Genomic Characterization of Fluoroquinolone-Resistant Thermophilic Campylobacter Strains Isolated from Layer Chicken Feces in Gangneung, South Korea by Whole-Genome Sequencing.

Thermophilic Campylobacter species of poultry origin have been associated with up to 80% of human campylobacteriosis cases. Layer chickens have received less attention as possible reservoirs of Campylobacter species. Initially, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of two archived Campylobacter isolates (Campylobacter jejuni strain 200605 and Campylobacter coli strain 200606) from layer chickens to five antimicrobials (ciprofloxacin, nalidixic acid, erythromycin, tetracycline, and gentamicin) were determined using broth microdilution while the presence of selected antimicrobial resistance genes was performed by polymerase chain reaction (PCR) using specific primers. Whole-genome sequencing (WGS) was performed by the Illumina HiSeq X platform. The analysis involved antimicrobial resistance genes, virulome, multilocus sequence typing (MLST), and phylogeny. Both isolates were phenotypically resistant to ciprofloxacin (MIC: 32 vs. 32 µg/mL), nalidixic acid (MIC: 128 vs. 64 µg/mL), and tetracycline (MIC: 64 vs. 64 µg/mL), but sensitive to erythromycin (MIC: 1 vs. 2 µg/mL) and gentamicin (MIC: 0.25 vs. 1 µg/mL) for C. jejuni strain 200605 and C. coli strain 200606, respectively. WGS confirmed C257T mutation in the gyrA gene and the presence of cmeABC complex conferring resistance to FQs in both strains. Both strains also exhibited tet(O) genes associated with tetracycline resistance. Various virulence genes associated with motility, chemotaxis, and capsule formation were found in both isolates. However, the analysis of virulence genes showed that C. jejuni strain 200605 is more virulent than C. coli strain 200606. The MLST showed that C. jejuni strain 200605 belongs to sequence type ST-5229 while C. coli strain 200606 belongs to ST-5935, and both STs are less common. The phylogenetic analysis clustered C. jejuni strain 200605 along with other strains reported in Korea (CP028933 from chicken and CP014344 from human) while C. coli strain 200606 formed a separate cluster with C. coli (CP007181) from turkey. The WGS confirmed FQ-resistance in both strains and showed potential virulence of both strains. Further studies are recommended to understand the reasons behind the regional distribution (Korea, China, and Vietnam) of such rare STs.

Antimicrobial Resistance Profiles, Virulence Genes, and Genetic Diversity of Thermophilic Campylobacter Species Isolated From a Layer Poultry Farm in Korea.

Thermophilic Campylobacter species are among the major etiologies of bacterial enteritis globally. This study aimed at assessing the antimicrobial resistance (AMR) profiles, virulence genes, and genetic diversity of thermophilic Campylobacter species isolated from a layer poultry farm in South Korea. One hundred fifty-three chicken feces were collected from two layer poultry farms in Gangneung, South Korea. The Campylobacter species were isolated by cultural techniques, while PCR and sequencing were used for species confirmation. Antimicrobial susceptibility testing for six antimicrobials [ciprofloxacin (CIP), nalidixic acid (NAL), sitafloxacin (SIT), erythromycin (ERY), tetracycline (TET), and gentamicin (GEN)] was carried out by broth microdilution. Three AMR and nine virulence genes were screened by PCR. Genotyping was performed by flaA-restriction fragment length polymorphism (RFLP) and multilocus sequence typing (MLST). Of the 153 samples, Campylobacter spp. were detected in 55 (35.9%), with Campylobacter jejuni and Campylobacter coli being 49 (89.1%) and six (10.9%), respectively. High-level resistance was observed for CIP (100%), NAL (100%), and TET (C. jejuni, 93.9%; C. coli: 83.3%). No resistance was observed for SIT. The missense mutation (C257T) in gyrA gene was confirmed by sequencing, while the tet(O) gene was similar to known sequences in GenBank. The rate of multidrug-resistant (MDR) strains was 8.2%, and they all belonged to C. jejuni. All Campylobacter isolates possessed five virulence genes (cdtB, cstII, flaA, cadF, and dnaJ), but none possessed ggt, while the rates for other genes (csrA, ciaB, and pldA) ranged between 33.3 and 95.9%. The flaA-RFLP yielded 26 flaA types (C. jejuni: 21 and C. coli: five), while the MLST showed 10 sequence types (STs) for C. jejuni and three STs for C. coli, with CC-607 (STs 3611) and CC-460 (ST-460) being predominant. Among the 10 STs of C. jejuni, three were newly assigned. The findings of this study highlight the increased resistance to quinolones and TET, the virulence potential, and the diverse genotypes among Campylobacter strains isolated from the layer poultry farm.

Susceptibility of Campylobacter Strains to Selected Natural Products and Frontline Antibiotics.

Campylobacter species have developed resistance to existing antibiotics. The development of alternative therapies is, therefore, a necessity. This study evaluates the susceptibility of Campylobacter strains to selected natural products (NPs) and frontline antibiotics. Two C. jejuni strains (ATCC® 33560TM and MT947450) and two C. coli strains (ATCC® 33559TM and MT947451) were used. The antimicrobial potential of the NPs, including plant extracts, essential oils, and pure phytochemicals, was evaluated by broth microdilution. The growth was measured by spectrophotometry and iodonitrotetrazolium chloride. Antibiotic resistance genes (tet(O) and gyrA) were characterized at the molecular level. The minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs) ranged from 25 to 1600 µg/mL. Cinnamon oil, (E)-Cinnamaldehyde, clove oil, eugenol, and baicalein had the lowest MIC and MBC values (25-100 µg/mL). MT947450 and MT947451 were sensitive to erythromycin and gentamicin but resistant to quinolones and tetracycline. Mutations in gyrA and tet(O) genes from resistant strains were confirmed by sequencing. The findings show that NPs are effective against drug-sensitive and drug-resistant Campylobacter strains. The resistance to antibiotics was confirmed at phenotypic and genotypic levels. This merits further studies to decipher the action mechanisms and synergistic activities of NPs.

Improvement in host metabolic homeostasis and alteration in gut microbiota in mice on the high-fat diet: A comparison of calcium supplements.

Despite the previously reported health benefits of calcium intake for the attenuation of metabolic disease, few studies have investigated the relationships among calcium intake, gut microbiota, and host metabolism. In this study, we assessed the effects of calcium supplementation on host microbial community composition and metabolic homeostasis. Mice were fed a high-fat diet with different calcium concentrations (4 and 12 g/kg) of 2 calcium supplements, calcium carbonate and calcium citrate. Supplementation with the higher concentration of calcium citrate significantly prevented body weight gain and decreased plasma biomarkers for metabolic disorder compared to calcium carbonate supplementation. Both calcium supplementation led to changes in microbial composition, increased propionate production and increased anorexigenic GLP-1 gene expression. The calcium citrate groups also experienced less metabolic endotoxemia. Our findings suggested that calcium supplementation could ameliorate host metabolic disorder caused by a high-fat diet, due to gut microbiota changes as well as decreased intestinal inflammation.

Brazilein Suppresses Inflammation through Inactivation of IRAK4-NF-κB Pathway in LPS-Induced Raw264.7 Macrophage Cells.

The medicinal herbal plant has been commonly used for prevention and intervention of disease and health promotions worldwide. Brazilein is a bioactive compound extracted from Caesalpinia sappan Linn. Several studies have showed that brazilein exhibited the immune suppressive effect and anti-oxidative function. However, the molecular targets of brazilein for inflammation prevention have remained elusive. Here, we investigated the mechanism underlying the inhibitory effect of brazilein on LPS-induced inflammatory response in Raw264.7 macrophage cells. We demonstrated that brazilein decreased the expression of IRAK4 protein led to the suppression of MAPK signaling and IKKß, and subsequent inactivation of NF-κB and COX2 thus promoting the expression of the downstream target pro-inflammatory cytokines such as IL-1ß, MCP-1, MIP-2, and IL-6 in LPS-induced Raw264.7 macrophage cells. Moreover, we observed that brazilein reduced the production of nitrite compared to the control in LPS-induced Raw264.7. Thus, we suggest that brazilein might be a useful bioactive compound for the prevention of IRAK-NF-κB pathway associated chronic diseases.

Role of the Red Ginseng in Defense against the Environmental Heat Stress in Sprague Dawley Rats.

Global temperature change causes heat stress related disorders in humans. A constituent of red ginseng has been known the beneficial effect on the resistance to many diseases. However, the mechanism of red ginseng (RG) against heat stress still remains unclear. To determine the effect of RG on heat stress, we examined the effect of the RG on the gene expression profiles in rats subjected to environmental heat stress. We evaluated the transcripts associated with hepatic lipid accumulation and oxidative stress in rats subjected to heat stress. We also analyzed the reactive oxygen species (ROS) contents. Our results suggested RG inhibited heat stress mediated altering mRNA expressions include HSPA1, DEAF1, HMGCR, and FMO1. We also determined RG attenuated fat accumulation in the liver by altering C/EBPß expression. RG promoted to repress the heat stress mediated hepatic cell death by inhibiting of Bcl-2 expression in rats subjected to heat stress. Moreover, RG administered group during heat stress dramatically decreased the malondialdehyde (MDA) contents and ROS associated genes compared with the control group. Thus, we suggest that RG might influence inhibitory effect on environmental heat stress induced abnormal conditions in humans.

Oleuropein suppresses LPS-induced inflammatory responses in RAW 264.7 cell and zebrafish.

Oleuropein is one of the primary phenolic compounds present in olive leaf. In this study, the anti-inflammatory effect of oleuropein was investigated using lipopolysaccharide (LPS)-stimulated RAW 264.7 and a zebrafish model. The inhibitory effect of oleuropein on LPS-induced NO production in macrophages was supported by the suppression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In addition, our enzyme immunoassay showed that oleuropein suppressed the release of pro-inflammatory cytokines such as interleukin-1ß (IL-1ß) and interleukin-6 (IL-6). Oleuropein inhibited the translocation of p65 by suppressing phosphorylation of inhibitory kappa B-α (IκB-α). Oleuropein also decreased activation of ERK1/2 and JNK, which are associated with LPS-induced inflammation, and its downstream gene of AP-1. Furthermore, oleuropein inhibited LPS-stimulated NO generation in a zebrafish model. Taken together, our results demonstrated that oleuropein could reduce inflammatory responses by inhibiting TLR and MAPK signaling, and may be used as an anti-inflammatory agent.

Low molecular weight fucoidan from the sporophyll of Undaria pinnatifida suppresses inflammation by promoting the inhibition of mitogen-activated protein kinases and oxidative stress in RAW264.7 cells.

The suppression of MAPK and oxidative stress could be an important anti-inflammatory mechanism. Fucoidan regulates MAPK activity in several cell lines. However, the mechanism for the anti-inflammatory and oxidative stress effect of low molecular weight fucoidan (LMWF) is poorly understood in RAW264.7 cells. The objective of this study was to examine the critical role of LMWF during LPS-induced inflammation in RAW264.7 cells. To determine the potential role of LMWF, we analysed pro-inflammatory cytokine, transcription factor, inflammation-related and oxidative stress related protein expression in vitro during LPS-induced inflammation in RAW264.7 cells. In this study, we demonstrated the anti-inflammatory effects of LMWF on LPS-induced inflammation in macrophages through the regulation of signalling pathways, including its effect on the attenuation of inflammatory cytokines, such as IL-1ß, IL-1 and TNF-α, and the degradation of phosphorylated p38 MAPK, ERK1/2 and JNK. This study also demonstrates that LMWF might block NO as well as the expression of reactive oxygen species (ROS), which subsequently inhibits the iNOS and COX-2 expression induced by LPS. Based on these findings, we suggest that LMWF might have great potential as an external pathogen prevention and intervention agent for inflammatory diseases.

Fucoidan regulate blood glucose homeostasis in C57BL/KSJ m+/+db and C57BL/KSJ db/db mice.

Type 2 diabetes mellitus is a multisystem disease that is characterized by hyperglycemia and is associated with the dysfunction and failure of various organs. The control of postprandial hyperglycemia is important in the prevention and intervention of type 2 diabetes. Fucoidan has several biological activities in vitro and in vivo. However, the effect of fucoidan on hyperglycemia in non-diabetic and diabetic mice has not been investigated. This study was undertaken to study the effects of different molecular weight forms (5 kilodalton (k), 5-30 k and crude) of fucoidan on oral glucose tolerance tests in non-diabetic mice and on food intake, weight gain, fasting blood glucose and blood biochemistry of db/db mice. Treatment with 200 mg/mL 5 k, 5-30 k and crude fucoidan substantially prevented hyperglycemia according to oral glucose tolerance tests in non-diabetic mice. In addition, fucoidan fractions significantly reduced blood glucose levels in diabetic mice.

Schisandra chinensis prevents hepatic lipid peroxidation and oxidative stress in rats subjected to heat environmental stress.

Increases in temperature cause a proliferation of heat-stress-related disorders by disrupting the body's homeostasis system, particularly when excessive levels of reactive oxygen species disrupt the balance of antioxidant defence systems. Thus, controlling oxidative stress is important for the regulation of body homeostasis. Schisandra chinensis (SC) has a potential effect on antioxidants and is resistant to high temperatures. However, the mechanism of SC during heat stress is unclear. Therefore, we evaluated the effect of SC on heat stress by performing several bioactive genetic assays on Sprague Dawley (SD) rats. The results demonstrated that heat stress significantly increased in heat-stress-related gene expression whereas it was dramatically reduced in the gene expression of the SC group. The genes related to oxidative stress were also significantly suppressed in the SC group compared with those of the heat stress group. Furthermore, there was a greater decrease in the MDA content of the SD rats in the orally administered SC group than in the heat exposure group. Thus, we demonstrate that SC has a protective effect on heat stress as a result of its strong antioxidant properties and the prevention of lipid peroxidation.

SEAWEED EXTRACTS AS A POTENTIAL TOOL FOR THE ATTENUATION OF OXIDATIVE DAMAGE IN OBESITY-RELATED PATHOLOGIES1.

Recent studies suggest that seaweed extracts are a significant source of bioactive compounds comparable to the dietary phytochemicals such as onion and tea extracts. The exploration of natural antioxidants that attenuate oxidative damage is important for developing strategies to treat obesity-related pathologies. The objective of this study was to screen the effects of seaweed extracts of 49 species on adipocyte differentiation and reactive oxygen species (ROS) production during the adipogenesis in 3T3-L1 adipocytes, and to investigate their total phenol contents and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities. Our results show that high total phenol contents were observed in the extracts of Ecklonia cava (see Table 1 for taxonomic authors) (681.1 ± 16.0 µg gallic acid equivalents [GAE] · g-1 ), Dictyopteris undulata (641.3 ± 70.7 µg GAE · g-1 ), and Laurencia intermedia (560.9 ± 48.1 µg GAE · g-1 ). In addition, DPPH radical scavenging activities were markedly higher in Sargassum macrocarpum (60.2%), Polysiphonia morrowii (55.0%), and Ishige okamurae (52.9%) than those of other seaweed extracts (P < 0.05). Moreover, treatment with several seaweed extracts including D. undulata, Sargassum micracanthum, Chondrus ocellatus, Gelidium amansii, Gracilaria verrucosa, and Grateloupia lanceolata significantly inhibited adipocyte differentiation and ROS production during differentiation of 3T3-L1 preadipocytes. Furthermore, the production of ROS was positively correlated with lipid accumulation (R2 = 0.8149). According to these preliminary results, some of the seaweed extracts can inhibit ROS generation, which may protect against oxidative stress that is linked to obesity. Further studies are required to determine the molecular mechanism between the verified seaweeds and ROS, and the resulting effects on obesity. [Table: see text].