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编号:11200919
Direct Detection of Cardiobacterium hominis in Ser
     ABSTRACT

    Bacterial DNA was detected directly in the serum of a patient with endocarditis by broad-range 16S rRNA PCR followed by sequencing and analysis of the results by the BLAST search. Using these methods, Cardiobacterium hominis was identified in 2 days from the date of serum collection. The microorganism was also isolated and identified using conventional methods (bacterial culture and biochemical tests) 17 days from the date of sample collection. This is the first report showing the direct detection of C. hominis in a patient's serum using molecular-based methods, emphasizing their potential usefulness as additional and rapid diagnostic tools for the detection and identification of fastidious bacteria.

    CASE REPORT

    A 62-year-old man with a past history of arterial hypertension was admitted to our department because of a 3-week history of poor dietary intake, fatigue, weakness, diffuse abdominal discomfort, chills, and night sweats. His symptoms began after a short period of diarrhea and fever. He also reported a history of toothache and gum bleeding 4 weeks prior to admission. One week prior to his admission he was seen by his primary physician, who diagnosed viral infection. He had no previous history of cardiac murmur, heart disease, intravenous drug use, recent dental work, skin injury, or previous hospitalizations.

    On admission, physical examination revealed a temperature of 39°C, a heart rate of 105 beats per minute, 25 respirations per minute, and blood pressure of 160/70 mm Hg. He appeared pale and moderately ill but was not in acute distress. He had no subconjunctival, palatal, or subungual splinter hemorrhages, and his ocular fundi were normal. Dental caries was noted. There was no palpable peripheral adenopathy, but a spleen tip and a palpable liver were felt. Auscultation of the heart revealed a grade 2 of 6 at the apex of heart, without S3 sound or signs of heart failure. The remainder of the physical examination was unremarkable.

    A complete blood count revealed a white-blood-cell count of 14,400 cells/mm3, with 87% neutrophils, 5.5% lymphocytes, and 7.5% monocytes. Hemoglobin was 11.5 g/dl, and the platelet count was 262,000/mm3. Renal function testing revealed normal creatinine and blood urea levels. At that time, the erythrocyte sedimentation rate (ESR) was 62 mm in the first hour, C-reactive protein was 14.33 mg/dl, and fibrinogen was 597 mg/dl. Arterial blood gas analysis showed respiratory alkalosis with pH 7.503, 69 mm Hg PaO2, 26.2 mm Hg PaCO2, and 20.8 mmol/liter HCO3. A urine specimen was positive for hemoglobin (+++) and protein (+); the sediment contained 4 to 6 red blood cells, 1 to 2 white cells per high-power field, and granular casts. Mild proteinuria (636 mg/24 h) was also present. His electrocardiogram showed sinus tachycardia. At entry, a two-dimensional trans-thoracic echocardiography showed normal valves without any obvious sign of valve vegetations and a left ventricular ejection fraction of 65 to 70%. Additional investigation included an abdominal ultrasonography, which confirmed the presence of mild splenomegaly. Serology tests for brucellosis, leptospirosis, viral infections, and leishmaniasis tested negative, as did cultures for tuberculosis bacillus.

    Infective endocarditis was suspected, and a series of six blood cultures was taken before starting empirical treatment with intravenous teicoplanin (400 mg every 12 h [q12h] for the first day and 400 mg q24h thereafter), ceftriaxone (2 g q12h), and ampicillin-sulbactam (3 g q6h). Twenty milliliters of whole blood from three different sites was obtained; samples were collected in 2-h intervals. Ten milliliters of blood from each syringe was injected into an aerobic blood culture bottle (BACTEC Plus+ Aerobic/F; Becton Dickinson, Sparks, MD), and the remaining 10 ml was injected into an anaerobic bottle (BACTEC Plus+ Anaerobic/F; Becton Dickinson, Sparks, MD) by using the BACTEC 9120 System (Becton Dickinson). Furthermore, 10 ml of whole blood was collected in rubber-sealed pyrogen-free tubes for direct detection of bacterial DNA (Endo Tube ET; Chromogenix AB, Vienna, Austria). According to our protocol, molecular methods detecting bacterial DNA directly in the serum of patients with serious infections (such as meningitis, endocarditis, etc.) are applied on a routine basis (6, 8, 24). Serum was obtained by centrifuging blood at 3,500 rpm for 10 min. DNA was extracted using a QIAmp DNA Mini kit (QIAGEN, Hilden, Germany) according to the instructions of the manufacturer. The yield and purity of DNA were measured by reading A260 and A260/A280 in a BioPhotometer (Eppendorf). PCR for the complete amplification of the 16S rRNA gene was carried out using the universal primers 5'-AGAGTTTGATCATGGCTCA-3' (forward; located at positions 8 to 27) and 5'-ACGGCGACTGCTGCTGGCAC-3' (reverse; positions 531 to 514) (18). Five microliters of template, corresponding to 200 ng of total DNA, was analyzed for the presence of bacterial DNA. As a positive control, 5 μl of template infected by a known concentration of Escherichia coli DNA (50 pg) was used; the positive results demonstrate that this protocol prevents PCR inhibitors. PCR was carried out through the cycles as follows: an initial cycle of 95°C for 4 min was followed by 35 cycles of 94oC for 30 s, 55oC for 30 s, and 72oC for 90 s, with a final extension period at 72°C for 10 min. Total PCR volume was filtered through QIAquick Spin Columns to remove primers and nucleotides. Purified products (5 μl) were visualized on 1.5% agarose gels stained with ethidium bromide. A band of approximately 520 bp was obtained, corresponding to the specific amplification of the prokaryotic 16S rRNA gene. PCR amplicons were sequenced in both directions using the pair of primers described above by the ABI PRISM BigDye Terminator v3.1 Cycle Sequencing kit and ABI PRISM 310 automated sequencer according to the manufacturer's instructions, and the obtained sequences (approximately 460 bp) were compared with 16S rRNA sequences available both in the Ribosomal Database Project and the GenBank and EMBL databases obtained from the National Center for Biotechnology Information by an advanced BLAST search (18, 19). The isolated bacterial DNA sequence was compared with all the available sequences of microorganisms and demonstrated the following results: 100% identity with C. hominis 16S rRNA gene sequence (GenBank accession no. M35014), 99% with C. hominis (GenBank accession no. AY360343), 97% with Cardiobacterium sp. A (GenBank accession no. AF144697), 97% with Cardiobacterium sp. B (GenBank accession no. AF144696), 94% with Cardiobacterium valvarum sp. nov. (GenBank accession no. AY596468), 93% with Cardiobacterium valvarum (GenBank accession no. AY596470), 93% with Cardiobacterium valvarum (GenBank accession no. AY596469), and 93% with Cardiobacterium valvarum (GenBank accession no. AF506987) (9, 10, 11). Organisms with less than 97.5% identity in 16S rRNA sequences are usually considered separate species. Of note, the results were available within 2 days from the date of sample collection. The analytical sensitivity of this method was sufficient; after DNA extraction from serial dilutions of the serum (up to 100,000), PCR showed a positive band of up to 10,000-fold dilution, which corresponded to 20 pg of total DNA.

    C. hominis is rarely reported to be resistant to penicillin, and penicillin or ampicillin has been considered the drug of choice for treating infective endocarditis caused by this organism (14). However, the emergence of beta-lactamase-producing C. hominis isolates (14) led us to continue antibiotic therapy with ceftriaxone and ampicillin-sulbactam, and 4 days later the patient's symptoms improved considerably, with absolute resolution of the patient's fever and night sweats. However, a new transesophageal echocardiography during the second week of hospitalization revealed the presence of small vegetation in the aortic valve but without significant hemodynamic insufficiency.

    Blood cultures, simultaneously obtained with the serum sample, remained negative after 5 days of routine incubation; however, after a prolonged incubation of 7 more days (12 days total incubation), a positive reading was obvious in the aerobic vials, indicating the presumptive presence of viable microorganisms. Subcultures from the positive vials on Mueller-Hinton agar supplemented with 5% sheep blood (BioMerieux, La Balme les Grottes, France) at 37°C in an atmosphere of 5% carbon dioxide (CO2) showed the growth of a gram-negative bacillus after 3 days of incubation (15 days total from the date of collection). The colony was tiny, opaque, and circular. Biochemical tests including catalase, oxidase, indole, arginine dihydrolase, nitrate, esculin hydrolysis, ornithine decarboxylase, alkaline phosphatase, and fermentation of glycose, lactose, sucrose, xylose, maltose, and mannitol were performed in conventional tube media (BBL; Becton Dickinson). After 2 days, the microorganism showed a weak positive reaction to oxidase and indole and slight fermentation with glucose, sucrose, and maltose. These results were slightly different from those described for C. hominis (oxidase positive, indole positive, fermentation of glucose, sucrose, maltose, and mannitol). Along with the usual phenotypic studies, molecular identification based on 16S rRNA gene and sequencing analysis as described previously was performed using as template DNA extracted directly from four to five colonies of the microorganism. As anticipated, the identification verified that the microorganism isolated from the culture belonged to a C. hominis species. Although the conventional tests are easily applied in a clinical setting, they are rather time-consuming in cases of slow-growing bacteria because the positivity of the tests is/ significantly dependent on bacterial inocula. We note that in our case the final bacterial culture results were available 17 days from the date of the collection of the samples.

    As mentioned above, C. hominis strains that produce beta-lactamase have been previously reported (14). In order to investigate whether our strain produces beta-lactamase or not, a nitrocefin-based test (Cefinase disk; BBL Microbiology Systems) was used; our strain was not a beta-lactamase producer. Unfortunately, so far neither criteria for the susceptibility breakpoints nor the correct medium or atmosphere and time of incubation have been established for this microorganism; however, in order to acquire some experience on the MICs of various antimicrobials agents for our strain, MIC determination was performed by E-test (Biodisk, Solna, Sweden) on nonstandardized blood Mueller-Hinton after 48 h of incubation in a CO2 chamber as described by Han and Falsen (10). The MICs of antibiotics for our strain were as follows: penicillin, 0.25 μg/ml; amikacin, 1.5 μg/ml; ceftazidime, 0.125 μg/ml; ciprofloxacin, 0.064 μg/ml; imipenem, 0.19 μg/ml; ticarcillin-clavulanate, 0.094 μg/ml; ceftriaxone, 0.125 μg/ml; ampicillin-sulbactam, 0.064 μg/ml; and trimethoprim-sulfamethoxazole, 0.023 μg/ml. According to the results obtained by E-test, we continued only ampicillin-sulbactam administration for a total of 6 weeks, with very good outcome for the patient (aortic valve replacement was not needed).

    We describe the first report of infective endocarditis due to C. hominis in Greece (13); after a prolonged blood culture incubation of 12 days, the microorganism was isolated in solid media 3 days later (a total of 15 days), while the final bacterial culture results after phenotypic tests (a further 2 days) were available in 17 days from the date of sample collection. However, using a broad-range 16S rRNA PCR assay followed by sequencing and analysis of the results by a BLAST search, we were able to identify the causative agent of the infection directly in the serum of the patient only 2 days after serum collection. To the best of our knowledge, this is the first report showing the direct and rapid detection of C. hominis in the serum of a patient with endocarditis using a molecular method.

    Cardiobacterium hominis is a small, gram-negative coccobacillus which is part of the normal human oropharyngeal flora, but upon disturbance of the mucosal integrity it can become invasive and pathogenic (21, 23). The organism is a known but rare cause of endocarditis; it is notorious for causing apparently culture-negative endocarditis, as are the other members of the HACEK group (i.e., Haemophilus species, Actinobacillus actinomycetemcomitans, Eikenella corrodens, and Kingella kingae), since their growth requires special media and prolonged incubation (2, 3, 4, 5, 13).

    C. hominis is rarely the cause of human infection and was first isolated from four patients with infective endocarditis in 1962 (22). Since then, fewer than 70 cases of C. hominis endocarditis have been reported in the literature. In addition, it is unusual for this organism to cause focal infections outside the vascular system, including bacterial meningitis, abdominal abscess, osteomyelitis, pericarditis, and septic arthritis (1, 7, 12, 17). In most of the cases of infective endocarditis caused by C. hominis, there is some form of preexisting cardiac disease and a history of dental manipulation, although for most cases no portal for entry can be identified (23). The illness has a characteristically insidious onset, with the presence of low-grade fever that often affects the middle-aged population, but stigmata of endocarditis such as splenomegaly, anemia, and hematuria, as found in our patient, are usually already present during examination, while the ESR is, typically, moderately raised (23). In our case, the presence of dental caries along with his past history of gum bleeding and the findings of physical and laboratory investigations prompted us to suspect infective endocarditis as the most likely diagnosis besides the absence of preexisting heart disease.

    C. hominis tends to form large, friable vegetation on endocardial surfaces which are associated with a significant risk of cerebral embolization (30%) or mycotic aneurysm formation (10%), both of which appear to be more common in C. hominis endocarditis than with endocarditis caused by other gram-negative bacilli (20, 23). However, in our case small vegetation in the aortic valve without significant hemodynamic insufficiency was found during the second week of hospitalization. In addition, aortic valve replacement was not needed and the above-mentioned frequent complications were not observed in our patient. It seems rational that suggesting infective endocarditis as the most likely diagnosis as well as the immediate administration of antibiotics against gram-negative and -positive microorganisms resulted in a favorable outcome for the patient.

    In conclusion, although the detection of C. hominis in the resected heart valve materials has already been reported (8, 15, 16), this is the first report of direct detection of the above-mentioned bacterium in the serum of a patient with endocarditis. C. hominis has historically been associated with culture-negative infective endocarditis due to its fastidious growth requirements, but recent improvements in blood culture systems and blood culture medium formulations have resulted in improved recovery of this pathogen. However, the sensitivity of the new blood culture systems probably depends on the number of bacterial cells that are circulating in the blood at the time of sample collection (3, 5). Nevertheless, even in our case the final bacterial culture results using improved culture systems were available 17 days from the date of sample collection. In this context, the use of 16S rRNA broad-range PCR combined with direct DNA sequencing from patient serum could be an alternative molecular-based procedure in an attempt to identify rapidly—in this case in 2 days—the causative pathogen in cases of difficult and serious infections due to fastidious microorganisms. In addition, this method can discriminate C. hominis from C. valvarum, which has recently been found to be responsible for endocarditis, but biochemical tests have failed to identify this species because of its phenotypic similarities with C. hominis (9, 11).

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    Department of Medicine

    1 Department of Microbiology, Medical School, University of Thessaly, Larissa, Greece(N. Gatselis, E. Malli, G.)