Cheng-Yen Kao

An integrated microfluidic system for diagnosis of the resistance of Helicobacter pylori to quinolone-based antibiotics

Helicobacter pylori (H. pylori) is a species of bacteria that can colonize the human stomach mucosa. It is closely associated with gastric diseases such as ulcer and inflammation. Recently, some H. pylori strains were found to express resistance to a family of antibiotics known as quinolones due to single-point mutations. Although traditional polymerase chain reaction (PCR) and molecular diagnostic-based approaches can be used to determine the presence and abundance of antibiotic-resistant H. pylori strains, such processes are relatively expensive, labor-intensive, and require bulky and costly equipment. This study therefore reports an advanced diagnostic assay performed on an integrated microfluidic system for rapid detection of antibiotic resistance in H. pylori. The assay features three components: (1) nucleic acid extraction by specific probe-conjugated magnetic beads, (2) amplification of the target deoxyribonucleic acid (DNA) fragments by using single-nucleotide-polymorphism polymerase chain reaction (SNP-PCR), and (3) optical detection of the PCR products. The device integrates several microfluidic components including micro-pumps, normally-closed micro-valves, and reaction chambers such that the entire diagnostic assay can be automatically executed on a single microfluidic system within one hour with detection limits of 10(0), 10(2), and 10(2) bacterial cells for H. pylori detection and two different SNP sites strains. Three PCR-based assays for determining presence of H. pylori infection and two DNA single-point mutation assays aimed at determining whether the infected strains were resistant to quinolone can be performed simultaneously on a single chip, suggesting that this microfluidic system could be a promising tool for rapid diagnosis of the presence of antibiotic-resistant H. pylori strains.

Length of thymidine homopolymeric repeats modulates promoter activity of sabA in Helicobacter pylori.

Background:  Helicobacter pylori uses SabA to interact with sialyl‐Lewis x on the gastric mucosal surface to establish persistent colonization. The number of CT repeats in sabA is variable and thus influences SabA translation, but the expression of SabA determined by Western blotting does not fully match with a CT sequence‐based prediction. Furthermore, a homopolymeric thymidine (polyT) tract located upstream of sabA has been observed, but its role in regulating sabA expression is still unknown.

Molecular characterization of extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella spp. isolates in Mongolia

BACKGROUND/PURPOSE The aim of this study was to determine the molecular characteristics of β-lactamase genes in extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae isolates from Mongolia. METHODS Fifty-six ESBL-producing Enterobacteriaceae isolates were collected, of which 46 were Escherichia coli, seven were Klebsiella pneumoniae, and three were K. oxytoca. Minimum inhibitory concentrations for selected antibiotics were tested using the agar dilution method, and the β-lactamase genes were determined using polymerase chain reaction combined with sequencing. Pulsed-field gel electrophoresis (PFGE) was used for genotyping all isolates, and phylogenetic grouping was performed on ESBL-producing E. coli isolates. Conjugation tests combined with plasmid digestion assays were used to determine whether there was a horizontal spread in Mongolia. RESULTS Among the 56 ESBL-producing isolates, 43 isolates (76.8%) were resistant to fluoroquinolones, but all isolates were susceptible to carbapenems and amikacin. The polymerase chain reaction sequencing results showed that the dominant CTX-M genotype was CTX-M-15 (19/46, 41.3%) in the ESBL-producing E. coli isolates. By contrast, CTX-M-14 and CTX-M-3 were the major genotypes found in Klebsiella spp. Phylogenetic analysis revealed that 21 ESBL-producing E. coli isolates belonged to group D (21/46, 45.6%), followed by group A (13/46, 28.3%), group B2 (11/46, 23.9%), and group B1 (1/46, 2.2%). Only four E. coli isolates (4/46, 8.7%) belonged to the ST131 clone. PFGE showed that the ESBL-producing Enterobacteriaceae were genetically unrelated. The conjugation assay showed that two plasmids harboring CTX-M-15 in E. coli isolates were genetic unrelated, whereas seven plasmids harboring CTX-M-14 (5/7 and 2/7) and four plasmids harboring CTX-M-55 (4/4) showed genetic relatedness, indicating the dissemination of resistance plasmids in this area.

Genome Sequence and Annotation of Helicobacter pylori Strain Hp238, Isolated from a Taiwanese Patient with Mucosa-Associated Lymphoid Tissue Lymphoma.

ABSTRACT We present the complete genome sequence of Helicobacter pylori strain Hp238, isolated from a Taiwanese patient with gastric mucosa-associated lymphoid tissue lymphoma. Importantly, H. pylori strain Hp238 can multiply in THP-1 cells after internalization through the induction of autophagosome formation. These genome data will help to identify genes associated with H. pylori intracellular multiplication and pathogenesis.

An integrated microfluidic system for diagnosis of quinolones resistance of helicobacter pylori

Helicobacter pylori (H. pylori) is a bacterium which can colonize the stomach mucosa and therefore play a crucial role in gastric diseases. Triple therapy treatment consisting of two antibiotics and a proton pump inhibitor has been routinely taken to eradicate H. pylori. Recently, some point mutations were found in gyrase genes against Quinolones. The highly frequent mutation sites were indicated as single amino acid substitution discovered in gyrase A subunit. The epsilometer test has been commonly used to confirm the antibiotic resistance after triple therapy treatment. However, the method is time-consuming and false negative results with trace amounts of H. pylori could be easily induced. Alternatively, conventional molecular diagnostic techniques such as polymer chain reaction (PCR) could be used to confirm the antibiotic resistance of H. pylori. However, this diagnostic process is relatively labor-intensive and requires expensive and bulky apparatus. In this study a new method was therefore developed to perform molecular diagnostic techniques of SNP-PCR on an integrated microfluidic system to detect the Quinolones resistance of H. pylori.