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Klebsiella pneumoniae (KP) remains the most prevalent nosocomial pathogen and carries the carbapenemase (KPC) gene which confers resistance towards carbapenem. Thus, it is necessary to discover novel antimicrobials to address the issue of antimicrobial resistance in such pathogens. Natural products such as essential oils are a promising source due to their complex composition. Essential oils have been shown to be effective against pathogens, but the overall mechanisms have yet to be fully explained. Understanding the molecular mechanisms of essential oil towards KPC-KP cells would provide a deeper understanding of their potential use in clinical settings. Therefore, we aimed to investigate the mode of action of essential oil against KPC-KP cells from a proteomic perspective by comparing the overall proteome profile of KPC-KP cells treated with cinnamon bark (Cinnamomum verum J. Presl) essential oil (CBO) at their sub-inhibitory concentration of 0.08% (v/v). A total of 384 proteins were successfully identified from the non-treated cells, whereas only 242 proteins were identified from the CBO-treated cells. Proteins were then categorized based on their biological processes, cellular components and molecular function prior to pathway analysis. Pathway analysis showed that CBO induced oxidative stress in the KPC-KP cells as indicated by the abundance of oxidative stress regulator proteins such as glycyl radical cofactor, catalase peroxidase and DNA mismatch repair protein. Oxidative stress is likely to oxidize and disrupt the bacterial membrane as shown by the loss of major membrane proteins. Several genes selected for qRT-PCR analysis validated the proteomic profile and were congruent with the proteomic abundance profiles. In conclusion, KPC-KP cells exposed to CBO undergo oxidative stress that eventually disrupts the bacterial membrane possibly via interaction with the phospholipid bilayer. Interestingly, several pathways involved in the bacterial membrane repair system were also affected by oxidative stress, contributing to the loss of cells viability.

Recently, the antibacterial properties of Carvacrol (Carv) have been significantly reported. However, due to the unstable properties of Carv under various environment conditions, research approaches tailored towards its widespread and efficient use in various antimicrobial applications are scarce. Here, we discuss progress towards overcoming this challenge by utilising the encapsulation of Carv in gellan gum hydrogels to form thin films (GG-Carv) containing different concentrations of Carv (0.01–0.32 M). FTIR spectrum of GG-Carv revealed that both functional groups from GG and Carv existed. The carbon, hydrogen and nitrogen elemental analysis further supported the encapsulation of Carv with the changes in the element percentage of GG-Carv. Both swelling and degradation percentage increased with time and the decreasing patterns were observed as the concentration of Carv increased. In an antibacterial study, GG-Carv exhibited significant antibacterial activity against E. coli with the clear inhibition zone of 200 mm and the detection of bacterial growth showed enhancement with continuous decline throughout the study as compared to free-standing Carv.

Leptospirosis is one of the most widespread zoonotic diseases and can infect both humans and animals worldwide. Healthy cat, as a potential source of exposure to humans, are likely underestimated owing to the lack of overt clinical signs associated with Leptospira spp. infection in this species. The aim of the study was to determine the exposure, shedding, and carrier status of leptospires in shelter cats in Malaysia by using serological, molecular, and bacteriological methods. For this study, 82 healthy cats from two shelters were sampled. The blood, urine, and kidneys were tested using the microscopic agglutination test (MAT), polymerase chain reaction (PCR), and bacterial culture. On the basis of serological, molecular, and/or culture techniques, the total detection of leptospiral infection was 29.3% (n = 24/82). Through culture techniques, 16.7% (n = 4/24) of the cats that tested positive were carriers with positive kidney cultures, and one cat was culture positive for both urine and kidney. The Leptospira spp. isolates were identified as pathogenic L. interrogans serovar Bataviae through serological and molecular methods. Through serological techniques, 87.5% (n = 21/24) had positive antibody titers (100-1600) and most of the Bataviae serogroup (n = 19/21). Using PCR, 16.7% (n = 4/24) of cats were shown to have pathogenic Leptospira spp. DNA in their urine. Furthermore, three out of four culture positive cats were serology negative. The present study reports the first retrieval of pathogenic leptospires from urine and kidneys obtained from naturally infected cats. The results provide evidence of the potential role of naturally infected cats in the transmission of leptospires. Additionally, leptospiral infection occurs sub-clinically in cats. The culture isolation provides evidence that healthy cats could be reservoirs of leptospiral infection, and this information may promote the development of disease prevention strategies for the cat population.

Mining of plant-derived antimicrobials is the major focus at current to counter antibiotic resistance. This study was conducted to characterize the antimicrobial activity and mode of action of linalyl anthranilate (LNA) against carbapenemase-producing Klebsiella pneumoniae (KPC-KP). LNA alone exhibited bactericidal activity at 2.5% (V/V), and in combination with meropenem (MPM) at 1.25% (V/V). Comparative proteomic analysis showed a significant reduction in the number of cytoplasmic and membrane proteins, indicating membrane damage in LNA-treated KPC-KP cells. Up-regulation of oxidative stress regulator proteins and down-regulation of oxidative stress-sensitive proteins indicated oxidative stress. Zeta potential measurement and outer membrane permeability assay revealed that LNA increases both bacterial surface charge and membrane permeability. Ethidium bromide influx/efflux assay showed increased uptake of ethidium bromide in LNA-treated cells, inferring membrane damage. Furthermore, intracellular leakage of nucleic acid and proteins was detected upon LNA treatment. Scanning and transmission electron microscopies again revealed the breakage of bacterial membrane and loss of intracellular materials. LNA was found to induce oxidative stress by generating reactive oxygen species (ROS) that initiate lipid peroxidation and damage the bacterial membrane. In conclusion, LNA generates ROS, initiates lipid peroxidation, and damages the bacterial membrane, resulting in intracellular leakage and eventually killing the KPC-KP cells. [Display omitted] •Novel antimicrobial, LNA kills KPC-KP cells effectively at 2.5%.•LNA disrupts bacterial membrane by generating reactive oxygen species.•LNA causes intracellular leakage of nucleic acids and proteins, killing the cells.

Objective: The livestock handler attitude and their handling of animals is crucial for improving animal welfare standards, minimizing stress, improving productivity and meat quality. The present study was undertaken to assess the effect of training livestock handlers on behavioral, physiological, and hormonal responses during preslaughter handling in goats.Methods: A total of 6 handlers were divided into trained (trained in basic animal handling practices, animal behavior, and animal welfare), contact trained (not trained directly but interacted and saw the working of trained handlers), and untrained groups (no formal training). The handling experiment was conducted on 18 male goats by following a crossover design. The goats were moved from lairage to slaughter point by trained, contact-trained, and untrained handlers. Various behavioral, physiological, and hormonal parameters were recorded at the lairage before handling and at the slaughter point after handling the goats.Results: The training of livestock handlers had a significant effect on behavioral, physiological, and hormonal responses in goats. The goats handled by untrained and contacttrained handlers were recorded with intense vocalization, significant (p<0.05) increase in heart rate and blood glucose, and catecholamines (adrenaline and nor-adrenaline), thereby indicating stress and poor animal welfare. The trained handlers were observed to use visual interactions (waving of hands or objects, blocking, hand raising, etc), and lower stress responses were recorded in the goats handled by this group.Conclusion: The present study highlights the importance of training to livestock handlers in improving animal welfare and minimizing stress in goats during pre-slaughter stress.

Canine leptospirosis has always been a differential diagnosis in dogs presenting with clinical signs and blood profiles associated with kidney and/or liver disease. The conventional polymerase chain reaction (PCR) provides diagnoses, but real-time PCR-based tests provide earlier confirmation and determine the severity of infection, especially in the acute stage, allowing early detection for immediate treatment decisions. To our knowledge, real-time PCR has not been routinely adopted for clinical investigation in Malaysia. This study evaluated TaqMan real-time PCR (qPCR) assays diagnosing leptospirosis and compared their applicability to clinical samples from dogs with kidney and/or liver disease against a conventional PCR reference. The qPCR assays were validated using existing leptospiral isolates. Whole blood and urine samples were analysed using a conventional PCR, and qPCRs and a microscopic agglutination test. The sensitivity and specificity of the qPCRs were determined. The qPCR assay had more diagnostic value than the qPCR assay. Further evaluation of this assay revealed that it could detect as low as five DNA copies per reaction with high specificity for the tested leptospiral strains. No cross-amplification was observed with other organisms. Analysing the clinical samples, the qPCR assay had 100.0% sensitivity and >75.0% specificity. The qPCR assay is sensitive, specific and has the potential to be applied in future studies.

Theileriosis is a blood piroplasmic disease that adversely affects the livestock industry, especially in tropical and sub-tropical countries. It is caused by haemoprotozoan of the Theileria genus, transmitted by hard ticks and which possesses a complex life cycle. The clinical course of the disease ranges from benign to lethal, but subclinical infections can occur depending on the infecting Theileria species. The main clinical and clinicopathological manifestations of acute disease include fever, lymphadenopathy, anorexia and severe loss of condition, conjunctivitis, and pale mucous membranes that are associated with Theileria-induced immune-mediated haemolytic anaemia and/or non-regenerative anaemia. Additionally, jaundice, increases in hepatic enzymes, and variable leukocyte count changes are seen. Theileria annulata and Theileria parva induce an incomplete transformation of lymphoid and myeloid cell lineages, and these cells possess certain phenotypes of cancer cells. Pathogenic genotypes of Theileria orientalis have been recently associated with severe production losses in Southeast Asia and some parts of Europe. The infection and treatment method (ITM) is currently used in the control and prevention of T. parva infection, and recombinant vaccines are still under evaluation. The use of gene gun immunization against T. parva infection has been recently evaluated. This review, therefore, provides an overview of the clinicopathological and immunopathological profiles of Theileria-infected cattle and focus on DNA vaccines consisting of plasmid DNA with genes of interest, molecular adjuvants, and chitosan as the most promising next-generation vaccine against bovine theileriosis.

Activation of hepatic stellate cells has been recognized as one of the first steps in liver injury and repair. During activation, hepatic stellate cells transform into myofibroblasts with concomitant loss of their lipid droplets (LDs) and production of excessive extracellular matrix. Here we aimed to obtain more insight in the dynamics and mechanism of LD loss. We have investigated the LD degradation processes in rat hepatic stellate cells in vitro with a combined approach of confocal Raman microspectroscopy and mass spectrometric analysis of lipids (lipidomics). Upon activation of the hepatic stellate cells, LDs reduce in size, but increase in number during the first 7 days, but the total volume of neutral lipids did not decrease. The LDs also migrate to cellular extensions in the first 7 days, before they disappear. In individual hepatic stellate cells. all LDs have a similar Raman spectrum, suggesting a similar lipid profile. However, Raman studies also showed that the retinyl esters are degraded more rapidly than the triacylglycerols upon activation. Lipidomic analyses confirmed that after 7 days in culture hepatic stellate cells have lost most of their retinyl esters, but not their triacylglycerols and cholesterol esters. Furthermore, we specifically observed a large increase in triacylglycerol-species containing polyunsaturated fatty acids, partly caused by an enhanced incorporation of exogenous arachidonic acid. These results reveal that lipid droplet degradation in activated hepatic stellate cells is a highly dynamic and regulated process. The rapid replacement of retinyl esters by polyunsaturated fatty acids in LDs suggests a role for both lipids or their derivatives like eicosanoids during hepatic stellate cell activation.

Recent advances in emotions and cognitive science make it imperative to assess the emotional stress in goats at the time of slaughter. The present study was envisaged to study the electroencephalogram and physiological responses as affected by slaughter empathy in goats. A total of 12 goats were divided into two groups viz., E-group (goats exposed to slaughter environment, n = 6) and S-group (goat slaughtered in front of E-group, n = 6). The electroencephalogram and physiological responses in male Boer cross goats (E-group) were recorded in a slaughterhouse in two stages viz., control (C) without exposure to the slaughter of conspecifics and treatment (T) while visualizing the slaughter of conspecifics (S—slaughter group). The exposure of the goat to the slaughter of a conspecific resulted in a heightened emotional state. It caused significant alterations in neurobiological activity as recorded with the significant changes in the EEG spectrum (beta waves (p = 0.000491), theta waves (p = 0.017), and median frequency MF or F50 (p = 0.002)). Emotional stress was also observed to significantly increase blood glucose (p = 0.031) and a non-significant (p = 0.225) increase in heart rate in goats. Thus, slaughter empathy was observed to exert a significant effect on the electric activity of neurons in the cerebrocortical area of the brain and an increase in blood glucose content.

Canine mammary gland tumour (CMT) commonly affects the female dog. The objective of this study was to develop a doxorubicin-resistant CMT cell line and determine its in vitro and in vivo characteristics, including mRNA and microRNA (miRNA) expression profiles. Doxorubicin-resistant CMT-Star cells were developed from CMT-Stylo cells. The cells were characterized, including tumorigenicity in NOD/SCID mouse models. MiRNA and mRNA expression of the two cell lines were profiled and clustered. ATP binding cassette subfamily B member 1 (ABCB1) and subfamily G member 2 (ABCG2) expressions were significantly increased in the CMT-Star cell line. CMT-Star cells also had altered expression of 785 genes and 14 miRNAs. Downregulating plasminogen (PLG) and plasminogen activator urokinase (PLAU) while upregulating transforming growth factor beta receptor 3 (TGFBR3), epidermal growth factor receptor 1 (EGFR1) and ABCB1 rendered CMT-Star cells less proliferative, less invasive and more resistant to chemotherapeutic drugs. The upregulated miRNAs in CMT-Star cells include miRNA-191, -29a, -107, -99b, -874, -93 and -210, while the downregulated miRNAs include miRNAs-106a, -92a, -92b, -155 and -15b. TGFβR, EGF receptor 1 and Wnt signalling are enriched in doxorubicin-resistant CMT-Star cells and could be potential therapeutic targets in dogs with doxorubicin-resistant CMT.