The impacts of a fliD mutation on the biofilm formation of Helicobacter pylori

AbstractObjectiveTo investigate the impact of the fliD gene on the biofilm formation of Helicobacter pylori (H. pylori).MethodsH. pylori fliD mutant was constructed using inverse PCR mutagenesis. The mobility of the bacteria and its adhesion ability to human epithelial cells were assessed using a motility assay and a fluorescein isothiocyanate staining adhesion assay, respectively. The formation of biofilm was evaluated using a pellicle assay and a crystal violet staining assay. The cyto-architecture of the biofilm was documented with scanning electron microscopy.ResultsIt was found that there was no significant difference in the levels of bacterial adhesion and the biofilm formation between the wild-type ATCC 43504 and the fliD mutant. Apart from a poor motility, the fliD mutant had a slightly delayed formation of its biofilm and an incomplete cyto-architecture of its biofilm. The bacterial cells residing in the biofilm of the fliD mutant showed a loose accumulation with less apparent cross-linking fibrils. Most of the mutant cells had truncated flagella.ConclusionsThis study provides the preliminary evidences that fliD potentially regulates biofilm formation and is required for the motility of H. pylori. Further studies need to be performed in order to develop fliD as a novel target for vaccine or antimicrobial agent in future

Fresh garlic extract inhibits Staphylococcus aureus biofilm formation under chemopreventive and chemotherapeutic conditions

Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) are the leading aetiological pathogens of nosocomial infections worldwide. These bacteria form biofilms on both biotic and abiotic surfaces causing biofilm-associated infections. Within the biofilm, these bacteria might develop persistent and antimicrobial resistant characteristics resulting in chronic infections and treatment failures. Garlic exhibits broad pharmaceutical properties and inhibitory activities against S. aureus. We investigated the effects of aqueous fresh garlic extract on biofilm formation in S. aureus ATCC25923 and MRSA strains under chemopreventive and chemotherapeutic conditions. The viable bacteria and biofilm levels were quantified through colony count and crystal violet staining, respectively. The use of fresh garlic extract under both conditions significantly inhibited biofilm formation in S. aureus strains ATCC25923 and MRSA. Garlic could be developed as either a prophylactic or therapeutic agent to manage S. aureus biofilm-associated infections

Rapid detection of multidrug-resistant tuberculosis based on allele-specific recombinase polymerase amplification and colorimetric detection.

Multidrug-resistant tuberculosis (MDR-TB) poses a serious threat to TB control. Early diagnosis and proper treatment are essential factors to limit the spread of the disease. The existing molecular tests for MDR-TB usually require specific instruments, steady power supply, and routine maintenance, which might be obstacles for low-resource settings. This study aimed to develop allele-specific isothermal recombinase polymerase amplification (allele-specific RPA) to simultaneously detect the most common mutations in the rpoB gene at codons 516, 526, and 531, which are associated with rifampicin resistance, and in the katG gene at codon 315, which is related to isoniazid resistance. Allele-specific primers targeting four major mutations, rpoB516, rpoB526, rpoB531, and katG315, were constructed and used in individual RPA reactions. The RPA amplicons were endpoints detected by the naked eye immediately after applying SYBR Green I. The optimised RPA assay was evaluated with the Mycobacterium tuberculosis wild-type strain H37Rv and 141 clinical M. tuberculosis isolates. The results revealed that allele-specific RPA combined with SYBR Green I detection (AS-RPA/SYBR) detected these four major mutations with 100% sensitivity and specificity relative to DNA sequencing. The limits of detection for these particular mutations with AS-RPA/SYBR were 5 ng. As a result of the outstanding performance of AS-RPA/SYBR, including its easy setup, speed, lack of a specific instrument requirement, and lack of cross-reaction with other bacteria, this technique may be integrated for the molecular diagnosis of MDR-TB, especially in low-resource settings

Rapid detection of extended spectrum β-lactamase producing Escherichia coli isolated from fresh pork meat and pig cecum samples using multiplex recombinase polymerase amplification and lateral flow strip analysis.

The emergence and dissemination of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli is a global health issue. Food-producing animals, including pigs, are significant reservoirs of antimicrobial resistance (AMR), which can be transmitted to humans. Thus, the rapid detection of ESBLs is required for efficient epidemiological control and treatment. In this study, multiplex recombinase polymerase amplification (RPA) combined with a single-stranded tag hybridization chromatographic printed-array strip (STH-PAS), as a lateral flow strip assay (LFA), was established for the rapid and simultaneous detection of multiple bla genes in a single reaction. Visible blue lines, indicating the presence of the blaCTX-M, blaSHV, and blaOXA genes, were observed within 10 min by the naked eye. The limit of detection of all three genes was 2.5 ng/25 μL, and no cross-reactivity with seven commensal aerobic bacteria was observed. A total of 93.9% (92/98) and 96% (48/50) of the E. coli isolates from pork meat and fecal samples, respectively, expressed an ESBL-producing phenotype. Nucleotide sequencing of the PCR amplicons showed that blaCTX-M was the most prevalent type (91.3-95.83%), of which the main form was blaCTX-M-55. The sensitivity and specificity of the RPA-LFA were 99.2% and 100%, respectively, and were in almost perfect agreement (κ = 0.949-1.000) with the results from PCR sequencing. Thus, the RPA-LFA is a promising tool for rapid and equipment-free ESBL detection and may facilitate clinical diagnosis in human and veterinary medicine, as well as AMR monitoring and surveillance