Tatsuya Fukumoto

Dec1 and Dec2 Expression is Disrupted in the Suprachiasmatic Nuclei of Clock Mutant Mice

DEC1 and DEC2 are basic helix-loop-helix transcription factors that functionally resemble negative feedback components of the mammalian circadian clock. The genes Dec1 and Dec2 are expressed rhythmically in the rat suprachiasmatic nuclei, and Dec1 expression is stimulated by light in a timedependent manner with the kinetics of an immediate early gene. DEC1 and DEC2 can inhibit CLOCK:BMAL1 transactivation of the clock gene Per1, suggesting that these transcription factors may help regulate circadian timing. The authors present data on the expression pattern of Dec1 and Dec2 in wild-type and homozygous Clock mutant mice. In the suprachiasmatic nuclei, the Clock mutation significantly reduces the expression of Dec1 and Dec2. Dec1 becomes arrhythmic; Dec2 remains weakly rhythmic in a 12L:12D light-dark cycle but is arrhythmic in constant darkness. A robust attenuation of the Dec1 and Dec2 signals in Clock mutant mice was detected in all brain areas examined. These data point to up-regulation of Dec1 and Dec2 by Clock in vivo.

Ciliates rapidly enhance the frequency of conjugation between Escherichia coli strains through bacterial accumulation in vesicles.

The mechanism underlying bacterial conjugation through protozoa was investigated. Kanamycin-resistant Escherichia coli SM10λ+ carrying pRT733 with TnphoA was used as donor bacteria and introduced by conjugation into ciprofloxacin-resistant E. coli clinical isolate recipient bacteria. Equal amounts of donor and recipient bacteria were mixed together in the presence or absence of protozoa (ciliates, free-living amoebae, myxamoebae) in Pages amoeba saline for 24 h. Transconjugants were selected with Luria broth agar containing kanamycin and ciprofloxacin. The frequency of conjugation was estimated as the number of transconjugants for each recipient. Conjugation frequency in the presence of ciliates was estimated to be approximately 10⁻⁶, but in the absence of ciliates, or in the presence of other protozoa, it was approximately 10⁻⁸. Conjugation also occurred in culture of ciliates at least 2 h after incubation. Successful conjugation was confirmed by the polymerase chain reaction. Addition of cycloheximide or latrunculin B resulted in suppression of conjugation. Heat killing the ciliates or bacteria had no effect on conjugation frequency. Co-localization of green fluorescent protein-expressing E. coli and PKH-67-vital-stained E. coli was observed in the same ciliate vesicles, suggesting that both donor and recipient bacteria had accumulated in the same vesicle. In this study, the conjugation frequency of bacteria was found to be significantly higher in vesicles purified from ciliates than those in culture suspension. We conclude that ciliates rapidly enhance the conjugation of E. coli strains through bacterial accumulation in vesicles.

Host range of obligate intracellular bacterium Parachlamydia acanthamoebae

The obligate intracellular bacterium Parachlamydia acanthamoebae is a potential human pathogen, but the host range of the bacteria remains unknown. Hence, the growth of P. acanthamoebae Bn9 in protozoa (Tetrahymena, Acanthamoeba, Dictyostelium) and mammalian cells (HEp‐2, Vero, THP‐1, PMA‐stimulated THP‐1, Jurkat) was assessed using an AIU assay which had been previously established by the current authors. P. acanthamoebae grew in Acanthamoeba but not in the other cell types. The growth was also confirmed using DAPI staining, FISH and TEM. These results indicate that the host range of P. acanthamoebae is limited.

Ciliates promote the transfer of the gene encoding the extended-spectrum β-lactamase CTX-M-27 between Escherichia coli strains

OBJECTIVES The mechanism by which Escherichia coli acquires multidrug resistance genes from other bacteria in the natural environment or livestock is still unclear. The ability of ciliates to promote the transfer of genes encoding extended-spectrum β-lactamases (ESBLs) between the CTX-M-27 donor and clinically isolated recipient E. coli strains was investigated. METHODS Equal amounts (∼10(9) cfu) of donor cefotaxime-resistant E. coli and recipient ciprofloxacin-resistant E. coli strains were mixed together in the presence or absence of 10(5) ciliates in Pages amoeba saline for 24 h, in the presence or absence of certain drugs (cytochalasin D, cycloheximide and latrunculin B). RESULTS Gene transfer frequency in the presence of ciliates was estimated at ∼10(-6); in the absence of ciliates it was ∼10(-10). Protein synthesis (cycloheximide) or phagocytosis (cytochalasin D or latrunculin B) inhibitors significantly reduced the frequency of gene transfer. CONCLUSIONS Ciliates promote the transfer of genes encoding ESBLs between E. coli strains, implying that the presence of ciliates may provide a significant impact on emerging multidrug-resistant bacteria.

Impact of Free-Living Amoebae on Presence of Parachlamydia acanthamoebae in the Hospital Environment and Its Survival In Vitro without Requirement for Amoebae

ABSTRACT Parachlamydia acanthamoebae is an obligately intracellular bacterium that infects free-living amoebae and is a potential human pathogen in hospital-acquired pneumonia. We examined whether the presence of P. acanthamoebae is related to the presence of Acanthamoeba in an actual hospital environment and assessed the in vitro survival of P. acanthamoebae. Ninety smear samples were collected between November 2007 and March 2008 (trial 1, n = 52) and between October 2008 and February 2009 (trial 2, n = 38) from the floor (dry conditions, n = 56) and sink outlets (moist conditions, n = 34) of a hospital. The prevalences of P. acanthamoebae DNA in the first and second trials were 64.3% and 76%, respectively. The prevalences of Acanthamoeba DNA in the first and second trials were 48% and 63.1%, respectively. A statistical correlation between the prevalence of P. acanthamoebae and that of Acanthamoeba was found (trial 1, P = 0.011; trial 2, P = 0.022), and that correlation increased when samples from just the dry area (floor smear samples, P = 0.002) were analyzed but decreased when samples from a moist area were analyzed (P = 0.273). The in vitro experiment showed that, without Acanthamoeba, P. acanthamoebae could not survive in dry conditions for 3 days at 30°C or 15 days at 15°C. Thus, both organisms were coincidentally found in an actual hospital environment, with the presence of Acanthamoeba having a significant effect on the long-term survival of P. acanthamoebae, suggesting that this potential human pathogen could spread through a hospital environment via Acanthamoeba.

Successful Colistin Treatment of Multidrug-Resistant Pseudomonas aeruginosa Infection Using a Rapid Method for Determination of Colistin in Plasma: Usefulness of Therapeutic Drug Monitoring

A 56-year-old woman with systemic lupus erythematosus had bacteremia due to multidrug-resistant Pseudomonas aeruginosa (MDRP). She was initially treated with imipenem-cilastatin, tobramycin, and aztreonam; however, MDRP was still detected intermittently in her plasma. Multidrug-susceptibility tests demonstrated that MDRP was susceptible only to colistin. Therefore, in addition to these antibiotics, the administration of intravenous colistin methanesulfonate, a prodrug formula of colistin, was started at a daily dose of 2.5 mg/kg (as colistin base activity). The initial dose setting was based on the patients renal function (baseline creatinine clearance=32.7 mL/min). After initiating colistin, the patients C-reactive protein levels gradually decreased. Blood cultures showed no evidence of MDRP on days 8, 14, and 22 after colistin initiation. However, the patients renal function went from bad to worse owing to septic shock induced by methicillin-resistant Staphylococcus aureus (MRSA) infection. A few days later, the trough plasma levels of colistin were 7.88 mg/L, which appeared to be higher than expected. After decreasing the colistin dose, the patients renal function gradually improved. On the final day of colistin treatment, the plasma levels decreased to 0.60 mg/L. MDRP could not be detected in blood culture after colistin treatment. Therefore, we successfully treated a case of bloodstream infection due to MDRP by therapeutic drug monitoring (TDM) of colistin. It is suggested that the monitoring of blood colistin levels by liquid chromatography-tandem mass spectrometry can contribute to safer, more effective antimicrobial therapy of MDRP because TDM facilitates quick decisions on dose adjustments.

Acanthamoeba containing endosymbiotic chlamydia isolated from hospital environments and its potential role in inflammatory exacerbation

BackgroundEnvironmental chlamydiae belonging to the Parachlamydiaceae are obligate intracellular bacteria that infect Acanthamoeba, a free-living amoeba, and are a risk for hospital-acquired pneumonia. However, whether amoebae harboring environmental chlamydiae actually survive in hospital environments is unknown. We therefore isolated living amoebae with symbiotic chlamydiae from hospital environments.ResultsOne hundred smear samples were collected from Hokkaido University Hospital, Sapporo, Japan; 50 in winter (February to March, 2012) and 50 in summer (August, 2012), and used for the study. Acanthamoebae were isolated from the smear samples, and endosymbiotic chlamydial traits were assessed by infectivity, cytokine induction, and draft genomic analysis. From these, 23 amoebae were enriched on agar plates spread with heat-killed Escherichia coli. Amoeba prevalence was greater in the summer-collected samples (15/30, 50%) than those of the winter season (8/30, 26.7%), possibly indicating a seasonal variation (p = 0.096). Morphological assessment of cysts revealed 21 amoebae (21/23, 91%) to be Acanthamoeba, and cultures in PYG medium were established for 11 of these amoebae. Three amoebae contained environmental chlamydiae; however, only one amoeba (Acanthamoeba T4) with an environmental chlamydia (Protochlamydia W-9) was shown the infectious ability to Acanthamoeba C3 (reference amoebae). While Protochlamydia W-9 could infect C3 amoeba, it failed to replicate in immortal human epithelial, although exposure of HEp-2 cells to living bacteria induced the proinflammatory cytokine, IL-8. Comparative genome analysis with KEGG revealed similar genomic features compared with other Protochlamydia genomes (UWE25 and R18), except for a lack of genes encoding the type IV secretion system. Interestingly, resistance genes associated with several antibiotics and toxic compounds were identified.ConclusionThese findings are the first demonstration of the distribution in a hospital of a living Acanthamoeba carrying an endosymbiotic chlamydial pathogen.

Impact of anaerobic and oligotrophic conditions on survival of Alloiococcus otitidis, implicated as a cause of otitis media

The survival of Alloiococcus otitidis (NCFB2890) with different nutritional supplements, including brain–heart infusion broth (BHI), phosphate-buffered saline (PBS), distilled water (DW), and middle ear effusion (MEE), as well as various atmospheres (aerobic, microaerobic, anaerobic), was compared using cultures, LIVE/DEAD staining, and transmission electron microscopy. The bacterial morphological traits and viability were maintained in BHI and MEE under aerobic conditions but were rapidly lost in PBS and DW. In contrast, anaerobic conditions did not support viability at all. Thus, the bacteria critically required an aerobic atmosphere for its survival as well as the appropriate nutrients, implying that culture of this pathogen from clinical specimens would become more difficult through oxygen depletion depending on a slight change in the middle ear atmosphere.

Tetrahymena promotes interactive transfer of carbapenemase gene encoded in plasmid between fecal Escherichia coli and environmental Aeromonas caviae : Ciliates promote bacterial gene transfer

Tetrahymena can facilitate plasmid transfer among Escherichia coli or from E. coli to Salmonella Enteritidis via vesicle accumulation. In this study, whether ciliates promote the interactive transfer of plasmids encoding blaIMP‐1 between fecal E. coli and environmental Aeromonas caviae was investigated. Both bacteria were mixed with or without ciliates and incubated overnight at 30°C. The frequency of plasmid‐acquired bacteria was estimated by colony counts using an agar plate containing ceftazidim (CAZ) followed by determination of the minimum inhibitory concentration (MIC). Cultures containing ciliates interactively transferred the plasmid between E. coli and Aeromonas with a frequency of 10−4 to 10−5. All plasmid‐acquired bacteria showed a MIC against CAZ of >128 μg/mL and the plasmid transfer was confirmed by PCR amplification of the blaIMP‐1 gene. Fluorescent observation showed that both bacteria accumulated in the same vesicle and that transwell sequestering significantly decreased the transfer frequency. Although ciliates preferentially ingested E. coli rather than A. caviae, both bacteria were co‐localized into the same vesicles of ciliates, indicating that their meeting is associated with the gene transfer. Thus, ciliates interactively promote plasmid transfer between E. coli and A. caviae. The results of this study will facilitate control of the spread of multiple‐antibiotic resistant bacteria.

Ciliates promote the transfer of a plasmid encoding blaNDM-5 from Escherichia coli, isolated from a hospital in Japan, to other human pathogens

Title Ciliates promote the transfer of a plasmid encoding blaNDM-5 from Escherichia coli, isolated from a hospital in Japan, to other human pathogens Author(s) Okubo, Torahiko; Matushita, Mizue; Ohara, Yukiko; Matsuo, Junji; Oguri, Satoshi; Fukumoto, Tatsuya; Hayasaka, Kasumi; Akizawa, Kouzi; Shibuya, Hitoshi; Shimizu, Chikara; Yamaguchi, Hiroyuki Citation International Journal of Antimicrobial Agents, 49(3): 387-388