Detection and characterization of potential virulence determinants in Staphylococcus pseudintermedius and S. schleiferi strains isolated from canine otitis externa in Korea.

BACKGROUND: A recent increase in the occurrence of canine skin and soft tissue infections, including otitis externa and pyoderma, caused by antimicrobial-resistant Staphylococcus pseudintermedius and S. schleiferi has become a significant public and veterinary health issues. OBJECTIVE: We investigated the virulence potentials associated with the occurrence of canine otitis externa in S. pseudintermedius and S. schleiferi. METHODS: In this study, the prevalence of genes encoding leukocidins, exfoliative toxins, and staphylococcal enterotoxins (SEs) was investigated using previously characterized S. pseudintermedius (n = 26) and S. schleiferi (n = 19) isolates derived from canine otitis externa. Susceptibility to cathelicidins (K9CATH and PMAP-36) and hydrogen peroxide (H2O2) was also examined in both staphylococcal species. RESULTS: A high prevalence of genes encoding leukocidins (lukS/F-I, lukS1/F1-S, and lukS2/F2-S), exfoliative toxins (siet, expB, and sset), and SEs was identified in both S. pseudintermedius and S. schleiferi isolates. Notably, S. pseudintermedius isolates possessed higher number of SE genes, especially newer SE genes, than S. schleiferi isolates harboring egc clusters. Although no significant differences in susceptibility to K9CATH and H2O2 were observed between the two isolate groups, S. pseudintermedius isolates exhibited enhanced resistance to PMAP-36 compared to S. schleiferi isolates. CONCLUSIONS: These findings suggest that high a prevalence of various toxin genes together with enhanced resistance to cathelicidins may contribute to the pathogenicity of S. pseudintermedius and S. schleiferi in canine cutaneous infections.

Species Profiles and Antimicrobial Resistance of Non-aureus Staphylococci Isolated from Healthy Broilers, Farm Environments, and Farm Workers.

Non-aureus staphylococci (NAS), particularly antimicrobial-resistant NAS, have a substantial impact on human and animal health. In the current study, we investigated (1) the species profiles of NAS isolates collected from healthy broilers, farm environments, and farm workers in Korea, (2) the occurrence of antimicrobial-resistant NAS isolates, especially methicillin resistance, and (3) the genetic factors involved in the methicillin and fluoroquinolone resistance. In total, 216 NAS isolates of 16 different species were collected from healthy broilers (n=178), broiler farm environments (n=18), and farm workers (n=20) of 20 different broiler farms. The two most dominant broiler-associated NAS species were Staphylococcus agnetis (23.6%) and Staphylococcus xylosus (22.9%). Six NAS isolates were mecA-positive carrying staphylococcal cassette chromosome mec (SCCmec) II (n=1), SCCmec IV (n=1), SCCmec V (n=2), or non-typeable SCCmec element (n=2). While two mecA-positive Staphylococcus epidermidis isolates from farm workers had SCCmec II and IV, a mecA-positive S. epidermidis isolate from broiler and a Staphylococcus haemolyticus isolate farm environment carried SCCmec V. The occurrence of multidrug resistance was observed in 48.1% (104/216 isolates) of NAS isolates with high resistance rates to ß-lactams (>40%) and fusidic acid (59.7%). Fluoroquinolone resistance was confirmed in 59 NAS isolates (27.3%), and diverse mutations in the quinolone resistance determining regions of gyrA, gyrB, parC, and parE were identified. These findings suggest that NAS in broiler farms may have a potential role in the acquisition, amplification, and transmission of antimicrobial resistance.

High Prevalence of Clonal Complex 398 Methicillin-Susceptible and -Resistant Staphylococcus aureus in Pig Farms: Clonal Lineages, Multiple Drug Resistance, and Occurrence of the Staphylococcal Cassette Chromosome mec IX.

High prevalence of livestock-associated methicillin-susceptible and -resistant Staphylococcus aureus (LA-MSSA and LA-MRSA, respectively) in livestock farms, particularly pig farms, is an increasingly serious threat to food safety and public health. In this study, 173 S. aureus (84 MRSA and 89 MSSA) isolates from healthy pigs, farm environments, and farm workers in Korea were examined to determine the (1) genetic diversity of S. aureus isolates (sequence type [ST], spa, and agr types), (2) staphylococcal cassette chromosome mec (SCCmec) types of MRSA isolates, and (3) multidrug resistance (MDR) phenotypes of MRSA and MSSA isolates. Clonal complex 398 (CC398) genotypes of MRSA and MSSA isolates, particularly CC398-spa type t571-agr I lineages, displaying MDR phenotypes were highly prevalent in pig farms. High prevalence of CC398-t571 MRSA and MSSA was more frequently associated with weaning piglets and growing pigs. Moreover, the same clonal lineages of S. aureus isolates colonized both pigs and farm workers, suggesting the transmission of antimicrobial-resistant CC398 MRSA and MSSA between pigs and humans in the pig farms. Furthermore, two dominant SCCmec types, SCCmec V and SCCmec IX, were identified in CC398 MRSA isolates colonizing healthy pigs. To the best of our knowledge, this is the first report of a CC398 LA-MRSA isolate carrying SCCmec IX in Korea. Collectively, these results suggest widespread distribution of the CC398 lineage among MRSA and MSSA isolates in pigs, farm environments, and farm workers in Korea.

Clonal distribution and antimicrobial resistance of methicillin-susceptible and -resistant Staphylococcus aureus strains isolated from broiler farms, slaughterhouses, and retail chicken meat.

Colonization of food-producing animals by antimicrobial-resistant Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), has become a serious public health problem worldwide. In the current study, clonal diversities of livestock-associated S. aureus isolates collected from broiler farms, slaughterhouses, and retail chicken meat were examined. Two-hundred S. aureus isolates (43 MRSA and 157 methicillin-susceptible S. aureus [MSSA] isolates) were analyzed to determine 1) the genotypes of the isolates (multilocus sequence, agr, and spa types), 2) the methicillin resistance phenotype and staphylococcal cassette chromosome mec (SCCmec) types, 3) the antimicrobial resistance profiles, and 4) the mutational changes in gyrA, gyrB, parC, and parE in fluoroquinolone-resistant isolates. Fifteen different sequence types (STs) of MSSA strains displaying a relatively high degree of genetic diversity were detected in broiler farms, slaughterhouses, and retail chicken meat. In contrast to MSSA, 2 dominant genetic lineages of MRSA (ST692-SCCmecV with t2249 spa type, and ST188-SCCmecIVa with spa type t189) were found in healthy broilers. The high prevalence of ST692 and ST188 in healthy broilers is associated with high levels of multiple antimicrobial-resistance phenotypes, particularly fluoroquinolone resistance. All fluoroquinolone-resistant isolates carried double point mutations in gyrA (S84L) and parC (S80F), regardless of STs or methicillin resistance. Notably, only the ST188 lineage carried an additional third mutation in gyrB (D494N), correlating with enhanced ciprofloxacin minimum inhibitory concentration values versus the strains with double mutations. These results provide important insights into the genetic diversity of antimicrobial-resistant S. aureus strains associated with the chicken meat production chain, including healthy broilers, in Korea.

Profiles of Non-aureus Staphylococci in Retail Pork and Slaughterhouse Carcasses: Prevalence, Antimicrobial Resistance, and Genetic Determinant of Fusidic Acid Resistance.

As commensal colonizers in livestock, there has been little attention on staphylococci, especially non-aureus staphylococci (NAS), contaminating meat production chain. To assess prevalence of staphylococci in retail pork and slaughterhouse carcass samples in Korea, we collected 578 samples from Korean slaughterhouses (n=311) and retail markets (n=267) for isolation of staphylococci and determined antimicrobial resistance phenotypes in all the isolates. The presence of and prevalence of fusB-family genes (fusB, fusC, fusD, and fusF) and mutations in fusA genes were examined in fusidic acid resistant isolates. A total of 47 staphylococcal isolates of 4 different species (Staphylococcus aureus, n=4; S. hyicus, n=1; S. epidermidis, n=10; Mammaliicoccus sciuri, n=32) were isolated. Fusidic acid resistance were confirmed in 9/10 S. epidermidis and all of the 32 M. sciuri (previously S. sciuri) isolates. Acquired fusidic acid resistance genes were detected in all the resistant strains; fusB and fusC in S. epidermidis and fusB/C in M. sciuri. Multi-locus sequence type analysis revealed that ST63 (n=10, 31%) and ST30 (n=8, 25%) genotypes were most prevalent among fusidic acid resistant M. sciuri isolates. In conclusion, the high prevalence of fusB-family genes in S. epidermidis and M. sciuri strains isolated from pork indicated that NAS might act as a reservoir for fusidic acid resistance gene transmissions in pork production chains.

Effect of extracorporeal shock wave therapy on keratinocytes derived from human hypertrophic scars.

Hypertrophic scars represent a common complication in burn patients. In addition to cosmetic defects, they may cause serious sensory abnormalities such as pain and itching, severe dysfunction depending on the site, and emotional disorders such as anxiety and depression. The present study aimed to identify the molecular mechanisms underlying the use of extracorporeal shock wave therapy in keratinocytes. Keratinocytes derived from hypertrophic scar tissue were cultured and expression of proliferation markers (keratin 5 and 14), activation markers (keratin 6 and 17), differentiation markers (keratin 1, 10, and involucrin), apoptosis factors (Bax, Bcl2, and Caspase 14), and proliferation/differentiation regulators (p21 and p27) was investigated to compared with that of those in keratinocytes derived from normal skin tissue. Scar-derived keratinocytes were treated with extracorporeal shock waves under 1000 impulses at 0.1, 0.2, and 0.3 mJ/mm2. Shock waves altered the molecular pattern of proliferation, activation, differentiation, and apoptosis, as well as proliferation/ differentiation regulators, including Bax, Bcl2, ASK1, p21, p27, and Notch1. In summary, we show that extracorporeal shock wave therapy regulates the proliferation and differentiation of keratinocytes derived from hypertrophic scar to maintain normal epidermal integrity.

Species Distribution, Antimicrobial Resistance, and Enterotoxigenicity of Non-aureus Staphylococci in Retail Chicken Meat.

Non-aureus staphylococci (NAS), including coagulase-negative staphylococci, have emerged as important causes of opportunistic infections in humans and animals and a potential cause of staphylococcal food poisoning. In this study, we investigated (i) the staphylococcal species profiles of NAS in in retail chicken meat, (ii) the phenotypic and genotypic factors associated with antimicrobial resistance in the NAS isolates, and (iii) the prevalence of classical and newer staphylococcal enterotoxin (SE) genes. A total of 58 NAS of nine different species were isolated from retail raw chicken meat samples. The occurrence of multidrug resistance in the NAS, particularly S. agnetis and S. chromogenes, with high resistance rates against tetracycline or fluoroquinolones were confirmed. The tetracycline resistance was associated with the presence of tet(L) in S. chromogenes and S. hyicus or tet(K) in S. saprophyticus. The occurrence of fluoroquinolone resistance in S. agnetis and S. chromogenes was usually associated with mutations in the quinolone resistance determining regions (QRDR) of gyrA and parC. In addition, the frequent presence of SE genes, especially seh, sej, and sep, was detected in S. agnetis and S. chromogenes. Our findings suggest that NAS in raw chicken meat can have potential roles as reservoirs for antimicrobial resistance and enterotoxin genes.

Effect of Combining Low Temperature Plasma, Negative Pressure Wound Therapy, and Bone Marrow Mesenchymal Stem Cells on an Acute Skin Wound Healing Mouse Model.

Low-temperature plasma (LTP; 3 min/day), negative pressure wound therapy (NPWT; 4 h/day), and bone marrow mesenchymal stem cells (MSCs; 1×106 cells/day) were used as mono- and combination therapy in an acute excisional skin wound-healing ICR mouse model. These therapies have been beneficial in treating wounds. We investigated the effectiveness of monotherapy with LTP, NPWT, and MSC and combination therapy with LTP + MSC, LTP + NPWT, NPWT + MSC, and LTP + NPWT + MSC on skin wounds in mice for seven consecutive days. Gene expression, protein expression, and epithelial thickness were analyzed using real time polymerase chain reaction (RT-qPCR), western blotting, and hematoxylin and eosin staining (H&E), respectively. Wound closure was also evaluated. Wound closure was significantly accelerated in monotherapy groups, whereas more accelerated in combination therapy groups. Tumor necrosis factor-α (TNF-α) expression was increased in the LTP monotherapy group but decreased in the NPWT, MSC, and combination therapy groups. Expressions of vascular endothelial growth factor (VEGF), α-smooth muscle actin (α-SMA), and type I collagen were increased in the combination therapy groups. Re-epithelialization was also considerably accelerated in combination therapy groups. Our findings suggest that combination therapy with LPT, NPWT, and MSC exert a synergistic effect on wound healing, representing a promising strategy for the treatment of acute wounds.

Determination of Glucan Contents in the Fruiting Bodies and Mycelia of Lentinula edodes Cultivars.

Shiitake mushrooms (Lentinula edodes) containing ß-glucans may be beneficial for human health; they have been used in the treatment of cancer, hypertension, and high cholesterol levels. The objective of this study was to determine the ß-glucan content in different sections of the fruiting bodies and mycelia of ten shiitake mushroom cultivars. The measured ß-glucan content ranged from 20.06 ± 1.76% to 44.21 ± 0.13% in the pileus sections, and from 29.74 ± 1.40% to 56.47 ± 4.72% in the stipe sections. The results of this study indicate that the variance in ß-glucan content dependent on the shiitake cultivar, and that the ß-glucan content is higher in the stipe than in the pileus.

Sorghum extract exerts an anti-diabetic effect by improving insulin sensitivity via PPAR-γ in mice fed a high-fat diet.

This study investigated the hypothesis that a sorghum extract exerts anti-diabetic effects through a mechanism that improves insulin sensitivity via peroxisome proliferator-activated receptor gamma (PPAR-γ) from adipose tissue. Seven C57BL/6 mice were fed an AIN-93M diet with fat consisting of 10% of total energy intake (LF) for 14 weeks, and 21 mice were fed a high-fat AIN diet with 60% of calories derived from fat (HF). From week 8, the HF diet-fed mice were orally administered either saline (HF group), 0.5% (0.5% SE group), or 1% sorghum extract (1% SE group) for 6 weeks (n = 7/group). Perirenal fat content was significantly lower in the 0.5% SE and 1% SE groups than that in the HF mice. Levels of total and low-density lipoprotein cholesterol, triglycerides, glucose, and the area under the curve for glucose were significantly lower in mice administered 0.5% SE and 1% SE than those in HF mice. Serum insulin level was significantly lower in mice administered 1% SE than that in HF mice or those given 0.5% SE. PPAR-γ expression was significantly higher, whereas the expression of tumor necrosis factor-α was significantly lower in mice given 1% SE compared to those in the HF mice. Adiponectin expression was also significantly higher in mice given 0.5% SE and 1% SE than that in the HF mice. These results suggest that the hypoglycemic effect of SE may be related with the regulation of PPAR-γ-mediated metabolism in this mouse model.