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Actinomyces graevenitzii is a newly recognized Actinomyces species that is seldom isolated from clinical specimens. A case of multiple pulmonary abscesses mimicking acute pulmonary coccidioidomycosis is described in this study, and the findings indicate that this organism is an opportunistic human pathogen.

We report what is believed to be the first case of pulmonary Actinomyces graevenitzii infection presenting as organizing pneumonia. Fever and night sweats developed in a 69-year-old male. The only abnormal laboratory data were an elevated erythrocyte sedimentation rate and C-reactive protein level. On chest images, multiple consolidations with air bronchograms were seen in the bilateral lungs. Histological examination from lung biopsy revealed a pattern of organizing pneumonia with microabscesses, but definitive diagnosis was not obtained because culture from lung specimen was negative. A. graevenitzii was eventually identified in the lung biopsy specimen by detection of an Actinomyces-specific PCR product followed by 16S rRNA gene sequencing. The patient was treated with high-dose ampicillin intravenously for 1 month, followed by oral amoxicillin and clarithromycin for 6 months, and recovered. We suggest that actinomycosis can present as organizing pneumonia, and identification of infection by PCR analysis and rRNA gene sequencing is a useful strategy in cases that are difficult to diagnose.

We report an extremely rare case of urosepsis caused by Globicatella sanguinis and Corynebacterium riegelii coinfection in a 94-year-old Japanese man with nephrolithiasis. Prompt identification of this coinfection is important so that effective antimicrobial coverage can be initiated.

Purpose: To report the predisposing factors, patient background, treatment and outcomes of ocular infections caused by Paecilomyces spp. in Japan., Methods: We reviewed the medical records and the published literature in Japan on ocular infections caused by microbiologically-proven Paecilomyces spp. Nineteen eyes of 18 patients; 4 eyes of 4 of our patients and 15 eyes of 14 published Japanese patients were studied., Results: Ten men and 8 women (9 OD, 8 OS, 1 OU) were diagnosed with ocular infections caused by Paecilomyces spp. The mean age was 69 years (range 33 to 90 years). The initial diagnosis of ocular infection caused by Paecilomyces spp. was keratitis in 14 eyes and endophthalmitis in 5 eyes. The final diagnosis was keratitis in 2 eyes, keratitis with involvement of the anterior segment of the eye, i.e., hypopyon or corneal rupture, in 12 eyes, and endophthalmitis in 5 eyes. The predisposing factors had a history of ocular surgery, corneal trauma and soft contact lens use. Other factors were diabetes and old age. The infections developed more often in the fall and winter. The identified Paecilomyces spp. had a high sensitivity to micafungin and voriconazole, but low sensitivity to amphotericin B, flucytosine and fluconazole. The prognosis of Paecilomyces spp. ocular infections was very poor, viz., final vision was counting fingers or worse in 60%, ocular perforation in 42%, and eye enucleation in 11%. The percentage of eyes with a final visual acuity worse than counting fingers was 0% in those with keratitis, 50% in those with involvement of the anterior segment, and 100% in those with endophtalmitis (p = 0.0446). Among the antifungal agents, the percentage of cases with final vision of counting fingers or worse was 90% with fluconazole, 80% with itraconazole, 100% with miconazole, and 71% with voriconazole., Conclusion: The results show that if the Paecilomyces spp. infection spreads intraocularly, it is difficult to mitigate the clinical damage even with antifungal agents with high sensitivity.

Infective endocarditis (IE) is traditionally diagnosed by microbiological analysis of blood cultures, following which therapeutic antibiotics are chosen based on antimicrobial sensitivity tests. However, such conventional techniques do not always lead to an accurate etiological diagnosis. Recently, PCR analysis of the 16S rRNA gene has been employed to identify organisms isolated from excised heart valves. In this study, we analyzed 19 valve samples from patients with confirmed IE, as identified by Duke's criteria. Using broad-range PCR amplification, followed by direct gene sequencing, pathological agents were identified in all samples. Although blood cultures yielded negative results in 4 cases, PCR analysis of valve samples showed positive identification of causative organisms. In 3 cases, there was a difference between blood culture and PCR in identification of pathological agents, which are likely to be misidentified by the conventional method based on the phenotypic database. Postoperative antibiotics were chosen considering the severity of lesions and the results of PCR, Gram staining, and valve cultures. All patients were cured without relapse. The broad-range PCR method was therefore beneficial for the management of IE because it enabled us to identify pathogens directly from the site of infection, even organisms that were difficult to culture or likely to be misidentified by the conventional culture method. Identification of the agents provided precise knowledge of the microbiological spectrum involved in the cases of IE.

Nocardia elegans infection in humans is rare and is predominantly associated with pulmonary infections. We describe the first case of N. elegans infection associated with purulent arthritis in humans. The patient was a 66-year-old woman without underlying disease. She had swelling in her left ankle that was increasing in size, but it did not cause the patient substantial pain. Punctual discharge was collected for Gram staining and Kinyoun's acid-fast staining. The results of microscopic findings were suggestive of the genus Nocardia. The 16S rRNA sequence of the isolate was completely identical (100%) with that of N. elegans, indicating that the isolate was N. elegans. All the previously reported 4 cases of N. elegans infection in humans were associated with respiratory infections; we present the first case of the infection involving purulent arthritis.

We encountered a case of neonatal meningitis caused by Streptococcus gallolyticus subsp. pasteurianus. The patient was an 8-day-old boy. Gram staining of the cerebrospinal fluid (CSF) revealed gram-positive cocci in pairs or in short chains. In culture, γ-streptococcus-like colonies grew. The result of 16S rRNA sequence analysis identified S. gallolyticus subsp. pasteurianus. From these results, bacterial meningitis was diagnosed and, as a result of antimicrobial susceptibility testing, single-dose ampicillin therapy was given. Because inflammatory deterioration and spread was suspected from the CSF test results, this therapy was added by panipenem/betamipron. In response to his recovery, antibiotic treatment was stopped and the boy was discharged. This bacterium was classified as S. gallolyticus subsp. pasteurianus in the latest report in 2003. Since this change, there have only been a few cases of neonatal meningitis caused by this bacterium. Here we report this rare case.

We report a case of sepsis caused by Rhodococcus corynebacterioides, identified using 16S rRNA gene sequencing, in a myelodysplastic syndrome patient who had undergone hematopoietic stem cell transplantation. This is the first report of R. corynebacterioides infection in a human.

Malassezia furfur, an etiological agent of catheter-associated fungemia, requires long-chain fatty acids for in vitro growth. We examined the applicability of rDNA sequence analysis, autoaggregation testing in liquid culture, utilization of parenteral lipid emulsions, and phospholipase activity for discrimination of catheter-associated M. furfur strains. The rDNA sequence types of catheter-associated M. furfur strains were distinct from those of other isolates. All M. furfur isolates recovered from blood culture bottles and the tips of catheters from patients receiving fat emulsion therapy were type I-3. Only M. furfur isolate GIFU 01 from a blood culture bottle showed no autoaggregation in liquid culture. All strains of M. furfur examined grew well on Sabouraud's dextrose agar supplemented with Intralipid lipid emulsion as compared to individual Tweens (20, 40, 60, 80) and Cremophor EL. A high percentage of type I-3 M. furfur strains (80.0%) showed very high phospholipase activity compared to type I-1 and I-4 strains obtained from healthy skin of the same subjects or healthy control subjects (20.0% and 0.0%, respectively). The blood culture bottle isolate GIFU 01 showed very high lipolytic enzymes activity for Intralipid but no phospholipase activity. These results suggest that particular factors, such as non-autoaggregation and very high lipolytic enzyme activity for parenteral lipid emulsions, play important roles in the growth and pathogenicity of Malassezia-related sepsis.

A 62-year-old man presented with general fatigue. He was diagnosed with septic shock and severe pneumonia. The sputum at admission yielded methicillin-sensitive Staphylococcus aureus (MSSA) strain and methicillin-resistant S. aureus (MRSA) strain. Despite antibiotic treatment, he did not improve. A chest computed tomography (CT) revealed multilobar cavity lesions. Only MRSA strain was confirmed at that time. We diagnosed him with necrotizing pneumonia. Despite treatment with vancomycin, his pneumonia worsened and he died. At autopsy, many gram-positive cocci were observed in the lungs. The clinical presentation of our patient was different from typical CA-MRSA-mediated necrotizing pneumonia.

Aerococcus urinae is a endocarditis rare causative organism with low virulene. We report an A. urinae endocarditis case treated by aortic valve replacement. An 80-year-old woman hospitalized for urinary tract infection and hydronephrosis due to three-week renal calculi. Blood culture on admission isolated Streptococcus acidominimus. During the course, she was transferred to our care for surgical intervention after developing congestive heart failure due to severe aortic regurgitation. Echocardiographic findings indicated infective endocarditis. She underwent aortic valve replacement, and gram staining of the resected valve tissue showed gram-positive cocci, although valve culture was negative. PCR amplification and DNA sequencing using the valve material matched an A. urinae sequence. The woman recovered and was discharged six weeks after antibiotic treatment.

We describe a 35-year-old man with X-linked agammaglobulinemia who had refractory chronic pleurisy caused by a Helicobacter equorum-like bacterium. Broad-range bacterial PCR targeting the 16S and 23S rRNA genes and in situ hybridization targeting the 16S rRNA gene of H. equorum confirmed the presence of this pathogen in a human for the first time.

Seven isolates of a slowly growing, non-chromogenic Mycobacterium species were obtained from sputum and bronchial lavage fluid samples from elderly patients in different regions of Japan. These isolates were distinguished from related non-tuberculous species by colony morphology, positive results for Tween hydrolysis, catalase at 68 °C, nitrate reductase and pyrazinamidase and negative results for semi-quantitative catalase, urease and arylsulfatase. The mycolic acid pattern obtained by HPLC revealed a single cluster of late-eluting mycolic acids similar to but different from those of Mycobacterium malmoense ATCC 29571(T). The 16S rRNA gene, 16S-23S internal transcribed spacer (ITS), rpoB and hsp65 sequences were unique in comparison with those of other mycobacteria. Comparison of 16S rRNA gene sequences showed that the isolates were most closely related to Mycobacterium tuberculosis H37Rv(T) (21 base differences in 1508 bp; 98.6 % 16S rRNA gene sequence similarity). A representative strain, GTC 2738(T), showed 91.9 % rpoB sequence similarity with Mycobacterium marinum strain M, 95 % hsp65 sequence similarity with Mycobacterium kansasii CIP 104589(T) and 81.1 % 16S-23S ITS sequence similarity with Mycobacterium gordonae ATCC 14470(T). Phylogenetic analysis of concatenated sequences of the 16S rRNA, rpoB and hsp65 genes showed that strain GTC 2738(T) was located on a distinct clade adjacent to M. tuberculosis, M. ulcerans and M. marinum, with bootstrap values of 81 %. DNA-DNA hybridization demonstrated less than 70 % reassociation with type strains of genetically related species and supported the novel species status of the isolates. On the basis of this evidence, a novel species with the name Mycobacterium shinjukuense sp. nov. is proposed. The type strain, isolated from a sputum sample, is strain GTC 2738(T)( = JCM 14233(T) = CCUG 53584(T)).

An 82-year-old woman with a past history of a colectomy for colon cancer was admitted to our hospital because of fever and cough. Her chest radiologic images on admission showed bilateral pulmonary infiltration, which suggested community-acquired pneumonia. However, the results of gram and acid-fast staining demonstrated branching acid-bacilli, which suggested pulmonary nocardiosis. We administered sulfamethoxazole-trimethoprim, and her condition and radiology findings promptly improved. Nocardia cyriacigeorgica was identified from her sputum. One of the nocardial isolates, formerly identified as Nocardia asteroides, has recently been reclassified as Nocardia cyriacigeorgica by the 16S rRNA gene amplification method. Pulmonary nocardiosis may be misidentified as "aspiration pneumonia", especially in elderly people. It is important for patients with community-acquired pneumonia to be evaluated with sputum gram staining for accurate diagnosis.

Bacillus cereus is becoming an important nomosomial pathogen because of frequent isolation from blood cultures and from severe systemic infections. To differentiate highly pathogenic outbreak strain of B. cereus from other sources of the Bacillus cereus, we attempted to analyze their dnaJ sequences. Assays indicated that dnaJ sequence similarity of all of 52 blood culture isolates of B. cereus ranged from 92.8% to 100%. The distance between B. anthracis and B. cereus except six outbreak isolates ranged from 3.8% to 6.4%. The dnaJ sequences of six outbreak strains of B. cereus (GTC 02891, GTC 02896, GTC 02916, GTC 02917, GTC 03221, and GTC 03222) were closely related to those of B. anthracis (99.2%-99.5% sequence similarity). Ba813 sequences were only found in the six outbreak strains of B. cereus. The other pathogenic factors of B. anthracis were not found in these six outbreak strains, with the exception of GTC 02891 (cap-positive). The six outbreak strains formed clear β-hemolytic colonies on a sheep blood agar plate. Our findings suggest that outbreak strains of B. cereus isolated from blood cultures are likely to have the risk of causing serious infection, and dnaJ and Ba813 are important markers to identify such strains. Phylogenetic analysis of dnaJ and MLST revealed that the six outbreak strains of B. cereus are closely related to B. anthracis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Bartonella quintana, known to cause various clinical symptoms, is increasingly recognized as one important cause of culture-negative endocarditis. We report a case of infectious endocarditis with B. quintana on the prosthetic valve, accompanied by proteinase 3-antineutrophil cytoplasmic antibody-positive collagen vascular disease-like symptoms 1 year earlier., (Copyright © 2011 Elsevier Inc. All rights reserved.)

An 11-year-old girl with metastatic neuroblastoma developed recurrent bacteremia during sustained neutropenia after autologous peripheral blood transplantation. All febrile episodes of bacteremia were caused by single Delftia acidovorans strain revealed by ERIC-PCR. This strain became resistant to broad-spectrum penicillins and cephalosporins through antibiotic treatments. Removal of the indwelling vascular catheter resulted in resolution of the infection. So far as we know, this is the first report of vascular catheter-related D. acidovorans bacteremia in Japan.

Splenic abscess is relatively uncommon. Infections caused by lactobacilli, which are usually one of the constituents of normal flora of human gut and vagina, are also very uncommon. Here we report a case of splenic abscess caused by Lactobacillus paracasei. We performed a literature review for this rare entity. Immunocompromised status of the patient, who had uncontrolled diabetes, could have contributed to the pathogenesis of this rare disease.

A 44-year-old man presented with a very rare case of skull base osteomyelitis manifesting as persistent diplopia. He initially had the symptom with fever after dental extraction. Biopsy from the cervix and upper pharynx performed in a previous hospital had showed negative findings by histological and bacterial examinations. Magnetic resonance (MR) imaging disclosed enhanced lesions in the right cavernous sinus, clivus, and right cervical regions. Computed tomography revealed osteolysis, and fluorodeoxyglucose positron emission tomography (FDG-PET) showed areas of increased uptake. Bacteriological examination of the isolated clival lesion disclosed Actinomyces israelii, and he was treated with intravenous penicillin, 18 million units a day for 6 weeks. MR imaging revealed reduction of intensity in the enhanced areas, and FDG-PET showed disappearance of the increased uptake. After 6 months of oral antibiotics administration, MR imaging disclosed disappearance of the enhanced lesions, and the patient had no sign of neurological deficits. Skull base osteomyelitis resembles neoplasm or inflammatory disease of this region in neurological and radiographic findings. The biopsy specimen should be ideally obtained from an isolated region, and prepared to identify a wide range of organisms and to differentiate other diseases. The serum level of C-reactive protein and FDG-PET are useful to follow up the efficacy of antibiotic therapy.

We report three cases of acute conjunctivitis due to different types of human adenovirus (HAdV) in medical workers. Case 1: A 37-year-old man had epidemic keratoconjunctivitis and urethritis caused by human adenovirus type 37 (HAdV-37). Case 2: A 27-year-old woman had acute conjunctivitis due to human adenovirus type 15 (HAdV-15), Case 3:A 32-year-old woman had acute follicular conjunctivitis leading to a serotype of human adenovirus-53 serotype (HAdV-53). Note that HAdV-37 infection may cause keratoconjunctrivitis and urethritis. HAdV-15, -37, and -53 are important causative agents of nosocomial outbreak, and the application of a rapid diagnostic kit or PCR to a rapid diagnosis and proper infection-control measures can significantly reduce infection spread.

A 76-year-old man was admitted to our hospital with dyspnea. Chest CT revealed bronchial stenosis caused by mediastinal lymphadenopathy. An FDG-PET scan showed increased FDG uptake in the mediastinal lymph nodes, lumbar vertebrae and left ilium. Neither the first biopsy specimen from the left ilium nor the endobronchial lesion showed malignant cells, but both were positive on acid-fast bacillus staining. Genetic testing found the Mycobacterium avium (MAC) gene. Therefore, we diagnosed disseminated MAC infection, and started antibiotic therapy. However, he did not respond to the therapy, and his bronchial stenosis worsened. We performed a biopsy of the newly-appearing supraclavicular lymph node, and of the left ilium again, and confirmed a new diagnosis; diffuse large B cell lymphoma, coexistent with disseminated MAC infection. This case suggests not only the simultaneous occurrence of disseminated MAC infection and diffuse large B cell lymphoma, but also the coexistence of both diseases within the same organs, and that there may be some relationship between the two diseases.

A 53-year-old woman was admitted to our hospital with dysbasia and forgetfulness. Her past history included uveitis at age 39. Medical examinations led to a diagnosis of neurosarcoidosis. Although she was treated with prednisolone, her symptoms remained, so she received steroid pulse therapy twice, and administration of azathioprine. In early January 2007, a chest X-ray film showed nodules in the right upper lung that rapidly increased in size and number. A CT scan revealed multiple nodules including cavitary lesions in both lung fields. Examination of bronchial lavage fluid and a transbronchial lung biopsy showed a mycelium-like gram-negative filament. After the treatment with benzylpenicillin for 1 month, her laboratory data and radiological abnormalities markedly ima proved. However, switching to oral administration of amoxicillin caused the regrowth of the nodules. She was retreated with intravenous benzylpenicillin for 8 weeks, followed by oral administration of amoxicillin for 5 months, and her condition completely resolved. The causative organism was identified as Rothia aeria (described in 2004) by 16S rRNA gene sequencing. This is the first report of a case of pulmonary infection with this species.

Scedosporium prolificans has been increasingly recognized as an etiological agent of disseminated mycelial infections in profoundly immunocompromised patients. Reported herein is a case of disseminated S. prolificans infection in a patient undergoing anti-neoplastic chemotherapy for acute myeloid leukemia. Antemortem blood culture yielded S. prolificans, which was confirmed on conventional morphological examination and polymerase chain reaction-based DNA sequencing targeting internally transcribed spacer regions. Histopathology of autopsy specimens indicated fungal infection in the heart, lungs, liver, kidneys, spleen, pancreas and gastrointestinal tract, with the development of hemorrhagic and ischemic necrosis. The infecting fungus had developing septate hyphae and was identified as belonging to the genus Scedosporium, on in situ hybridization of tissue. The combination of haphazardly branching hyphae and lemon-shaped conidia appeared to be the most useful distinguishing features to allow differentiation of this fungus from other filamentous fungi in tissue. Three other unique histopathological characteristics of the fungus were noted: (i) parallel hyphae bridged at right angles to produce letter-H patterns; (ii) intravascular conidiation; and (iii) purple conidia in tissue, though these are usually described as brown in most text books. Precise histopathology, in addition to other techniques such as in situ hybridization, can aid in the identification of etiological fungi.

A 73-year-old female hemodialysis patient experienced fever, shortness of breath on effort, and chest discomfort. A decrease in breath sounds in the right lung field, leukocytosis, elevated CRP level, and a right massive pleural effusion were observed. The patient was diagnosed with bacterial pleuritis based on leukocyte-predominant exudative pleural effusion, and treated with ceftriaxone. Her symptoms, however, were not improved, so thoracic drainage was attempted. Campylobacter species were isolated from cultured pleural fluid samples, and Campylobacter jejuni subspecies jejuni was detected on the multiplex PCR assay. The antibiotic was therefore changed to minocycline following pazufloxacin, and her symptoms were improved.

To determine the prevalence of genital mycoplasmas and ureaplasmas in the pharynges of Japanese female sex workers practicing fellatio on their clients, vaginal swabs and throat washings were collected from 403 female sex workers attending a clinic in Kyoto, Japan, for regular screening of gonococcal and chlamydial infections. Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum in vaginal and throat specimens were tested by nucleic acid amplification tests. The prevalence of N. gonorrhoeae, C. trachomatis, M. genitalium, M. hominis, U. parvum, and U. urealyticum in the genitals was 1.7%, 7.2%, 1.7%, 19.6%, 40.4%, and 10.2%, respectively, whereas their prevalence in the pharynges was 4.0%, 2.0%, 0%, 1.2%, 0.2%, and 0.7%, respectively. Gonococcal infection in the pharynx was significantly associated with gonococcal infection in the genitals. Chlamydial infection in the pharynx was also significantly associated with chlamydial infection in the genitals. M. hominis, U. parvum, and U. urealyticum were all detected in vaginal swabs and in throat washings; however, M. genitalium was detected in vaginal swabs but not in throat washings. For each of these genital mycoplasmas and ureaplasmas, a positive test result in the pharynx was not significantly associated with a positive result in the genitals. M. hominis, U. parvum, and U. urealyticum were detected in throat washings, but M. genitalium was not. These findings do not necessarily rule out the transmission of M. genitalium from the pharynx to the urethra by orogenital sex.

Cellulomonas denverensis is a small and thin gram-positive rod-shaped bacterium that was proposed as a new species in 2005. Here we report a female case of acute cholecystitis and sepsis in which C. denverensis was determined to be causative.

We describe the first case of bloodstream infection caused by Rhodococcus erythropolis. The identification was performed using 16S rRNA sequencing. This case illustrates that non-equi Rhodococcus infections may be underdiagnosed due to difficulties in identification in the routine clinical microbiology laboratory.

A 27-year-old female patient underwent cesarean section and a postoperative hematoma occurred at the site of the uterine incision. The patient underwent relaparotomy to remove the hematoma. Four days later she developed a fever of over 39 degrees C and an abscess had formed at the site. Despite therapy with several antimicrobial agents, her fever persisted. Consequently, she underwent transvaginal abscess drainage, after which she promptly became afebrile. Mycoplasma hominis was considered to be the primary causative organism. There are two reasons that could explain why the wound infection became serious: (i) M. hominis is resistant to several antimicrobial agents that are usually used to treat obstetric infections; and (ii) a long time is required to identify the pathogen. In conclusion, M. hominis should be considered as a causative organism if an antimicrobial-resistant infection occurs at the surgical site after a cesarean section.

Rothia aeria, a gram-positive coccoid- to rod-shaped bacterium with irregular morphology, is an extremely rare causative organism of infections in humans. We report the first case of R. aeria sepsis clinically manifested in a female neonate soon after birth.

Infections caused by Mycobacterium wolinskyi have rarely been reported, and essentially all were cellulitis and/or osteomyelitis related with traumatic event or surgical wound. Here, we present the 1st case of septic complication due to this organism in a patient with chronic myelogenous leukemia of the 1st but late chronic phase.

Conventional polymerase chain reaction (PCR) used to identify mycobacterial species does not distinguish between Mycobacterium tuberculosis and M. bovis BCG, and several weeks or months may be needed to identify individual slow-growing Mycobacterial species. We report a 4-year-old girl who had subcutaneous abscess and sternal osteomyelitis after BCG vaccination at 4 month of age. We directly identified M. bovis BCG genome in the punctured abscess within a few days using PCR and PCR-restriction fragment length polymorphism. Such PCR is useful for rapidly diagnosing and managing of appropriate therapy in patients with infection due to M. bovis BCG.

A 58-year-old man was admitted to our hospital with fever. The vegetation was confirmed by echocardiography on the tricuspid valve and Erysipelothrix rhusiopathiae was isolated by blood culture. The patient died due to heart failure, and tricuspid valve vegetation was confirmed on autopsy and the sample of Gram's staining showed gram-positive microcolonies. Although about 60 cases of E. rhusiopathiae endocarditis have been reported, Japanese cases are extremely rare.

A multiplex polymerase chain reaction (PCR) method, specifically designed for application in routine diagnostic laboratories, was developed for identifying 5 human pathogen Vibrio species: Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio mimicus, and Vibrio alginolyticus. This assay directed toward the dnaJ gene was tested on a total of 355 strains representing 13 Vibrio species and 17 non-Vibrio species. Specific PCR fragments were produced in isolates belonging to the 5 target species and were absent from all strains other than these 5 species and non-Vibrio strains, indicating a high specificity of this multiplex PCR. The multiplex PCR for the detection of Vibrio pathogens in clinical specimens was experimentally applied to spiked stool samples. Only 1 specific amplicon was observed, corresponding to the pathogen spiked into the stool sample. The detection limitation was 10(5) to 10(6) cells per milliliter stool. Our data showed that this method represented a robust tool for the specific and rapid detection of the 5 major pathogenic Vibrio species.

We report two cases of suture-related keratitis following penetrating keratoplasty. In both cases, Corynebacterium macginleyi was isolated from corneal specimens. Scanning electron microscopy revealed that corynebacteria could aggregate and form a biofilm. The MICs of sulbenicillin and fluoroquinolones were high for both isolates. Our findings show that C. macginleyi can cause keratitis with biofilm formation.

Rapid and accurate identification of mycobacterial species is essential for patient management. We describe the use of the Invader assay in conjunction with the BACTEC MGIT 960 system that together provide an efficient procedure for clinical use. This assay discriminates single-base differences (e.g., genotyping single-nucleotide polymorphisms) under homogeneous and isothermal conditions and can measure directly on genomic DNA without prior target DNA amplification. To identify a wide variety of mycobacterial species, 20 Invader probes were designed to target the 16S rRNA gene and the 16S-23S rRNA gene internal transcribed spacer 1 (ITS-1) region. To validate the Invader probes, we used 78 ATCC strains, and 607 clinical mycobacterial strains, which were identified by DNA sequencing of the 16S rRNA gene and ITS-1. The Invader assay could accurately identify and differentiate these strains according to target sequences. Moreover, it could detect and identify 116 (95.1%) of 122 positive liquid cultures from the BACTEC MGIT 960 system and did not react to 83 contaminated MGIT cultures. Species identification takes 6.5 h by the Invader assay: 2.0 h for DNA extraction, 0.5 h for handling, and up to 4 h for the Invader reaction. The Invader assay has the speed, ease of use, and accuracy to be an effective procedure for the bacteriological diagnosis of mycobacterial infections.

We report the first case of septicemia caused by anaerobic spiral-shaped Gram negative bacilli, Anaerobiospirillum succiniciproducens in Japan. A 71-year-old male who had been suffered from terminal stage of liver cirrhosis and hepatocelluler carcinoma was admitted to our hospital for his symptoms of general malaise and increasing ascites on September 1, 2004. He developed diarrhea seven times a day on the eighth hospital day and had fever of 38.7 degrees C with WBC 12,600/microl and CRP 6.6 mg/dl on the next day. Blood culture grew Gram negative spiral bacilli. We initially could not identify the offending bacterium that resembled to Campyrobacter morphologically using commercially available indentification kits. However, 16SrRNA sequencing test revealed 100% compatibility with Anaerobiospirillum succiniciproducens.

The availability of the dnaJ1 gene for identifying Mycobacterium species was examined by analyzing the complete dnaJ1 sequences (approximately 1200 bp) of 56 species (54 of them were type strains) and comparing sequence homologies with those of the 16S rRNA gene and other housekeeping genes (rpoB, hsp65). Among the 56 Mycobacterium species, the mean sequence similarity of the dnaJ1 gene (80.4%) was significantly less than that of the 16S rRNA, rpoB and hsp65 genes (96.6%, 91.3% and 91.1%, respectively), indicating a high discriminatory power of the dnaJ1 gene. Seventy-one clinical isolates were correctly clustered to the corresponding type strains, showing isolates belonging to the same species. In order to propose a method for strain identification, we identified an area with a high degree of polymorphism, bordered by conserved sequences, that can be used as universal primers for PCR amplification and sequencing. The sequence of this fragment (approximately 350 bp) allows accurate species identification and may be used as a new tool for the identification of Mycobacterium species.

A strain isolated from pleural fluid of a patient with suppurative pleuritis (strain GTC 3021(T)) was characterized in terms of its phenotypic and biochemical features, cellular fatty acid profile and phylogenetic position based on 16S rRNA gene sequence analysis. 16S rRNA gene sequence analysis showed that the isolate was a member of the genus Prevotella. The isolate was related to Prevotella enoeca ATCC 51261(T) with about 92 % 16S rRNA gene sequence similarity. The strain was an obligately anaerobic, non-pigmenting, non-spore-forming, non-motile, Gram-negative rod. Although the phenotypic and biochemical characteristics of the strain were similar to those of P. enoeca JCM 12259(T), the cellular fatty acid composition of the isolate was significantly different from that of P. enoeca JCM 12259(T) (C(18 : 1) omega 9c and anteiso-C(15 : 0) fatty acid content). Based on these data, we propose a novel Prevotella species, Prevotella pleuritidis sp. nov., with the type strain GTC 3021(T) (=JCM 14110(T) =CCUG 54350(T)). The G+C content of the type strain is 45.4 mol%.

The interrelationships of 27 Aeromonas strains were investigated using dnaJ sequences and DNA-DNA hybridization. dnaJ sequence similarities showed a stronger relationship with DNA-DNA relatedness values than did 16S rRNA gene sequence similarities. Additionally, dnaJ sequence analysis, with interspecies divergence over 5.2 % in most cases, gave better resolution than 16S rRNA gene sequences for the differentiation of strains at the species level. Relationships among Aeromonas species were therefore elucidated on the basis of dnaJ sequences and DNA-DNA reassociation. Strains of Aeromonas encheleia and Aeromonas sp. HG11 were unquestionably grouped in the same genetic species, since they shared 98.7 % dnaJ sequence similarity and 82-85 % genomic relatedness. The phylogenetically close relationships obtained from dnaJ sequence analysis (1.7-3.3 % genetic distance) were corroborated by high DNA-DNA relatedness (73-97 %) to support the previous suggestion that Aeromonas culicicola and Aeromonas allosaccharophila are later heterotypic synonyms of Aeromonas veronii. Our findings will contribute to the clarification of controversial relationships in the genus Aeromonas and also demonstrate that analysis of dnaJ sequences can be a powerful tool for interspecies study of the genus.

Phylogenetic relations within the family Enterobacteriaceae were analyzed using partial dnaJ sequences of 165 strains belonging to 93 species from 27 enterobacterial genera. The dnaJ phylogeny was in relative agreement with that constructed by 16S rDNA sequences, but more monophyletic groups were obtained from the dnaJ tree than from the 16S rDNA tree. The degree of divergence of the dnaJ gene was approximately 6 times greater than that of 16S rDNA. Also, the dnaJ gene showed the most discriminatory power in comparison with tuf and atpD genes, facilitating clear differentiation of any 2 enterobacterial species by dnaJ sequence analysis. The application of dnaJ sequences to the identification was confirmed by assigning 72 clinical isolates to the correct enterobacterial species. Our data indicate that analysis of the dnaJ gene sequences can be used as a powerful marker for phylogenetic study and identification at the species level of the family Enterobacteriaceae.

The utility of the dnaJ gene for identifying Vibrio species was investigated by analyzing dnaJ sequences of 57 type strains and 22 clinical strains and comparing sequence homologies with those of the 16S rDNA gene and other housekeeping genes (recA, rpoA, hsp60). Among the 57 Vibrio species, the mean sequence similarity of the dnaJ gene (77.9%) was significantly less than that of the 16S rDNA gene (97.2%), indicating a high discriminatory power of the dnaJ gene. Most Vibrio species were, therefore, differentiated well by dnaJ sequence analysis. Compared to other housekeeping genes, the dnaJ gene showed better resolution than recA or rpoA for differentiating Vibrio coralliilyticus from Vibrio neptunius and Vibrio harveyi from Vibrio rotiferianus. Among the clinical strains, all 22 human pathogenic strains, including an atypical strain, were correctly identified by the dnaJ sequence. Our findings suggest that analysis of the dnaJ gene sequence can be used as a new tool for the identification of Vibrio species.