Comparison of the clinical and microbiological characteristics of Campylobacter and Helicobacter bacteremia: the importance of time to blood culture positivity using the BACTEC blood culture systems

Objective Campylobacter spp. and Helicobacter spp. are rare but important causes of bacteremia in humans. Distinguishing these bacteria is complicated because of their similar phenotypic profiles. We conducted clinical and microbiological investigations of Campylobacter spp. or Helicobacter spp. bacteremia. Patients diagnosed with bacteremia from 2008 to 2014 were included. The clinical and microbiological characteristics of Campylobacter spp. and Helicobacter spp. bacteremia were compared. The BACTEC system was used in blood cultures. A receiver operating characteristic curve was plotted based on the time to blood culture positivity. Results Sixteen cases of Helicobacter spp. bacteremia (patient age: 61 ± 18 years) and 14 cases of Campylobacter spp. bacteremia (patient age: 49 ± 21 years) were identified. Median time to blood culture positivity was longer for the Helicobacter spp. cases than the Campylobacter spp. cases (91.4 h vs 55.3 h, p < 0.01). A time to blood culture positivity > 75 h predicted Helicobacter spp. bacteremia with a sensitivity of 0.88 and a specificity of 0.93 (area under the receiver operating characteristic curve of 0.90). In conclusion, a time to blood culture positivity was useful in distinguishing Helicobacter spp. bacteremia from Campylobacter spp. bacteremia.


Introduction
Bacteremia due to enterohepatic Helicobacter species (HS) has frequently been reported [1][2][3][4][5][6]. Because HS resembles Campylobacter species (CS) on Gram staining, it is difficult to distinguish from CS based on positive blood cultures. Accordingly, HS have often been misidentified as CS [6]. Previous reports have noted substantial differences between HS and CS in terms of antibiotic susceptibility and clinical courses [2,7,8]; thus, microbiological identification is crucial to improve patient care. Although the use of matrix-assisted laser desorption/ ionization time-of-flight mass-spectrometry (MALDI-TOF) can help distinguish HS from CS by direct analysis of individual cultured colonies [9], HS are usually difficult to culture, and these species take about 2-3 days to grow. MALDI-TOF was used in directly identifying blood culture isolates from positive blood cultures [10]. However, no study on the identification of HS and CS on positive blood culture broths using MALDI-TOF is available. A

Open Access
BMC Research Notes *Correspondence: kyamamoto@hosp.ncgm.go.jp 1 Disease Control and Prevention Center, National Center for Global Health and Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan Full list of author information is available at the end of the article simple predictor is required in distinguishing HS from CS when blood cultures are positive of spiral-shaped bacteria. We conducted clinical and microbiological investigations of these two bacteremia, and compared HS with CS in terms of time to blood culture positivity (TTBP) to obtain a TTBP cut-off value that could be used to predict HS bacteremia.

Patients and definitions
The study used a retrospective, single-center, and case control design. The medical records of all patients at the National Center for Global Health and Medicine (779 beds, Tokyo, Japan) who had bacteremia due to HS or CS were reviewed between January 2008 and April 2014. We collected the following clinical information: age at diagnosis, sex, history of animal contact, hospitalacquired infections, underlying diseases, side effects of immunosuppressants, clinical manifestations, antibiotic treatments, and outcomes. We defined "persistent bacteremia" as constant positive blood cultures 48 h after the start of antibiotics. "Recurrence" was defined as the recurrence of bacteremia caused by the same species after the completion of an initial antibiotic course. Furthermore, cases in which the patient had stayed at the hospital for more than 2 days prior to bacteremia were classified as "hospital-acquired infections. "

Bacterial isolates
All blood culture samples were collected into standard aerobic and anaerobic culture bottles (92F, 93F, Becton-Dickinson Microbiology Systems, Sparks, MD, USA) and processed using the BACTEC 9240 or 9120 systems (Becton-Dickinson Microbiology Systems, Sparks, MD, USA). These samples were routinely monitored for at least 120 h (from January 2008 to March 2009) or 144 h (from April 2009 to April 2014). If a physician required a prolonged incubation of the blood culture, the duration was extended to 21 days (within 504-528 h). Because blood culture samples with extended incubation time were manually monitored manually and routinely checked on the 14th and 21st days, the exact TTBP in these cases cannot be determined. Instead, the TTBP was set to 144 h if the samples were positive during the extended incubation time.
A microaerobic culture was performed using modified Skirrow agar (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) and Chocolate agar (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan) at 35 °C, using Anaer-oPack for a microaerophilic environment (Mitsubishi Gas Chemical Company, Tokyo, Japan). When the bacteria could not grow on modified Skirrow agar, we used tryptic soy agar/broth with 5% sheep blood (own mixture) at 35 °C for 7 days. The grown colonies were identified by using API Campy systems (bioMérieux, Marcy l'Etoile, France), which can distinguish HS from CS through biochemical characterization.

DNA preparation and strain identification through 16S rRNA sequencing
The subspecies were further identified through molecular analysis under these two conditions: if no colony grew on the agar or if sheet-shaped colonies grew, which were suspected to be HS. DNA was extracted from fresh colonies grown on modified Skirrow agar using the hot extraction method. Bacterial 16S rRNA gene sequencing was performed as previously described [11], using the following primers: 5F (5′-TTG GAG AGT TTG ATC CTG GCT C-3′) and 1194R (

Statistical analysis
Fisher's exact test or the Mann-Whitney U test was carried out to compare the characteristics of HS and CS. The ROC curve was plotted, and the area under the ROC curve (AUC) was calculated using SPSS (version 24; IBM, New York, USA). The cut-off value was calculated by maximizing Youden's index (sensitivity + specificity − 1) [12].

Clinical characteristics
During this study period, 62,073 blood culture sets, of which 3028 sets were collected from children, were tested in our hospital. Fifty-seven (0.09%) blood cultures placed inside the bottles with aerobic organisms were positive of spiral-shaped bacilli. Thirty patients were diagnosed with bacteremia based on the presence of spiral-shaped bacilli. All patients' characteristics and outcomes are shown in Table 1. The most common underlying disease was solid organ cancer, particularly lung cancer. Skin lesions were common in HS bacteremia, whereas diarrhea was a common symptom of CS bacteremia. Other rare infectious clinical syndromes included graft vessel infection due to H. cinaedi/H. bilis, cellulitis due to C. jejuni, and acute obstructive cholangitis due to C. jejuni.
Twenty-eight (93%) patients were treated with antibiotic, of which 25 patients (89%) received antibiotics before identification of the bacteria. During the initial therapy, 93% of the patients were treated using monotherapy, particularly with beta-lactam agents or carbapenem. The combination therapy of amoxicillin and doxycycline was used in 10 patients with HS bacteremia (62.5%). The duration of antibiotic use was significantly longer for patients with HS bacteremia those with CS bacteremia (p < 0.001). Two patients had persistent HS bacteremia despite antibiotic treatment, and 3 patients experienced recurrence of HS bacteremia (Table 2). These 3 patients did not experience the third recurrence. Five patients with HS and 3 patients with CS died during the study period. However, none of these patients died because of infection-related causes.

Microbiological characteristics
Of 57 blood cultures, 36 were positive for HS and 21 were positive for CS. The HS were H. cinaedi (n = 30), In a subsequent analysis of the results of all isolations, we found that TTBP had an AUC of 0.88 (p < 0.001) for the prediction of HS bacteremia (Fig. 1b). Additionally, TTBP > 75 h predicted HS bacteremia with a sensitivity of 0.83 and a specificity of 0.91.

Discussion
The TTBPs of HS and CS significantly varied in our analyses of both the initial and all isolations included in our study. TTBP was a good parameter for distinguishing these 2 types of spiral-shaped bacteria. A 5-day median TTBP for H. cinaedi was reported in a recent study that used a BACTEC system for blood cultures [1]. Additionally, the median TTBP for C. jejuni was previously reported to be 5 days [13], which is longer than the TTBP in our study (median 2.3 days). Differences in the results of the present study may be due to the blood culture of C. jejuni that was implemented using an older BACTEC system as previously reported. Although TTBP was a good predictor for HS or CS bacteremia when blood culture becomes positive, MALDI-TOF is still among the most rapid and accurate identification methods. Grown bacterial colonies are used in identifying the type of bacteria using MALDI-TOF, and Winkler et al. reported that this method was useful for HS and CS [9]. However, bacterial colonies of both HS and CA take about 2-3 days to grow. HS was more difficult to grow on medium than CS [7]. Similarly, more than half of the HS were not grown in our study (data not shown). Several studies about the identification of microorganisms using MALDI-TOF from direct positive blood culture broths [10] or direct positive blood culture subsequent to short incubation on solid medium are available [14]. Although HS may be distinguished from CS when blood culture becomes positive, the efficacy of these methods used in identifying HS and CS have not been verified. Moreover, microbiological laboratories in several hospitals do not use MALDI-TOF because of its cost. In conclusion, a TTBP cut-off of > 75 h was useful in distinguishing bacteremia due to HS from bacteremia due to CS when using the BACTEC systems.
Although there are no guidelines for the treatment for HS bacteremia, many antibiotic agents have been used successfully, both alone and in combination [7]. Although the breakpoint of H. cinaedi has not been established, it has been reported that the MICs of tetracyclines, carbapenems, and aminoglycosides are relatively low, and that those of ampicillin and cephalosporins are moderate. In contrast, MICs of macrolides and fluoroquinolones are relatively high for H. cinaedi [3,4,7]. Unfortunately, drug susceptibility testing for Helicobacter spp. could be performed for only one strain that was isolated from the patient without recurrent or persistent bacteremia. It was only possible to test a single strain because it was difficult to culture Helicobacter spp. on the plate and to interpret the susceptibility test result. In all cases of recurrent or persistent HS bacteremia, patients had only been treated with beta-lactam agents prior to recurrence or persistent bacteremia. Although we suspect that the MICs of the antibiotics were related to the prognosis of HS, further studies are needed in order to clarify this relation.

Limitations
This study had two major limitations. First, our assessment of TTBP as a means of distinguishing HS from CS is only applicable to the BACTEC systems. Other automated blood culture systems, such as the BacT/Alert system, have early HS TTBPs and lower detection rates than the BACTEC systems [7]. Consequently, the TTBPs of HS and CS may differ from the results of our study when other automated blood culture systems are used. Second, C. fetus was not detected in our institute. The clinical manifestations of bacteremia due to C. fetus differ from those of bacteremia due to other CS [8]. Therefore, the clinical manifestations that were reported in the present study may differ from those of other institutes where C. fetus is frequently detected. However, because C. fetus do not have a longer TTBP than other CS, the usefulness of TTBP in distinguishing spiral-shaped bacteria may not be affected. Authors' contributions KY1 analyzed and interpreted the patient data. KM and KY2 performed the microbiological examination and collected the data of all the strains. MN, KS, and TK performed the molecular microbiological examination. KY1, KH, SK1, and NO were the major contributors in writing the manuscript. All authors read and approved the final manuscript. We designated Kei Yamamoto, Koji Yamada, Satoshi Kutsuna, and Shuzo Kanagawa as KY1, KY2, SK1, and SK2, respectively. Fig. 1 ROC curve demonstrating the TTBP used to diagnose bacteremia caused by spiral-shaped bacteria. ROC obtained from the results that were positive for spiral-shaped bacteria, of the first isolation from each patient (a) and those of all isolations (b) for TTBP to diagnose bacteremia due to Helicobacter spp. AUC, area under the receiver operating characteristic curve