Prevalence and characterization of antimicrobial-resistant Escherichia coli isolated from veterinary staff, pets, and pet owners in Thailand.

BACKGROUND: Companion animals may act as antimicrobial resistance (AMR) reservoirs. This study investigated the prevalence and AMR patterns of Escherichia coli in pets and people in close contact with pets. METHODS: A total of 955 samples were collected from veterinary clinics across Thailand by rectal and skin or ear swabs from dogs and cats and fecal swabs from veterinarians, veterinary assistants, and pet owners. The minimum inhibitory concentrations (MICs) of the obtained isolates were investigated using Sensititre™ MIC plates against 21 different antimicrobial drugs. RESULTS: Escherichia coli from pets was frequently resistant to ampicillin (100%) and amoxicillin-clavulanic acid (100%), whereas E. coli from pet owners, veterinarians, and veterinary assistants was mostly resistant to tetracycline. The multiple antibiotic resistance index revealed that multidrug-resistant E. coli isolates were frequently found in dogs (34.92%), cats (62.12%), veterinarians (61.11%), veterinarian assistants (36.36%), and pet owners (47.62%). The most common AMR genes identified in this study were blaCTX-M, blaTEM, tetA, and tetB, which were associated with the antimicrobial susceptibility results. Additionally, extended-spectrum beta-lactamase (ESBL)-associated genes (i.e., blaCTX-M, blaTEM, and blaSHV) were found in 21.69%, 71.97%, 27.78%, and 21.43% of E. coli isolated from dogs, cats, veterinarians, and pet owners, respectively. CONCLUSIONS: Our findings demonstrated the presence of AMR genes, particularly ESBL-associated genes, in E. coli isolated from healthy pets and veterinarians. This implies that these sources of E. coli could potentially be reservoirs for antibiotic resistance, thereby increasing the risk of harm to both humans and animals. These findings highlight the importance of implementing effective AMR control measures in veterinary practices, as bacteria resistant to commonly used antimicrobials are present in humans and animals.

Kaempferia parviflora Extracts Protect Neural Stem Cells from Amyloid Peptide-Mediated Inflammation in Co-Culture Model with Microglia.

The existence of neuroinflammation and oxidative stress surrounding amyloid beta (Aß) plaques, a hallmark of Alzheimer's disease (AD), has been demonstrated and may result in the activation of neuronal death and inhibition of neurogenesis. Therefore, dysregulation of neuroinflammation and oxidative stress is one possible therapeutic target for AD. Kaempferia parviflora Wall. ex Baker (KP), a member of the Zingiberaceae family, possesses health-promoting benefits including anti-oxidative stress and anti-inflammation in vitro and in vivo with a high level of safety; however, the role of KP in suppressing Aß-mediated neuroinflammation and neuronal differentiation has not yet been investigated. The neuroprotective effects of KP extract against Aß42 have been examined in both monoculture and co-culture systems of mouse neuroectodermal (NE-4C) stem cells and BV-2 microglia cells. Our results showed that fractions of KP extract containing 5,7-dimethoxyflavone, 5,7,4'-trimethoxyflavone, and 3,5,7,3',4'-pentamethoxyflavone protected neural stem cells (both undifferentiated and differentiated) and microglia activation from Aß42-induced neuroinflammation and oxidative stress in both monoculture and co-culture system of microglia and neuronal stem cells. Interestingly, KP extracts also prevented Aß42-suppressed neurogenesis, possibly due to the contained methoxyflavone derivatives. Our data indicated the promising role of KP in treating AD through the suppression of neuroinflammation and oxidative stress induced by Aß peptides.

Development of a Rapid Reverse Transcription-Recombinase Polymerase Amplification Couple Nucleic Acid Lateral Flow Method for Detecting Porcine Epidemic Diarrhoea Virus.

Porcine epidemic diarrhea virus (PEDV) infection is an important acute diarrheal disease of swine that results in economic and industrial losses worldwide. The clinical manifestations in infected piglets are severe diarrhea, dehydration with milk curd indigestion, leading to death. The diagnosis of PEDV is essential for monitoring and managing the disease. PEDV can be detected and identified by serology and the nucleic acid of the virus in clinical samples. Therefore, a novel isothermal amplification and detection technique, reverse transcription-recombinase polymerase amplification couple nucleic acid lateral flow (RT-RPA-NALF) was developed for the rapid detection of PEDV. Qualitative reverse transcription-polymerase chain reaction (RT-qPCR) was established as the gold standard assay to compare results. Specific primer pairs and probes were designed, and RT-RPA conditions were optimized to amplify the M gene of PEDV. The established RT-RPA-NALF assay could finish in 25 min at a temperature of 42 °C and the amplicon interpreted by visual detection. The developed RT-RPA-NALF assay was specific to the M gene of PEDV, did not detect other common swine diarrhea pathogens, and showed minimal detection at 102 TCID50/mL PEDV. The RT-RPA-NALF assay can detect PEDV in 5 simulated fecal samples. Furthermore, in 60 clinical fecal samples, the results of RT-RPA-NALF correlated with RT-qPCR assay, which provides sensitivity of 95.65% and specificity of 100%, with a coincident rate of 98.33%. The rapid RT-RPA-NALF is simple and rapid, increases high sensitivity, and can be used in the field.

Occurrence of antimicrobial resistance and antimicrobial resistance genes in methicillin-resistant Staphylococcus aureus isolated from healthy rabbits.

Background and Aim: Methicillin-resistant globally, Staphylococcus aureus (MRSA) is a major cause of disease in both humans and animals. Several studies have documented the presence of MRSA in healthy and infected animals. However, there is less information on MRSA occurrence in exotic pets, especially healthy rabbits. This study aimed to look into the antimicrobial resistance profile, hidden antimicrobial-resistant genes in isolated bacteria, and to estimate prevalence of MRSA in healthy rabbits. Materials and Methods: Two-hundreds and eighteen samples, including 42 eyes, 44 ears, 44 oral, 44 ventral thoracic, and 44 perineal swabs, were taken from 44 healthy rabbits that visited the Prasu-Arthorn Animal Hospital, in Nakornpathom, Thailand, from January 2015 to March 2016. The traditional methods of Gram stain, mannitol fermentation, hemolysis on blood agar, catalase test, and coagulase production were used to confirm the presence of Staphylococcus aureus in all specimens. All bacterial isolates were determined by antimicrobial susceptibility test by the disk diffusion method. The polymerase chain reaction was used to identify the antimicrobial-resistant genes (blaZ, mecA, aacA-aphD, msrA, tetK, gyrA, grlA, and dfrG) in isolates of MRSA with a cefoxitin-resistant phenotype. Results: From 218 specimens, 185 S. aureus were isolated, with the majority of these being found in the oral cavity (29.73%) and ventral thoracic area (22.7%), respectively. Forty-seven (25.41%) MRSAs were found in S. aureus isolates, with the majority of these being found in the perineum (16, 34.04%) and ventral thoracic area (13, 27.66%) specimens. Among MRSAs, 29 (61.7%) isolates were multidrug-resistant (MDR) strains. Most of MRSA isolates were resistant to penicillin (100%), followed by ceftriaxone (44.68%) and azithromycin (44.68%). In addition, these bacteria contained the most drug-resistance genes, blaZ (47.83%), followed by gyrA (36.17%) and tetK (23.4%). Conclusion: This study revealed that MRSA could be found even in healthy rabbits. Some MRSAs strains were MDR-MRSA, which means that when an infection occurs, the available antibiotics were not effective in treating it. To prevent the spread of MDR-MRSA from pets to owners, it may be helpful to educate owners about effective prevention and hygiene measures.

Curcuminoid supplementation in canine diabetic mellitus and its complications using proteomic analysis.

Introduction: Inflammation and oxidative stress contribute to diabetes pathogenesis and consequences. Therapeutic approaches for canine diabetes remain a challenge. Curcumin has anti-inflammatory and anti-oxidative effects and is beneficial for humans with diabetes mellitus (DM); however, data on its impact on canine diabetes is limited. This study aimed to evaluate the potential for causing adverse effects, anti-inflammatory effects, anti-oxidative effects and proteomic patterns of curcuminoid supplementation on canine DM. Methods: Altogether, 18 dogs were divided into two groups: DM (n = 6) and healthy (n = 12). Curcuminoid 250 mg was given to the DM group orally daily for 180 days. Blood and urine sample collection for hematological parameters, blood biochemistry, urinalysis, oxidative stress parameters, inflammatory markers and proteomics were performed every 6 weeks. Results and discussion: Curcuminoid supplementation with standard therapy significantly decreased oxidative stress with the increased glutathione/oxidized glutathione ratio, but cytokine levels were unaffected. According to the proteomic analysis, curcuminoid altered the expression of alpha-2-HS-glycoprotein, transthyretin, apolipoprotein A-I and apolipoprotein A-IV, suggesting that curcuminoid improves insulin sensitivity and reduces cardiovascular complications. No negative impact on clinical symptoms, kidneys or liver markers was identified. This study proposed that curcuminoids might be used as a targeted antioxidant strategy as an adjunctive treatment to minimize diabetes complications in dogs.

Staphylococcus argenteus from rabbits in Thailand.

Staphylococcus argenteus, a novel species of the genus Staphylococcus or a member of the S. aureus complex, is closely related to S. aureus and is usually misidentified. In this study, the presence of S. argenteus in isolated S. aureus was investigated in 67 rabbits with abscess lesions during 2014-2016. Among 19 S. aureus complex isolates, three were confirmed to be S. argenteus by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, nonribosomal peptide synthetase gene amplification, and multilocus sequence type. All S. aureus complex isolates, including the S. aureus isolates, were examined for their antimicrobial resistance phenotype by disk diffusion and for their resistance genotype by PCR assays. Among the S. argenteus isolates, one was susceptible to all antimicrobial drugs and the other two were resistant to penicillin and doxycycline. In contrast, most S. aureus isolates were resistant to penicillin (37.5%), and gentamicin (12.5%). Moreover, S. aureus isolates harbored the blaZ, mecA, aacA-aphD, and mrs(A) as well as mutations of gyrA and grlA, but S. argenteus isolates carried solely the blaZ. S. argenteus isolates were investigated for enterotoxin (sea-sed) and virulence genes by PCR. One isolate carried sea, sec, and sed, whereas the other two isolates carried only sea or sed. No isolate carried seb and see. All three S. argenteus isolates carried hla, hlb, and clfA, followed by pvl, whereas coa, spa (IgG-binding region), and spa (x region) were not detected in the three isolates. This paper presents the first identification of S. argenteus from rabbits in Thailand. S. argenteus might be pathogenic because the isolates carried virulence genes. Moreover, antimicrobial resistance was observed. Investigations of this new bacterial species should be conducted in other animal species as well as in humans.

Detection and drug resistance profile of Escherichia coli from subclinical mastitis cows and water supply in dairy farms in Saraburi Province, Thailand.

Subclinical mastitis is a persistent problem in dairy farms worldwide. Environmental Escherichia coli is the bacterium predominantly responsible for this condition. In Thailand, subclinical mastitis in dairy cows is usually treated with various antibiotics, which could lead to antibiotic resistance in bacteria. E. coli is also a reservoir of many antibiotic resistance genes, which can be conveyed to other bacteria. In this study, the presence of E. coli in milk and water samples was reported, among which enteropathogenic E. coli was predominant, followed by enteroaggregative E. coli and enterohemorrhagic E. coli, which was found only in milk samples. Twenty-one patterns of antibiotic resistance were identified in this study. Ampicillin- and carbenicillin-resistant E. coli was the most common among the bacterial isolates from water samples. Meanwhile, resistance to ampicillin, carbenicillin, and sulfamethoxazole-trimethoprim was the pattern found most commonly in the E. coli from milk samples. Notably, only the E. coli from water samples possessed ESBL phenotype and carried antibiotic resistance genes, blaTEM and blaCMY-2. This indicates that pathogenic E. coli in dairy farms is also exposed to antibiotics and could potentially transfer these genes to other pathogenic bacteria under certain conditions.

The Intriguing Correlation between Undercarboxylated Osteocalcin and Vitamin D.

Osteocalcin (OC) or bone Gla protein (BGP), a bone specific protein, is one of circulating biomarkers that can be used to determine the quality of bone tissues. It is synthesized by the osteoblast and the odontocyte. The process of OC production requires two types ofvitamin: vitamin D and vitamin K. Vitamin D is requiredfor the production of immature OC or undercarboxylated OC (UcOC) whereas vitamin K is requiredfor the transformation of UcOC to mature OC. In the past, several researchers believed that UcOC was the sensitive marker to determine vitamin K status. In fact, the level of UcOC also depends on vitamin D. This study aims to know the correlation between the levels ofvitamin D and UcOC. The collected data of UcOC and vitamin D levels from 95 healthy volunteers were analyzed. The finding was that there was a weak correlation between UcOC and vitamin D (r = 0.238; p = 0.020) together with UcOC and reciprocal ofvitamin D (r = 0.267; p = 0.009). From this correlation, the serum level of UcOC may be not the real marker for vitamin K.

Prediction of cadmium (Cd) toxicity in cattle.

Hepato- and nephrotoxicity can be induced by the exposure to cadmium (Cd). This toxicity can be detected by the elevation of blood biomarkers such as ALT AST, ALP BUN and creatinine. These elevations are found in small animals, e.g. mice and rats. However the alteration of biomarkers did not investigate in large animals, e.g. cattle. Hepato- and nephrotoxicity induced by cadmium can also be examined by the alterations of metallothionein (MT) and metal transcription factor-1 (MTF-1). To present study the expressions of these markers, the cattle were classified into five groups according to the levels of cadmium in the kidneys. ALT, AST andALP were analyzed to determine liver damage whereas BUN and creatinine were examined for kidney damage. The results showed that blood biomarkers were not sensitive enough to be correlated markers to cadmium induced hepato- and nephrotoxicity in cattle. The expressions of MT and MTF-1 protein were investigated by immunofluorescence method. The expressions of MT and MTF-1 proteins were firstly found in the cattle group which had low cadmium concentration in tissues (< 0.5 mg/kg). Thus, these proteins could be used as the sensitive markers to determine the cadmium exposure. The MT and MTF-1 gene expressions were also studied. However, there was no correlation between the level of RNA and the protein expressions due to the concentration of protein levels bearing unclear relationship with the mRNA level. The investigation of these protein expressions is very useful because the result can be used as a protective method to prevent consumption of cadmium-contaminated beef.

Calcium supplement: humanity's double-edged sword.

The principle aim of the present study is to investigate the dark side of calcium, pollutions in calcium preparation especially lead (Pb), mercury (Hg) and cadmium (Cd). The collected samples were the different calcium salts in the market and 18 preparations which were classified into 3 groups: Calcium carbonate salts, Chelated calcium and natural-raw calcium. All samples were analyzed for lead, cadmium and mercury by inductively Coupled Plasma Mass Spectrometry (ICP-MS) technique, in house method based on AOAC (2005) 999.10 by ICP-MS. The calcium carbonate and the natural-raw calcium in every sample contained lead at 0.023-0.407 mg/kg of calcium powder. Meanwhile, the natural-raw calcium such as oyster, coral and animal bone showed amount of lead at 0.106-0.384 mg/kg with small amounts of mercury and cadmium. The chelated calcium such as calcium gluconate, calcium lactate and calcium citrate are free of lead.

Study of validity of pyridinoline and correlation of pyridinoline and beta crosslap in postmenopausal women.

This research studied the validity and correlation of beta-CTx (Betacrosslap) and Pyridinoline (PYD) in 71 cases of postmenopausal Thai women who attended at the osteoporosis clinic. The validity consisted of sensitivity, specificity and accuracy of PYD. They were 53.44, 84.6 and 59.15% respectively. So, the validity of pyridinoline test was rather poor and the correlation between betacrosslap and pyridinoline was weak (r = 0.123, p = 0.0001) which was not significant in practice. In conclusion, the pyridinoline test was not proper for bone resorption even pyridinoline was abundant in bone.

The reality of osteocalcin as a marker of vitamin K2.

The present study showed the correlation between undercarboxylated osteocalcin and vitamin D had a role in production of the undercarboxylated osteocalcin, when the amount of vitamin D 25 (OH) D, (below 30 ng/ml) was decreased, the linear correlation (r) between the vitamin D and undercarboxylated osteocalcin were also decreased r = 0.032 when the level of vitamin D was risen, the linear correlation was 0.274,unfortunately the study-population had little group of the normal level of vitamin D due to most of them were vitamin D insufficiency so the authors could not show the more value of 'r'. However, the present study confirmed that the role of vitamin D information of undercarboxylated osteocalcin (UcOC) which we recognized the UcOC at the low level of vitamin D will misinterpretation of the level of vitamin K2.

Correlation between the circulating bone markers and vitamin D in the healthy Thai people.

There are several circulating bone markers that are useful for physicians in determining the bone quality. The markers are composed of N-terminal propeptide of type I collagen (PINP) which determines the bone formation, undercarboxylated osteocalcin (UcOC) and N-mid fragment of osteocalcin (N-MID) which determines the bone remodeling, C-telopeptide of type I collagen (betaPCTx) or betacrosslap (betaCTx), pyridinoline (PYD) and deoxypyridinoline (DPD) which determines the bone resorption. The vitamin named vitamin D (VitD) also affects the bone property. However there is little information about the relationship of these biomarkers. In this experiment, the researchers investigated the correlation of the circulating biomarkers and found the correlations between UcOC and betaCTx (r = 0.471, p = 0.011), between UcOC and Vit D (r = 0.39, p = 0.04), between N-MID and PINP (r = 0.833, p = 0.000), between N-MID and PCTx (r = 0.641, p = 0.000) and lastly between PINP and betaCTx (r = 0.657, p = 0.000) in the male group whereas the correlations between UcOC and PYD (r = 0.318, p = 0.043), between UcOC and DPD (r = 0.551, p = 0.008), between N-MID and PINP (r = 0.721, p = 0.000), between N-MID and PCTx (r = 0.719, p = 0.000), between N-MID and PYD (r = 0.485, p = 0.001), between N-MID and Vit D (r = 0.347, p = 0.026), between PINP and JCTx (r = 0.632, p = 0.000), between PINP and PYD (r = 0.312, p = 0.047), between bCTx and PYD (r = 0.365, p = 0.019), between PYD and DPD (r = 0.567, p = 0.006) and lastly between PYD and Vit D (r = 0.409, p = 0.008) were found in the females. In addition, the new biomolecule named matrix Gla protein (MGP), a small protein produced by bone tissues was also investigated. The authors found the correlation between MGP and PYD (r = 0.468, p = 0.012) in the males and found the correlations between UcOC and MGP (r = 0.421, p = 0.006), between N-MID and MGP (r = 0.333, p = 0.033), between MGP and PYD (r = 0.471, p = 0.002), between MGP and DPD (r = 0.472, p = 0.026) in the female group.

Bone markers in the healthy Thai people.

Osteoporosis is a systemic skeletal disease characterized by low bone mineral density (BMD) and micro-architectural deterioration of the bone tissues resulting in bone fragility and susceptibility to fracture. It is caused by the decrease of bone formation and the increase of bone resorption. Both bone formation and resorption can be examined by the circulating biomolecules. The biomolecules using in determining the bone formation are composed of osteocalcin (OC) in form of N-terminal midmolecule fragment (N-MID) and undercarboxylated osteocalcin (UcOC). The other bone formation markers are matrix Gla protein (MGP) and N-terminal propeptide of Type I collagen (PINP) whereas the biomolecules using in determining the bone resorption comprises of C-terminal cross-linking telopeptide of Type I collagen (betaCTx), collagen cross-links molecules which are pyridinoline (PYD) and deoxypyridinoline (DPD). Nevertheless, some vitamins such as vitamin D (Vit D) and some hormones e.g. parathyroid hormone (PTH) are also affected to the bone quality. To monitor and assess the bone mass, the normal values of bone markers as well as the relevant biomolecules are important and should be established. The researchers aimed to investigate the normal values of the interesting bone biomarkers and the relevant biomolecules in the adult volunteers.

Investigation of sensitive biomarkers to determine cadmium inducing hepato- and nephro-toxicity in cattle by immunofluorescence method.

Cadmium inducing hepato- and nephro-toxicity can cause the alteration of protein expression such as metallothionein (MT), i.e. cadmium binding protein, and the metal transcription factor-1 (MTF-1), which plays a significant role in cellular responses to the heavy metal stress. To study the expression of these proteins, the cattle were classified into five groups resulting from the levels of cadmium in the kidneys. Next, the blood biochemical profiles were analyzed to estimate the liver and kidney functions. The expressions of MT and MTF-1 proteins both in the liver and kidneys were investigated by immunofluorescence method. This study found that the blood biochemical profiles were not correlated with the level of cadmium in these tissues; however, the expressions of MT and MTF-1 proteins were earlier detected in the bovine which had a low level of cadmium contamination (0.5 mg/kg). Thus, these proteins could be used as the sensitive markers to determine the cadmium in tissue.

Immunohistochemical determination of estrogen and progesterone receptors in canine mammary tumors.

Mammary gland tumors are by far the most commonly found tumors in domestic dogs. Effective therapeutic procedures with prompt accurate diagnoses are of prime importance for this life threatening neoplasm. Although immunohistochemical methods provide valuable information such as the location and semi-quantitative data of the interested antigens in particular tumors, conventional methods like histopathological diagnosis remain useful and necessary for identification and classification of tumors. In the present study, we combined histopathology with immunohistochemical staining of estrogen receptors (ER) and progesterone receptors (PR) in canine mammary gland tumors. Fifty dogs with primary mammary tumors underwent surgery at the Veterinary Teaching Hospital of Mahidol University during 2005 to 2007. Three of them were diagnosed with precancerous lesions and negatively stained for ER or PR antibody. Twenty-one were diagnosed with benign tumors classified as adenomas and benign mixed mammary gland tumors. Nearly 60% of thelesions were negatively stained for ER or PR. PR positively stained, both PR and ER stained and ER stained tumors accounted for 19%, 19% and 5%, respectively. Of the malignant tumors, eighty-six percent were adenocarcinomas and 14% were malignant mixed mammary gland tumors. Nearly 70% were negatively stained for ER or PR, 14% were PR positively stained, 14% were both PR and ER stained and 5% were ER stained. Four dogs had unidentified lesions. In summary, more than half of of our benign and malignant canine mammary tumors were negatively stained for ER and PR. This indicates a lack of correlation with estrogen and/or progesterone receptor expression.

In vitro osteogenesis from human skin-derived precursor cells.

Embryonic tissue and organ development are initiated from three embryonic germ layers: ectoderm (skin and neuron), mesoderm (blood, bone, muscle, cartilage and fat) and endoderm (respiratory and digestive tract). In former times, it was believed that cell types in each germ layer are specific and do not cross from one to another throughout life. A new finding is that one tissue lineage can differentiate across to another tissue lineage, and this is termed transdifferentiation. We were interested in studying the transdifferentiation of skin-derived precursor cells (ectoderm layer) to osteoblastic cells (mesoderm layer). Human skin-derived precursor cells (hSKP) were isolated and induced into an osteoblastic lineage using osteogenic induction medium (alpha-MEM plus 10% fetal bovine serum supplemented with ascorbic acid, beta-glycerophosphate and dexamethasone). The specific characteristics of osteoblastic cells, including the expression of enzyme alkaline phosphatase, the deposition of mineral and the expression of osterix, bone sialoprotein and osteocalcin, were detected only from the inductive group. The results in our study show that SKP from human skin are a practically available source for osteogenesis. The samples are easily obtainable for autologous use with a high expansion capacity.