Fluorescent nucleic acid probe in droplets for bacterial sorting (FNAP-sort) as a high-throughput screening method for environmental bacteria with various growth rates.

We have developed a new method for selectively sorting droplets containing growing bacteria using a fluorescence resonance energy transfer (FRET)-based RNA probe. Bacteria and the FRET-based RNA probe are encapsulated into nanoliter-scale droplets, which are incubated to allow for cell growth. The FRET-based RNA probe is cleaved by RNase derived from the bacteria propagated in the droplets, resulting in an increase in fluorescence intensity. The fluorescent droplets containing growing bacteria are distinguishable from quenching droplets, which contain no cells. We named this method FNAP-sort based on the use of a fluorescent nucleic acid probe in droplets for bacterial sorting. Droplets containing the FRET-based RNA probe and four species of pure cultures, which grew in the droplets, were selectively enriched on the basis of fluorescence emission. Furthermore, fluorescent droplets were sorted from more than 500,000 droplets generated using environmental soil bacteria and the FRET-based RNA probe on days 1, 3, and 7 with repeated incubation and sorting. The bacterial compositions of sorted droplets differed on days 1, 3, and 7; moreover, on day 7, the bacterial composition of the fluorescent droplets was drastically different from that of the quenching droplets. We believe that FNAP-sort is useful for high-throughput cultivation and sorting of environmental samples containing bacteria with various growth rates, including slow-growing microbes that require long incubation times.

The first reported case of central venous catheter-related fungemia caused by Cryptococcus liquefaciens.

We describe a case of central venous catheter-related fungemia caused by Cryptococcus liquefaciens, a non-neoformans and non-gattii Cryptococcus, in a non-HIV patient. A 71-year-old man with diffuse large B-cell lymphoma receiving antineoplastic chemotherapy was febrile approximately 30 weeks after central venous port insertion, and C. liquefaciens was isolated from all three performed blood cultures as well as a central venous catheter tip culture. In vitro antifungal susceptibility tests showed that this yeast isolate was susceptible to low concentrations of amphotericin B, fluconazole, itraconazole and voriconazole yet was resistant to 5-fluorocytosine (MIC: >64 µg/ml), unlike Cryptococcus neoformans. Treatment of the patient with oral and intravenous voriconazole was effective and consistent with the susceptibility tests. Although non-neoformans and non-gattii Cryptococcus spp. are considered non-pathogenic environmental yeast, they may rarely be the causative agents of serious infections in humans, as in the present case.

Factors predicting the cause and prognosis of central line-associated bloodstream infections.

The increased use of indwelling catheters has led to an increased number of deaths due to central line-associated bloodstream infection (CLABSI). Improving CLABSI outcomes requires the identification of clinical characteristics affecting drug selection and factors associated with poor prognosis. The medical records of inpatients admitted to St. Marianna University School of Medicine between April 1, 2010 and March 31, 2013 were evaluated for the results of catheter tip cultures. The clinical characteristics of these cases and the characteristics of the pathogens involved were investigated to identify prognostic factors. Of the 1629 catheter cultures investigated, 183 were CLABSIs. Among them, 105 were caused by gram-positive bacteria, 43 by gram-negative bacteria, and 35 by fungi. Gram-negative CLABSIs were more common in cases with prior colonization by gram-negative bacteria and post-surgical cases. Fungal CLASBIs were more common in the cases with prior colonization by fungi, high-calorie infusion enforcement, broad-spectrum antibiotic treatment, and post-surgical cases. Death was significantly more likely in cases with findings of inflammation at the catheter insertion site and in those with abnormal body temperature, tachycardia, or abnormal white blood cell count. Thus, when treating CLABSI in post-surgical cases and in cases with prior colonization by gram-negative bacteria, therapy should include anti-pseudomonal agents. Considering the factors predicting poor prognostic identified in this study, clinicians must check the vital signs and catheter insertion site in patients with indwelling catheters.

[A case of Streptococcus gallolyticus subsp. gallolyticus infective endocarditis with colon adenoma].

An 80-year-old Japanese man had a fall presented with a 3-week history of right lumbago exacerbated by body movement as well as a 1-week history of anomalous behavior and appetite loss. He visited our hospital complaining of difficulty in standing up. He had a history of mitral prolapse due to an unknown rupture of the chordae tendineae 3 years earlier, which resulted in moderate mitral valve regurgitation and atrial fibrillation. Upon visiting the hospital, he had petechial hemorrhage and jaundice of the conjunctiva, a systolic murmur (Levine II/VI) at the apex and 4th interspace of the left sternal border, and a positive right straight leg raising test result. Moderate bilirubinemia and disseminated intravascular coagulation which were considered to have been produced secondarily were observed. Infective endocarditis was suspected, and 3 sets of blood culture were extracted. The patient was admitted on the same day. Blood cultures were positive for Streptococcus gallolyticus subsp. gallolyticus (6/6) on the following day. Transesophagela echocardiography was carried out on the same day, and vegetation with a diameter of 4mm was observed in the anterior mitral leaflet; the patient was subsequently diagnosed as having infective endocarditis. Colonic endoscopy was performed after hospitalization. Twelve colonic adenomata were found, and endoscopic mucosal resection was performed on one polyp. The bacterium found in the culture was classified as Streptococcus bovis type I, which causes infective endocarditis and bacteremia. Furthermore, this bacteria is a relatively rare causative organism of infective endocarditis. Tolerance to macrolide and tetracycline are reported in the literature. Moreover, the cell wall of this bacterium may have low pathogenicity as well as cause chronic inflammation in the large intestine mucous membrane, colonic polyps, and colorectal cancer. Several colonic adenomata and a partial shift to a malignant pathology were observed in this case. When this bacterium is detected, searching for a pathological change in the large intestine is believed to be indispensable.

Determination of epidemiology of clinically isolated Cryptococcus neoformans strains in Japan by multilocus sequence typing.

Cryptococcus neoformans and Cryptococcus gattii are the causative agents of cryptococcosis. Despite its importance, our knowledge of the epidemiology of cryptococcosis in Japan remains limited. To establish an epidemiological database on cryptococcosis in Japan, we determined the genetic variability of 44 Japanese clinical isolates of C. neoformans (var. grubii: serotype A) by multilocus sequence typing (MLST). The strains were clinically isolated from 1992 to 2011 in 5 different areas of Japan (the Hokkaido region [n = 1], Kanto region [n = 32], Chubu region [n = 1], Kansai region [n = 1], and Kyushu region [n = 9]). According to the method recommended by the International Society for Human and Animal Mycology cryptococcal genotyping working group, 36 isolates (82%) were identified as sequence type (ST)46. The remaining strains belonged to ST45 (n = 1) and ST47 (n = 1), and 6 isolates belonged to novel independent STs. There was little geographic difference in the ST population. Our present data are still limited; however, because most clinical isolates showed the same MLST profile in Japan, applying the current MLST scheme for Cryptococcus may at times be insufficient for investigating the infection route among outbreak cases. To solve this problem, it may be necessary to investigate other gene loci or develop a novel method with greater discriminatory power. However, in cases in which a strain belongs to a minor ST, our data may serve as useful epidemiological information in Japan.

Neonatal meningitis caused by Streptococcus gallolyticus subsp. pasteurianus.

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.

[A case of acute septic osteomyelitis onset due to Streptococcus dysgalactiae subsp. equisimilis in an elderly diabetic patient].

Group C streptococci are increasingly causing invasive infections such as that we report here. A 70-year-old man being treated for diabetes and seen at the emergency room for neck pain and fever was hospitalized for possible sepsis. His temperature was 39.8 degrees C, regular pulse 101 bpm, and pain reinforced in flexing and cervical rotation. Streptococcus dysgalactiae subsp. equisimilis (SDSE) was cultured from blood. Neck pain gradually decreased with of 2 million units PCG 6 times/day. Magnetic resonance imaging (MRI) of the cervical spine showed high-intensity areas in fat-suppression imaging at C7, Thl and intervertebral disks plus enhancement around the vertebral body, yielding a diagnosis of cervicothoracic vertebral osteomyelitis. Antimicrobial intravenous therapy continuede 6 weeks. The man was discharged after 45 days without relapse.

[Antimicrobial susceptibility and beta-lactamase producibility of bacteria clinically isolated during the period from December 1999 to February 2000].

Antimicrobial susceptibility and beta-lactamase producibility were tested in 848 clinical strains collected at 8 hospitals in Kanagawa prefecture during the period from December 1999 to February 2000. Positive rates of beta-lactamase used the nitrocefin method (Cefinase) were 21.9% of Staphylococcus aureus, 10.0% of Haemophilus influenzae, and 99.0% of Moraxella catarrhalis. Furthermore, on the acidometric method (P/Case test) penicillinase (PCase), cephalosporinase (CEPase), and both of PCase and CEPase were found to be positive in 19.0%, 16.0%, and 16.0% for Escherichia coli, 6.2/0/3.1% for Klebsiella pneumoniae, 0/66.3/26.5% for Enterobacter cloacae, 2.8/57.7/15.5% for Serratia marcescens, and 4.0/15.0/22.0% for Pseudomonas aeruginosa, respectively. Based on the assessment of minimal inhibitory concentrations (MICs) of antibacterial agents among beta-lactamase producing strains, there were 5 strains (4 strains of K. pneumoniae and 1 strain of E. coli) that may be ESBLs producing bacteria out of a total of 466 strains of Enterobacteriaceae and P. aeruginosa. During this process, 1 strain of class-B beta-lactamase-producing E. cloacae was isolated. MRSA were found in 79.2% of S. aureus, and BLNAR were found in 8.9% of H. influenzae.