Shih-Kuang Hsu

Genotypic detection and molecular epidemiology of extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae in a regional hospital in central Taiwan

This study was conducted to detect the genes encoding extended-spectrum beta-lactamases (ESBLs) and determine the epidemiological relatedness of 69 Escherichia coli and 33 Klebsiella pneumoniae isolates collected from a regional hospital in central Taiwan, mostly from inpatients (E. coli 87.0%; K. pneumoniae 88.0%). The phenotypes of these isolates were examined according to the combination disc method recommended by the Clinical and Laboratory Standards Institute. Most of the ESBL-producing E. coli and K. pneumoniae isolates (98.6% and 97%, respectively) could be detected using cefotaxime discs with and without clavulanate. Genotyping was performed by PCR with type-specific primers. CTX-M-14 type (53.6%) was the most prevalent ESBL among E. coli isolates while SHV type (57.6%) was the most dominant among K. pneumoniae isolates. Six E. coli and three K. pneumoniae isolates did not carry genes encoding ESBLs of types TEM, SHV, CTX-M-3, CTX-M-14, CMY-2 and DHA-1. The co-existence of two or more kinds of ESBL in a single isolate was common, occurring in 40.6% and 72.7% of E. coli and K. pneumoniae isolates, respectively. PFGE analysis revealed that ESBL producers isolated in this setting were genetically divergent.

Effects of Near-Field Ultrasound Stimulation on New Bone Formation and Osseointegration of Dental Titanium Implants In Vitro and In Vivo

A near-field ultrasound stimulation system was designed for use in in vitro and in vivo trials. The intensity of ultrasound was studied to optimize the osseointegration of the dental titanium implant into the adjacent bone. MG63 osteoblast-like cells were seeded on commercial purity titanium (CP-Ti) plate, and then sonicated for 3 min/day at a frequency of 1 MHz and intensities of 0.05, 0.15 and 0.30 W/cm(2), using either pulsed or continuous ultrasound. Cells were analyzed to determine viability (inhibition of (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction) and alkaline phosphatase (ALP). Tissue culture was performed in vitro by placing a CP-Ti plate in a cultured rat neonatal calvarial defect in response to ultrasound stimulation. In the in vivo trial, screw-shaped CP-Ti implants were inserted into the metaphysis of rabbit tibia, and then stimulated by ultrasound for 10 min daily for 30 d. All samples were processed for histomorphometric evaluation and analyzed by image system. Color Doppler ultrasonography was inspected to evaluate the supply of blood flow. Pulsed ultrasound groups had higher MTT and ALP than control. Tissue culture indicated that pulsed ultrasound groups promoted cell migration and new bone regeneration more effectively than in the control. In animal study, blood flow and mature type I collagen fibers were more prevalent around titanium implants, and bone formation was accelerated in pulsed ultrasound groups. In conclusion, low-intensity pulsed ultrasound at 0.05-0.3 W/cm(2) may accelerate cell proliferation and promote the maturation of collagen fibers and support osteointegration.

A novel hydantoinase process using recombinant Escherichia coli cells with dihydropyrimidinase and l-N-carbamoylase activities as biocatalyst for the production of l-homophenylalanine

A dihydropyrimidinase gene (pydB) was cloned from the moderate thermophilic Brevibacillus agri NCHU1002 and expressed in Escherichia coli. The purified dihydropyrimidinase exhibited strict d-enantioselectivity for D,L-p-hydroxyphenylhydantoin and D,L-5-[2-(methylthio)ethyl]hydantoin, and non-enantiospecificity for D,L-homophenylalanylhydantoin (D,L-HPAH). The hydrolytic activity of PydB was enhanced notably by Mn2+, with a maximal activity at 60 degrees C and pH 8.0. This enzyme was completely thermostable at 50 degrees C for 20 days. A whole cell biocatalyst for the production of L-homophenylalanine (L-HPA) from D,L-HPAH by coexpression of the pydB gene and a thermostable L-N-carbamoylase gene from Bacillus kaustophilus CCRC11223 in E. coli JM109 was developed. The expression levels of dihydropyrimidinase and L-N-carbamoylase in the recombinant E. coli cells were estimated to be about 20% of the respective total soluble proteins. When 1% (w/v) isopropyl-beta-D-thiogalactopyranoside-induced cells were used as biocatalysts, a conversion yield of 49% for L-HPA with more than 99% ee could be reached in 16 h at pH 7.0 from 10mM D,L-HPAH. The cells can be reused for at least eight cycles at a conversion yield of more than 43%. Our results revealed that coexpression of pydB and lnc in E. coli might be a potential biocatalyst for L-HPA production.

Asymmetrically simultaneous synthesis of L-homophenylalanine and N6-protected-2-oxo-6-amino-hexanoic acid by engineered Escherichia coli aspartate aminotransferase

L‐Homophenylalanine (L‐HPA) and N6‐protected‐2‐oxo‐6‐amino‐hexanoic acid (N6‐protected‐OAHA) can be used as building blocks for the manufacture of angiotensin‐converting enzyme inhibitors. To synthesize L‐HPA and N6‐protected‐OAHA simultaneously from 2‐oxo‐4‐phenylbutanoic acid (OPBA) and N6‐protected‐L‐lysine, several variants of Escherichia coli aspartate aminotransferase (AAT) were developed by site‐directed mutagenesis and their catalytic activities were investigated. Three kinds of N6‐protected‐L‐lysine were tested as potential amino donors for the bioconversion process. AAT variants of R292E/L18H and R292E/L18T exhibited specific activities of 0.70±0.01 U/mg protein and 0.67±0.02 U/mg protein to 2‐amino‐6‐tert‐butoxycarbonylamino‐hexanoic acid (BOC‐lysine) and 2‐amino‐6‐(2,2,2‐trifluoro‐acetylamino)‐hexanoic acid, respectively. E. coli cells expressing R292E/L18H variant were able to convert OPBA and BOC‐lysine to L‐HPA and 2‐oxo‐6‐tert‐butoxycarbonylamino‐hexanoic acid (BOC‐OAHA) with 96.2% yield in 8 h. This is the first report demonstrating a process for the simultaneous production of two useful building blocks, L‐HPA and BOC‐OAHA.

The Effect of Visible Light-Activated TiO2 Thin Film on Nosocomial Pathogens

Many methods had been developed to disinfect or sterilize the medical equipment or hospital environment for controlling the spread of nosocomial pathogens. This study was aimed to evaluate the effect of titanium dioxide (TiO2) thin film on preventing nosocomial infection. Firstly, TiO2 thin film was prepared by using the modified impregnation method and further analysis by field-emission scanning electron micrographs and X-ray diffractometer. The nano-size and Anatase crystal structure were confirmed. By using visible light activation of TiO2 thin film for 30 min, the inhibition of Enterococcus faecalis, Streptococcus bovis, Streptococcus agalactiae, and Staphylococcus aureus by TiO2 thin film treatment were 27.4%, 73.3%, 56.6%, and 9.4%, respectively. In addition, several important Gram negative bacteria causing nosocomial infection were also investigated. The inhibition rate of Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli were 32.5%, 31.3%, and 22.4%, respectively. The results revealed that TiO2 thin film activated by visible light may be useful for decreasing nosocomial infection.