tree of the isolates was constructed based on the nucleotide sequences of the 16S rRNA genes (Fig. 1). The analysis demonstrates a high degree of sequence homology among the strains isolated.

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FIG. 1.

Phylogenetic tree based on 16S rRNA gene sequences. High degrees of sequence homology among the strains are demonstrated.

Incidence of H. cinaedi bacteremia and clinical characteristics.We finally detected six cases of bacteremia due to H. cinaedi during the surveillance study. The patients with H. cinaedi bacteremia were detected in only three hospitals (23%) of the total number of participating institutions. The rate of H. cinaedi bacteremia in the 13 institutions ranged from 0 to 0.104%. The overall rate of H. cinaedi bacteremia was 0.036% of all blood culture samples (Table 1).

Clinical characteristics.The clinical characteristics of the six cases are shown in Table 3. All patients except one (83%) were female, and the median age was 52 years (range, 17 to 71 years). At the time of diagnosis of bacteremia, all patients were hospitalized. Underlying diseases of the patients were as follows: cancer or hematologic disorder was documented for three patients (50%), and one of these patients was undergoing chemotherapy with immunosuppressant drugs. Two other patients were undergoing hemodialysis for chronic renal failure. Another patient underwent a cesarean section because of threatened premature delivery. All patients with H. cinaedi bacteremia had fever, but colitis was not seen in any of these patients. Neutropenia was present in only one case (16.6%). Bacteremia was generally a late complication during the hospital stay. All patients recovered after antimicrobial therapy, including expanded-spectrum cephalosporin and carbapenem.

Nucleotide sequence accession numbers.The 16S rRNA gene sequences determined in this study have been deposited in GenBank under the accession numbers listed in Table 2.