We previously reported an outbreak in a neurosurgery ward of catheter-associated urinary tract infection with multidrug-resistant (MDR) Pseudomonas aeruginosa strain IMCJ2.S1, carrying the 6-N-aminoglycoside acetyltransferase gene [aac(6)-Iae]. For further epidemiologic studies, 214 clinical isolates of MDR P. aeruginosa showing resistance to imipenem (MIC > 16 g/ml), amikacin (MIC > 64 g/ml), and ciprofloxacin (MIC > 4 g/ml) were collected from 13 hospitals in the same prefecture in Japan. We also collected 70 clinical isolates of P. aeruginosa that were sensitive to one or more of these antibiotics and compared their characteristics with those of the MDR P. aeruginosa isolates. Of the 214 MDR P. aeruginosa isolates, 212 (99%) were serotype O11. We developed a loop-mediated isothermal amplification (LAMP) assay and a slide agglutination test for detection of the aac(6)-Iae gene and the AAC(6)-Iae protein, respectively. Of the 212 MDR P. aeruginosa isolates, 212 (100%) and 207 (98%) were positive in the LAMP assay and in the agglutination test, respectively. Mutations of gyrA and parC genes resulting in amino acid substitutions were detected in 213 of the 214 MDR P. aeruginosa isolates (99%). Of the 214 MDR P. aeruginosa isolates, 212 showed pulsed-field gel electrophoresis patterns with >70% similarity to that of IMCJ2.S1 and 83 showed a pattern identical to that of IMCJ2.S1, indicating that clonal expansion of MDR P. aeruginosa occurred in community hospitals in this area. The methods developed in this study to detect aac(6)-Iae were rapid and effective in diagnosing infections caused by various MDR P. aeruginosa clones.Pseudomonas aeruginosa causes nosocomial infections as a result of its ubiquitous nature, ability to survive in moist environments, and resistance to many antibiotics and antiseptics. A serious problem is the emergence of multidrug-resistant (MDR) P. aeruginosa strains resistant to ␤-lactams, aminoglycosides, and quinolones (34,39,46 We previously reported a nosocomial outbreak of catheterassociated urinary tract infection involving new MDR P. aeruginosa strain IMCJ2.S1, which occurred in a neurosurgery ward of a hospital located in the Tohoku area of Japan (46). This strain showed broad-spectrum resistance to aminoglycosides, ␤-lactams, fluoroquinolones, tetracyclines, sulfonamide, and chlorhexidine. We found that IMCJ2.S1 harbored a novel class 1 integron, In113, containing an array of three gene cassettes of the metallo-␤-lactamase (MBL) bla IMP-1 gene, aminoglycoside 6Ј-acetyltransferase aac(6Ј)-Iae gene, and aminoglycoside 3Ј-adenylyltransferase aadA1 gene (46). This strain possessed mutations of the gyrA (83Thr3Ile) and parC (87Ser3Leu) genes involving amino acid substitutions, resulting in high-level resistance to fluoroquinolones.In the geographic area where the MDR P. aeruginosa outbreak occurred (46), hospitals and a commercial clinical laboratory were surveyed for similar organisms. Because 99% of the MDR P. aeruginosa isolates analyzed were found to harbor the aac(6Ј)-Iae gene, we d...

Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the rapid detection of the major periodontal pathogens Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. The LAMP method amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand displacement activity. In this study, we initially designed the primers for LAMP assays to detect these bacteria and evaluated the specificity and sensitivity of these assays. The specificities of the primers for these bacteria were examined using various oral bacteria and various reaction times. The lower detection limits of the 60-min LAMP reaction without loop primers were 1 g/tube for P. gingivalis, 10 fg/tube for T. forsythia, and 1 ng/tube for T. denticola. Addition of the loop primers for each bacterium improved the detection specificities and sensitivities by several magnitudes. Furthermore, LAMP assays were applied to the rapid detection of these periodontal pathogens in clinical specimens, and the results were compared with those of conventional PCR detection. The results of the LAMP assays corresponded to those of conventional PCR assays. These results indicate that the LAMP assay is an extremely rapid, highly sensitive, specific method. This method is very useful for the rapid detection of periodontopathic bacteria and the diagnosis of periodontal disease.

Abstract. It has been suggested that the maturational stage of oocytes at time of insemination influences the sex ratio of resulting embryos. However, there are very few reports concerning the relationship between the maturation culture period of oocytes and the sex ratio of resulting embryos. The objective of this study was to investigate the effects of in vitro maturation culture period for bovine oocytes on the sex ratio of in vitro produced blastocysts using a novel technique of loopmediated isothermal amplification (LAMP). Cumulus-oocyte complexes were collected from the ovaries of slaughtered cows, and then matured in vitro for various periods (16, 22, 28, and 34 h). After maturation culture for each period, the oocytes were inseminated with frozen-thawed spermatozoa, and then cultured in vitro. Blastocysts were harvested on Day 7 after insemination, and the sex of the embryos was examined using the LAMP method. The rates of oocytes matured to the metaphase II stage were significantly lower (P<0.05) in the 16-h maturation group than in the other groups. The proportion of blastocyst formation after insemination was significantly higher (P<0.05) in the 22-h maturation group than in the other groups. The proportion of male blastocysts increased with the increase in maturation culture period. The proportion of male blastocysts derived from oocytes matured for 34 h was significantly higher (P<0.05) than from oocytes matured for 16 and 22 h. These results indicate that the sex ratio of in vitro fertilized embryos is apparently influenced by the maturation culture period of the oocytes Key words: In vitro fertilization (IVF), Loop-mediated isothermal amplification (LAMP), Maturation culture, Sex ratio (J. Reprod. Dev. 52: [123][124][125][126][127] 2006) ertilization of a mammalian oocyte establishes the sex of the embryo and initiates a cascade of events that leads to sexual differentiation. If the timing of insemination in vitro can be used to control the gender of offspring in cattle, it would have an immense economic benefit and would be applied to livestock production. To date, various factors, including embryo culture conditions [1][2][3] and the duration of sperm-oocyte coincubation [4], have been shown to influence the sex ratio of bovine embryos produced in vitro. It appears that a high concentration of glucose in the culture medium accelerates development of male embryos [1]. Kochhar et al. [4] suggest that a short duration of sperm-oocyte coincubation leads to a skewed sex

These results indicate that the loop-mediated isothermal amplification assay is very useful for rapid detection of E. faecalis and diagnosis of endodontic infection.

Our results suggest that the LAMP-based assay is very useful for the rapid detection of A. actinomycetemcomitans.