Detection of plasmid-mediated quinolone resistance genes in ß-lactamase-producing Escherichia coli isolates from layer hens.

This study was conducted to investigate the presence of plasmid-mediated quinolone resistance (PMQR) genes in ß-lactamase-producing Escherichia coli isolates from layer hens and to characterize their molecular background. Among 142 E. coli isolates, 86 (60.6%) showed multidrug resistance and 15 (10.6%) were found to be ß-lactamase-producing E. coli. Extended-spectrum ß-lactamase (ESBL) and plasmid-mediated AmpC (pAmpC) ß-lactamase genes, blaCTX-M-14 and blaCMY-2, were identified in three and six E. coli isolates, respectively. The non-ESBL or pAmpC gene, blaTEM-1, was found in eight of the isolates. Two isolates had both genes, blaCTX-M-14 and blaTEM-1. Among the 15 ß-lactamase-producing E. coli, six PMQR genes, qnrS1 (n = 3) and qnrB4 (n = 3), were identified. Among the six PMQR-positive E. coli isolates, four exhibited double amino acid exchanges at both gyrA and parC with ciprofloxacin and enrofloxacin minimum inhibitory concentrations of ≥32 and ≥16 µg/mL, respectively. Additionally, five transconjugants (33.3%) showed a transferability of ß-lactamase and PMQR genes. Pulsed-field gel electrophoresis (PFGE) analysis was conducted to investigate the 15 ß-lactamase-producing E. coli isolates. In PFGE, E. coli included three PFGE patterns showing the same farms and in accordance with both ß-lactamase and PMQR genes and the antimicrobial resistance pattern. Layer hens may act as a reservoir of antibiotic-resistant bacteria, and the PMQR gene in ß-lactamase-producing E. coli isolates from layer hens has the potential to enter the food chain. Therefore, our findings suggest that comprehensive surveillance of antimicrobial use in laying operation systems is necessary.

Characteristics of the antimicrobial resistance of Staphylococcus aureus isolated from chicken meat produced by different integrated broiler operations in Korea.

Vertical integration of the broiler industry allows producers to combine different biosecurity and sanitation practices, housing technologies, and feeding regimens to improve food safety. The purpose of this study was to investigate the prevalence of Staphylococcus aureus (S. aureus) and to characterize the antimicrobial-resistant isolates recovered from 7 different integrated broiler operation systems in Korea. Among 200 chicken meat samples, 94 were observed to be positive for S. aureus. However, the prevalence varied from 25.0 to 58.3% in chicken meats, indicating variation in S. aureus occurrence among the operations. Four methicillin-resistant S. aureus isolates (MRSA) were recovered from 3 different operations. A high proportion of the S. aureus isolates were resistant to penicillins (51.2%), tetracycline (38.8%), and ciprofloxacin (CIP; 33.9%). Especially, 3 different operations showed a high number of CIP resistance (45.5∼100%) and multidrug resistance (50.0∼100%). Among 41 CIP-resistant S. aureus isolates, 75.6% showed a double amino-acid exchange of both gyrA and parC, with CIP minimum inhibitory concentrations (MIC) of ≥32 µg/mL. Four MRSA isolates showed resistance to 5 or 7 classes of antimicrobial agents, exhibiting oxacillin, CIP, and enrofloxacin MIC ranges of 16 to 128, 32 to 64, and 8 to 128 µg/mL, respectively, and had double mutations of S84L/S80F in gyrA/parC. Our findings suggest that S. aureus with resistance to important antimicrobial compounds can now be found in association with integrated broiler operations, providing the data to support the development of a monitoring and prevention program in integrated operations.

Comparison of body surface area-based and weight-based dosing format for oral prednisolone administration in small and large-breed dogs.

This study compared the pharmacokinetics of Prednisolone (PDS) in small- and large breed dogs with a dosing format based on body surface area (BSA) or body weight (BW). The maximum concentration and area under the curve in large-breed dogs orally administered 2 mg/kg PDS were significantly greater than those in small-breed dogs given 2 mg/kg and in large-breed dogs given 40 mg/m2. The higher blood concentrations that result from BW-based dosing of oral PDS in large-breed dogs can be more than required for effect. Meanwhile, BSA dosing at 40 mg/m may be suboptimal. These findings confirm important differences between standard PDS dosing schemes in dogs while highlighting the need to further optimize PDS dosing in large-breed dogs.

Long-Term Management with Adipose Tissue-Derived Mesenchymal Stem Cells and Conventional Treatment in a Dog with Hepatocutaneous Syndrome.

Hepatocutaneous syndrome (HS) is an uncommon skin disorder that occurs in conjunction with liver disease and is diagnosed based on decreased plasma concentrations of amino acids and the histopathology of skin lesions. The survival period generally is <6 months. A 10-year-old castrated male Maltese dog was presented for evaluation of lethargy, polyuria, polydipsia, and skin lesions including alopecia, erythema, and crusts. Based on increased liver enzyme activity, low plasma amino acid concentrations, and findings from liver cytology and skin biopsy, the dog was diagnosed with HS. In addition to administration of antioxidants, hepatoprotective agents, and amino acids IV, allogenic adipose tissue-derived mesenchymal stem cells were infused 46 times over a 30-month period: 8 times directly into the liver parenchyma guided by ultrasonography and the remainder of the times into peripheral veins. After commencing stem cell therapy, the dog's hair re-grew and the skin lesions disappeared or became smaller. During ongoing management, the patient suddenly presented with anorexia and uncontrolled vomiting, and severe azotemia was observed. The dog died despite intensive care. On necropsy, severe liver fibrosis and superficial necrolytic dermatitis were observed. The dog survived for 32 months after diagnosis. A combination of amino acid and stem cell therapy may be beneficial for patients with HS.

Pharmacokinetics and Acid Suppressant Efficacy of Esomeprazole after Intravenous, Oral, and Subcutaneous Administration to Healthy Beagle Dogs.

BACKGROUND: Esomeprazole is an S-enantiomer of omeprazole that has favorable pharmacokinetics and efficacious acid suppressant properties in humans. However, the pharmacokinetics and effects on intragastric pH of esomeprazole in dogs have not been reported. OBJECTIVE: To determine the pharmacokinetics of esomeprazole administered via various routes (PK study) and to investigate the effect of esomeprazole on intragastric pH with a Bravo pH monitoring system (PD study). ANIMALS: Seven adult male Beagle dogs and 5 adult male Beagle dogs were used for PK and PD study, respectively. METHODS: Both studies used an open, randomized, and crossover design. In the PK study, 7 dogs received intravenous (IV), subcutaneous (SC), and oral doses (PO) of esomeprazole (1 mg/kg). Each treatment period was separated by a washout period of at least 10 days. Esomeprazole plasma concentrations were measured by HPLC/MS/MS. In the efficacy study, intragastric pH was recorded without medication (baseline pH) and following IV, SC, and PO esomeprazole dosing regimens (1 mg/kg) in 5 dogs. RESULTS: The bioavailability of esomeprazole administered as PO enteric-coated granules and as SC injections was 71.4 and 106%, respectively. The half-life was approximately 1 hour. Mean ± SD percent time intragastric pH was ≥3 and ≥4 was 58.9 ± 21.1% and 40.9 ± 17.3% for IV group, 75.8 ± 16.4% and 62.7 ± 17.7% for SC group, 88.2 ± 8.9% and 82.5 ± 7.7% for PO group, and 12.5 ± 3.6% and 3.7 ± 1.8% for baseline. The mean percent time with intragastric pH was ≥3 or ≥4 was significantly increased regardless of the dosing route (P < .05). CONCLUSION: The PK parameters for PO and SC esomeprazole administration were favorable, and esomeprazole significantly increased intragastric pH after IV, PO, and SC administration. IV and SC administration of esomeprazole might be useful when PO administration is not possible. No significant adverse effects were observed.

Enrofloxacin enhances the effects of chemotherapy in canine osteosarcoma cells with mutant and wild-type p53.

Adjuvant chemotherapy improves survival time in dogs receiving adequate local control for appendicular osteosarcoma, but most dogs ultimately succumb to metastatic disease. The fluoroquinolone antibiotic enrofloxacin has been shown to inhibit survival and proliferation of canine osteosarcoma cells in vitro. Others have reported that fluoroquinolones may modulate cellular responses to DNA damaging agents and that these effects may be differentially mediated by p53 activity. We therefore determined p53 status and activity in three canine osteosarcoma cell lines and examined the effects of enrofloxacin when used alone or in combination with doxorubicin or carboplatin chemotherapy. Moresco and Abrams canine osteosarcoma cell lines contained mutations in p53, while no mutations were identified in the D17 cells or in a normal canine osteoblast cell line. The addition of enrofloxacin to either doxorubicin or carboplatin resulted in further reductions in osteosarcoma cell viability; this effect was apparent regardless of p53 mutational status or downstream activity.

Pharmacokinetic study of meropenem in healthy beagle dogs receiving intermittent hemodialysis.

Meropenem, a second carbapenem antimicrobial agent with a broad spectrum of activity, is used to treat sepsis and resistant-bacterial infections in veterinary medicine. The objective of this study was to identify the pharmacokinetics of meropenem in dogs receiving intermittent hemodialysis (IHD) and to determine the proper dosing in renal failure patients receiving IHD. Five healthy beagle dogs were given a single i.v. dose of 24 mg/kg of meropenem and received IHD. The blood flow rate, dialysate flow, and ultrafiltration rate were maintained at 40 mL/min, 300 mL/min, and 40 mL/h, respectively. Blood samples were collected for 24 h from the jugular vein and from the extracorporeal arterial and venous line. Urine samples and dialysate were also collected. The concentrations of meropenem were assayed using HPLC/MS/MS determination. The peak plasma concentration was 116 ± 37 µg/mL at 15 min. The systemic clearance was 347 ± 117 mL/h/kg, and the steady-state volume of distribution was 223 ± 67 mL/kg. Dialysis clearance was 71.1 ± 34.3 mL/h/kg, and the extraction ratio by hemodialysis was 0.455 ± 0.150. The half-life (T1/2 ) in dogs with IHD decreased compared with those without IHD, and the reduction in T1/2 was greater in renal failure patients than in normal patients. Sixty-nine percent and 21% of the administered drug were recovered by urine and dialysate in the unchanged form, respectively. In conclusion, additional dosing of 24 mg/kg of meropenem after dialysis could be necessary according to the residual renal function of the patient based on the simulated data.

The pharmacokinetics of the ß2-adrenoceptor agonist, tulobuterol, in Beagle dogs following transdermal and intravenous administration.

Tulobuterol is a ß2-adrenergic agonist that was the first bronchodilator approved as a transdermal patch for humans. Previous studies have examined the pharmacokinetics of tulobuterol in humans but not in the veterinary species. In this study, the pharmacokinetics of tulobuterol was examined in healthy Beagle dogs after transdermal and intravenous administration. The Cmax was 2.09 ng/mL at 16.0 h for a 0.2 mg/kg patch and 4.85 ng/mL at 13.6 h for a 0.4 mg/kg patch. The effective blood level in humans is 1-3 ng/mL, a concentration achieved using the 0.2 mg/kg patch in dogs. In conclusion, application of a 0.2 mg/kg tulobuterol patch to healthy dogs led to an apparently effective blood concentration for 24 h.

Association of obesity with serum leptin, adiponectin, and serotonin and gut microflora in beagle dogs.

BACKGROUND: Serotonin (5-hydroxytryptamine, 5HT) is involved in hypothalamic regulation of energy consumption. Also, the gut microbiome can influence neuronal signaling to the brain through vagal afferent neurons. Therefore, serotonin concentrations in the central nervous system and the composition of the microbiota can be related to obesity. OBJECTIVE: To examine adipokine, and, serotonin concentrations, and the gut microbiota in lean dogs and dogs with experimentally induced obesity. ANIMALS: Fourteen healthy Beagle dogs were used in this study. METHODS: Seven Beagle dogs in the obese group were fed commercial food ad libitum, over a period of 6 months to increase their weight and seven Beagle dogs in lean group were fed a restricted amount of the same diet to maintain optimal body condition over a period of 6 months. Peripheral leptin, adiponectin, 5HT, and cerebrospinal fluid (CSF-5HT) levels were measured by ELISA. Fecal samples were collected in lean and obese groups 6 months after obesity was induced. Targeted pyrosequencing of the 16S rRNA gene was performed using a Genome Sequencer FLX plus system. RESULTS: Leptin concentrations were higher in the obese group (1.98 ± 1.00) compared to those of the lean group (1.12 ± 0.07, P = .025). Adiponectin and 5-hydroytryptamine of cerebrospinal fluid (CSF-5HT) concentrations were higher in the lean group (27.1 ± 7.28) than in the obese group (14.4 ± 5.40, P = .018). Analysis of the microbiome revealed that the diversity of the microbial community was lower in the obese group. Microbes from the phylum Firmicutes (85%) were predominant group in the gut microbiota of lean dogs. However, bacteria from the phylum Proteobacteria (76%) were the predominant group in the gut microbiota of dogs in the obese group. CONCLUSIONS AND CLINICAL IMPORTANCE: Decreased 5HT levels in obese group might increase the risk of obesity because of increased appetite. Microflora enriched with gram-negative might be related with chronic inflammation status in obese dogs.

Serum adipokine concentrations in dogs with acute pancreatitis.

BACKGROUND: Limited information is available about the role of adipokines in the development and progression of acute pancreatitis (AP) in dogs. OBJECTIVES: To determine whether the circulating concentrations of adipokines differed between healthy dogs and dogs with AP, and whether the circulating concentrations differed between AP survivors and AP nonsurvivors. ANIMALS: Twenty-eight healthy dogs and 25 client-owned dogs with AP. METHODS: Prospective observational cohort study of 25 client-owned dogs with newly diagnosed AP and 28 otherwise healthy dogs with similar body condition scores. The serum concentrations of leptin, adiponectin, resistin, visfatin, interleukin (IL)-1ß, IL-6, IL-10, IL-18, and tumor necrosis factor (TNF)-α were measured. RESULTS: The serum concentrations of leptin (P = .0021), resistin (P = .0010), visfatin (P < .0001), IL-1ß (P < .0001), IL-6 (P = .0002), IL-10 (P < .0001), and IL-18 (P < .0001) were significantly higher in dogs with AP than healthy dogs, whereas the adiponectin concentration (P = .0011) was significantly lower. There were significant differences in the serum concentrations of leptin (P = .028) and adiponectin (P = .046) in survivors and nonsurvivors. After the disappearance of clinical signs, the concentrations of resistin (P = .037) and IL-1ß (P = .027) decreased significantly, whereas the serum concentrations of leptin (P > .999), adiponectin (P = .11), visfatin (P = .83), IL-6 (P = .82), IL-10 (P = .82), IL-18 (P = .56), and TNF-α (P = .94) did not differ significantly. CONCLUSION AND CLINICAL IMPORTANCE: This study showed that dysregulation of adipokines might be involved in the pathogenesis of AP. In addition, leptin and adiponectin are likely to be associated with mortality rate in AP.

Pharmacokinetics and pharmacodynamics (PK/PD) of irbesartan in Beagle dogs after oral administration at two dose rates.

Irbesartan (Irb) is an angiotensin II type 1 receptor antagonist widely used in humans to treat hypertension. Age-related diseases such as hypertension are increasingly being diagnosed in dogs and there is the need for new drugs. The PK/PD of Irb was tested in Beagle dogs. Ten healthy Beagles were orally administered two dose rates (2 and 5 mg/kg), according to a cross over study design. Blood collections for PK analysis and systolic blood pressure (SBP), heart and respiratory rate, mucous membranes colour, capillary refill time and temperature evaluations were performed at scheduled intervals. The drug plasma concentration was dose dependent. The dogs administered 5 mg/kg showed a significant reduction in SBP, while in those receiving 2 mg/kg, this parameter was minimally affected. A counter clockwise hysteresis showed no direct correlation between SBP and plasma concentrations. The minimum effective concentration was theorized to be within the range 550-800 ng/mL. Although further studies are necessary, 5 mg/kg seems to be the more appropriate dose to obtain a hypotensive effect in Beagle dogs.

Homocysteine induces COX-2 expression in macrophages through ROS generated by NMDA receptor-calcium signaling pathways.

Homocysteine (Hcy) at elevated levels is a putative risk factor for many cardiovascular disorders including atherosclerosis. In the present study, we investigated the effect of Hcy on the expression of cyclooxygenase (COX)-2 in murine macrophages and the mechanisms involved. Hcy increased the expression of COX-2 mRNA and protein in dose- and time-dependent manners, but did not affect COX-1 expression. Hcy-induced COX-2 expression was attenuated not only by the calcium chelators, EGTA and BAPTA-AM, but also by an antioxidant, N-acetylcysteine. Calcium chelators also attenuated Hcy-induced reactive oxygen species (ROS) production in macrophages, indicating that Hcy-induced COX-2 expression might be mediated through ROS generated by calcium-dependent signaling pathways. In another series of experiments, Hcy increased the intracellular concentration of calcium in a dose-dependent manner, which was attenuated by MK-801, an N-methyl-D-aspartate (NMDA) receptor inhibitor, but not by bicuculline, a gamma-aminobutyric acid receptor inhibitor. Molecular inhibition of NMDA receptor using small interfering RNA also attenuated Hcy-induced increases in intracellular calcium. Furthermore, both ROS production and Hcy-induced COX-2 expression were also inhibited by MK-801 as well as by molecular inhibition of NMDA receptor. Taken together, these findings suggest that Hcy enhances COX-2 expression in murine macrophages by ROS generated via NMDA receptor-mediated calcium signaling pathways.

Zonisamide monotherapy for idiopathic epilepsy in dogs.

AIM: To evaluate the efficacy of zonisamide as a monotherapy in dogs with idiopathic epileptic seizure. METHODS: The experiment was conducted on 10 dogs with idiopathic epilepsy that were treated at the Seoul National University Hospital for Animals. A diagnosis was conducted based on physical and neurologic examination, complete blood count and chemical analysis, magnetic resonance imaging and cerebrospinal fluid analyses. Idiopathic epilepsy was diagnosed when all of these examinations were normal. Oral zonisamide was administrated to 10 dogs with idiopathic epilepsy at 5-15 mg/kg per os every 12 h to achieve a concentration of zonisamide in serum of 10-40 µg/mL. The frequency of seizures before and after the administration of zonisamide therapy was recorded and the concentrations of zonisamide in serum were measured. RESULTS: Six (60%) of the dogs were favourable responders to treatment, showing a ≥50% reduction in monthly frequency of seizures. Of the remaining four, two dogs did not show a reduction and the other two showed an increase in frequency of seizures. The mean dosage of zonisamide for favourable responders was 7.92 (SD 3.79) mg/kg, which was administered orally twice a day. Only one dog, which was one of the unfavourable responders in the whole study, experienced mild side effects. CONCLUSIONS: Among the dogs treated with oral zonisamide, 60% responded favourably. The effect of zonisamide as an anticonvulsant drug was demonstrated in this study. CLINICAL RELEVANCE: Based on these results, zonisamide monotherapy is effective in some dogs with idiopathic epilepsy.

SOX2 has a crucial role in the lineage determination and proliferation of mesenchymal stem cells through Dickkopf-1 and c-MYC.

SOX2 is a well-known core transcription factor in embryonic stem cells (ESCs) and has an important role in the maintenance of pluripotency. Recently, SOX2 expression has also been reported in adult stem cells (ASCs), but the role of SOX2 in ASCs remains unknown. In this study, we examined the molecular mechanisms of SOX2 in human mesenchymal stem cells (hMSCs), a type of ASCs, by performing inhibition studies. SOX2 inhibition resulted in altered cell growth and differentiation capabilities. These changes coincided with a decrease in Dickkopf-1 (DKK1), a soluble inhibitor of WNT signaling. Chromatin immunoprecipitation and luciferase assays showed that SOX2 binds to DKK1 and has a positive regulatory role in transcription. The enforced expression of DKK1 in SOX2-inhibited hMSCs reversed the differentiation deformities, but could not abrogate the cell proliferation defect. Proliferation was regulated by c-MYC, whose expression can also be controlled by SOX2. Our study shows that SOX2 directly regulates DKK1 expression and, as a consequence, determines the differentiation lineage of hMSCs. Moreover, SOX2 also regulates proliferation by affecting c-MYC. Therefore, these results suggest that SOX2 might have a specific function by regulating DKK1 and c-MYC in the differentiation and growth of ASCs, which is separate from its roles in ESCs.

Intra-operative warming with a forced-air warmer in preventing hypothermia after tourniquet deflation in elderly patients.

This randomized, single-blind study aimed to explore the effects of intra-operative warming with a forced-air warmer in the prevention of hypothermia after tourniquet deflation in elderly patients undergoing unilateral total knee replacement arthroplasty under general anaesthesia. Patients were randomized to receive either intra-operative warming using a forced-air warmer with an upper body blanket (warming group; n = 12) or no intra-operative warming (nonwarming group; n = 12). Oesophageal temperature was measured as core body temperature. At 30 min following tourniquet inflation, the core body temperature started to increase in the warming group whereas it continued to drop in the non-warming group. This difference was statistically significant. The final core body temperature after tourniquet deflation was significantly higher in the warming group (mean +/- SD 36.1 +/- 0.2 degrees C) than in the non-warming group (35.4 +/- 0.3 degrees C). Intra-operative forced-air warming increased the core body temperature before tourniquet deflation and prevented subsequent hypothermia in elderly patients under general anaesthesia.

Protective role of interleukin-10 promoter gene polymorphism in the pathogenesis of invasive pulmonary aspergillosis after allogeneic stem cell transplantation.

The current study attempted to evaluate the association between the IL-10 promoter gene single nucleotide polymorphism (SNP) and invasive pulmonary aspergillosis (IPA) after allogeneic stem cell transplantation (SCT) in 105 patients. Three single-nucleotide polymorphisms were investigated in the proximal region of the IL-10 promoter gene (-1082/-819/-592). Two haplotypes (1082*A/819*T/592*A [ATA] and 1082*A/819*C/592*C [ACC]) were found in the current study. The overall incidence of IPA was estimated as 14.1+/-4.5% with a median onset at 186 days post-transplant (62 approximately 405 days). An increased occurrence of IPA was noted dependent on the IL-10 haplotype (0% vs 11.5+/-6.4% vs 19.7+/-7.7% for ACC/ACC vs ATA/ACC vs ATA/ATA haplotype, P=0.0307 when comparing ACC with non-ACC haplotype). In a multivariate survival analysis using Cox's proportional hazard model, the IL-10 promoter gene SNPs were identified as an independent predictive factor for the development of IPA (P=0.012, hazard ratio (HR) 9.3), along with an histocompatibility leukocyte antigen (HLA)-identical donor (P=0.005, HR 16.3), the CD34+ cell dose transplanted (P=0.004, HR 26.5), and time-dependent chronic graft-versus-host disease (GVHD; P=0.049, HR 16.0). The IL-10 ACC haplotype was found to have an apparent protective role in the development of IPA after allogeneic transplantation, regardless of HLA-disparity or chronic GVHD.

Mizoribine-mediated apoptotic signaling pathway in human T-Cell line.

Mizoribine (MZR), an inhibitor of inosine monophosphate dehydrogenase, which depletes cellular guanadine triphosphate, is an immunosuppressive drug. The aim of this study was to evaluate the mechanism by which MZR exerts cytotoxic effects on human Jurkat T cells. Our study showed that MZR-induced apoptotic death of human Jurkat T cells is dose-dependent and time-dependent, as revealed by chromatin condensation and H2AX phosphorylation. Furthermore, MZR increased the catalytic activity of caspase family cysteine proteases, including caspase-3, caspase-8, and caspase-9, in human Jurkat T cells. In conclusion, MZR induces the apoptotic death of human Jurkat T cells via activation of caspase family proteases as well as by mitochondrial dysfunction.