Na-Hye Park

Impact of phenolic compounds in the acyl homoserine lactone-mediated quorum sensing regulatory pathways

Quorum sensing (QS) is a cell density-dependent regulation of virulent bacterial gene expression by autoinducers that potentially pertains in the epidemic of bacterial virulence. This study was initially designed to evaluate the effect of 5 phenolic compounds in the modulation of QS and virulence factors of Chromobacterium violaceum and Pseudomonas aeruginosa, and to determine the mechanisms of their effects. Biosensor strains were used to assess antibacterial and anti-QS effect of these compounds. Only methyl gallate (MG) among these compounds demonstrated profound anti-QS effect in the preliminary study, and thus only MG was utilized further to evaluate the effects on the synthesis and activity of acyl homoserine lactone (AHL) in C. violaceum and on the modulation of biofilm, motility, proteolytic, elastase, pyocyanin, and rhamnolipid activity in P. aeruginosa. Finally, the effect of MG on the expression of QS-regulated genes of P. aeruginosa was verified. MG suppressed both the synthesis and activity of AHL in C. violaceum. It also restricted the biofilm formation and other QS-associated virulence factor of P. aeruginosa. MG concentration-dependently suppressed the expression of lasI/R, rhlI/R, and pqsA of P. aeruginosa and was non-toxic in in vitro study. This is the first report of the anti-QS mechanism of MG.

In silico analysis of putative drug and vaccine targets of the metabolic pathways of Actinobacillus pleuropneumoniae using a subtractive/comparative genomics approach

Actinobacillus pleuropneumoniae is a Gram-negative bacterium that resides in the respiratory tract of pigs and causes porcine respiratory disease complex, which leads to significant losses in the pig industry worldwide. The incidence of drug resistance in this bacterium is increasing; thus, identifying new protein/gene targets for drug and vaccine development is critical. In this study, we used an in silico approach, utilizing several databases including the Kyoto Encyclopedia of Genes and Genomes (KEGG), the Database of Essential Genes (DEG), DrugBank, and Swiss-Prot to identify non-homologous essential genes and prioritize these proteins for their druggability. The results showed 20 metabolic pathways that were unique and contained 273 non-homologous proteins, of which 122 were essential. Of the 122 essential proteins, there were 95 cytoplasmic proteins and 11 transmembrane proteins, which are potentially suitable for drug and vaccine targets, respectively. Among these, 25 had at least one hit in DrugBank, and three had similarity to metabolic proteins from Mycoplasma hyopneumoniae, another pathogen causing porcine respiratory disease complex; thus, they could serve as common therapeutic targets. In conclusion, we identified glyoxylate and dicarboxylate pathways as potential targets for antimicrobial therapy and tetra-acyldisaccharide 4′-kinase and 3-deoxy-D-manno-octulosonic-acid transferase as vaccine candidates against A. pleuropneumoniae.

The phenotypic and molecular resistance induced by a single-exposure to sub-mutant prevention concentration of marbofloxacin in Salmonella Typhimurium isolates from swine

In the present study, the molecular mechanisms of antibiotic resistance in Salmonella Typhimurium clinical isolates from pigs were investigated using a single-step mutation model of exposure to sub-mutant prevention concentrations (MPCs) of marbofloxacin. The minimum inhibitory concentrations (MICs) of seven antibacterial drugs were evaluated against 30 S. Typhimurium clinical isolates from different pigs. MPCs of marbofloxacin were also determined. The mechanism of marbofloxacin-resistance was investigated by sequencing analysis of target gene mutations and quantifying the overexpression of efflux pumps and their regulators by quantitative RT-PCR. Marbofloxacin showed the highest potency against all isolates (23.3%), including multi-drug resistant isolates. The MPC50 (0.5μg/mL) and MPC90 (2μg/mL) of marbofloxacin were determined, as were MPC/MIC ratios of 2.5 to 8. A gyrA mutation (Ser83Phe or Asp87His) was detected in isolates with an MIC>0.06μg/mL and all single-step mutants. Moreover, expression of acrAB-tolC and marA/soxS/ramA increased following a single-step mutation, but only ramA expression showed a positive correlation with the resistance phenotype of clinical isolates and single-step mutants (p<0.05). Furthermore, the acrR mutation was detected in two clinical isolates and 50% of single-step mutants, regardless of whether the gyrA mutation was present. This is the first report of acrR mutations in S. Typhimurium isolates from pigs in Korea. Our findings suggest that a single-exposure to sub-MPCs of marbofloxacin was sufficient to reduce the susceptibility of Salmonella isolates. Therefore, optimized dosing based on application with the MPC concept is required to reduce the chances of marbofloxacin resistance.

Biofilm formation and determination of minimum biofilm eradication concentration of antibiotics in Mycoplasma hyopneumoniae

The study was aimed to investigate biofilm forming ability of Mycoplasma hyopneumoniae and to determine the minimum biofilm eradication concentrations of antibiotics. Biofilm forming ability of six strains of M. hyopneumoniae was examined using crystal violet staining on coverslips. The results demonstrated an apparent line of biofilm growth in 3 of the strains isolated from swine with confirmed cases of enzootic pneumonia. BacLight bacterial viability assay revealed that the majority of the cells were viable after 336 hr of incubation. Moreover, M. hyopneumoniae persists in the biofilm after being exposed to 10 fold higher concentration of antibiotics than the minimum inhibitory concentrations in planktonic cells. To the best of our knowledge, this is the first report of biofilm formation in M. hyopneumoniae. However, comprehensive studies on the mechanisms of biofilm formation are needed to combat swine enzootic pneumonia caused by resistant M. hyopneumoniae.

Evaluation of Fluoroquinolone Activity against Emergence of Resistant Salmonella enterica serovar Typhimurium Using In Vitro Dynamic Models

ABSTRACT The objectives of this study were to determine pharmacokinetic/pharmacodynamic (PK/PD) indices of fluoroquinolones that minimize the emergence of resistant Salmonella enterica serovar Typhimurium (S. Typhimurium) using in vitro dynamic models and to establish mechanisms of resistance. Three fluoroquinolones, difloxacin (DIF), enrofloxacin (ENR), and marbofloxacin (MAR), at five dose levels and 3 days of treatment were simulated. Bacterial killing-regrowth kinetics and emergence of resistant bacteria after antibacterial drug exposure were quantified. PK/PD indices associated with different levels of antibacterial activity were computed. Mechanisms of fluoroquinolone resistance were determined by analyzing target mutations in the quinolone resistance-determining regions (QRDRs) and by analyzing overexpression of efflux pumps. Maximum losses in susceptibility of fluoroquinolone-exposed S. Typhimurium occurred at a simulated AUC/MIC ratio (area under the concentration-time curve over 24 h in the steady state divided by the MIC) of 47 to 71. Target mutations in gyrA (S83F) and overexpression of acrAB-tolC contributed to decreased susceptibility in fluoroquinolone-exposed S. Typhimurium. The current data suggest AUC/MIC (AUC/mutant prevention concentration [MPC])-dependent selection of resistant mutants of S. Typhimurium, with AUC/MPC ratios of 69 (DIF), 62 (ENR), and 39 (MAR) being protective against selection of resistant mutants. These values could not be achieved in veterinary clinical areas under the current recommended therapeutic doses of the fluoroquinolones, suggesting the need to reassess the current dosing regimen to include both clinical efficacy and minimization of emergence of resistant bacteria.

Enhancement of Lipid Metabolism and Hepatic Stability in Fat-Induced Obese Mice by Fermented Cucurbita moschata Extract

The aim of this study was to evaluate the potentials of fermented Cucurbita moschata extract (FCME) in the treatment of obesity and nonalcoholic fatty liver disease (NAFLD). Five-week-old male C57BL/6 mice were assigned to 6 groups and treated for 8 weeks by feeding the normal diet (ND) and high fat diet (HFD) with and without FCME. Changes in body weight gain and consumption of feed and water were recorded. Major organs, adipose tissues, and blood samples were collected after the experimental period. The serum lipid profile, histological features of liver and adipose tissues, and mRNA expression of different adipogenic/lipogenic genes from liver tissue were evaluated. The supplementation of FCME in HFD significantly prevented HFD-induced increment of bodyweight. The adipose tissue mass, liver enzymes, and plasma lipids were also reduced significantly (p < 0.05) by the consumption of FCME. The mRNA expressions of adipogenic/lipogenic genes (PPARγ, C/EBPα, C/EBPβ, C/EBPγ, and SREBP-1C) in FCME-treated obese mice were considerably (p < 0.05) suppressed. FCME showed its antiobesity potential by suppressing the body weight gain and by modulating the plasma lipids and liver enzymes through the regulation of adipogenic/lipogenic transcriptional factors. Fermented Cucurbita moschata could be an opportunistic agent in controlling obesity and fatty liver changes.

Probiotic properties and adsorption of Enterococcus faecalis PSCT3-7 to vermiculite

The probiotic properties of Enterococcus (E.) faecalis PSCT3-7, a new strain isolated from the intestines of pigs fed dietary fiber containing 50% sawdust, were investigated. E. faecalis PSCT3-7 tolerated a pH range of 3 to 8 and 0.3% bile salts, and it inhibited the growth of Salmonella Typhimurium in a concentration-dependent manner. In addition, E. faecalis showed resistance to several antibacterial agents. Vermiculite, a nutrient and microbial carrier, increased the bile tolerance of the strain. Scanning electron microscope images revealed good adsorption of E. faecalis PSCT3-7 onto vermiculite. E. faecalis PSCT3-7 represents a potential probiotic candidate to administer with vermiculite to swine.

Spraying of swine buildings with lemon grass ( Cymbopogon citratus ) essential oil does not produce blood absorption in swine

Cymbopogon citratus (Lemon grass) essential oils have been used in swine buildings to reduce the offensive odor emanating from swine buildings. The present study was designed to investigate plasma residues of citral which is a major constituent of the essential oils of Cymbopogon citratus. An HPLC method was established, validated and used for the determination of citral in swine plasma harvested from blood samples taken at the 14 th , 21 st and 28 th day after spraying swine buildings with the 3% Lemon grass essential oil. Thereafter, analysis of the sample was conducted using HP ODS Hypersil column (200 × 4.6 mm, 5 µm) with a mobile phase consisting of methanol and 0.5% acetic acid; and a flow rate of 1mL/ min. The method was validated for parameters such as accuracy, precision, linearity and detection limits. Plasma spiked with standard citral (95%) revealed two chromatograms with retention times of about 10.7 and 12.2 minutes. The calibration curves for the citral isomers were found to be linear in the tested concentration ranges and mean recoveries wre 101% and 99.17%. This method was used to determine the residues of citral in swine plasma pretreated with methanol. Surprisingly, there was no any detectable level of citral in swine plasma within the 28 days of exposure. spraying of swine buildings with essential oils of lemon grass oil does not cause plasma residues of citral.