Green synthesized silver nanoparticles from Ocimum sanctum: A potent inhibitor of biofilm forming ability and efflux pumps in bacteria causing bovine mastitis.

Therapeutic management of mastitis faces significant challenges due to multidrug resistance. In the present study, multi-drug-resistant (MDR) Staphylococcus spp, Klebsiella pneumoniae, and Escherichia coli were isolated from bovine clinical mastitis cases and the phenotypic and genotypic multidrug resistance profiling was carried out. Silver nanoparticles (AgNPs) were biosynthesized using Ocimum sanctum leaf extracts and characterized via UV Vis absorption, Fourier Transform Infrared Spectroscopy, X-ray diffraction studies, Energy dispersive spectroscopy and Electron Microscopy. The determined minimum inhibitory concentration and minimum bactericidal concentration of the AgNPs against the recovered MDR isolates were 62.5 µg/ml and 125 µg/ml respectively. At a concentration of 50 µg/ml, the AgNPs demonstrated biofilm inhibitory activities of 80.35 % for MDR E. coli, 71.29 % for S. aureus and 60.18 % for MDR K. pneumoniae. Post-treatment observations revealed notable differences in biofilm formation across bacterial isolates. Furthermore, AgNP treatment led to significant downregulation of expression of the efflux pump genes acrB, acrE, acrF, and emrB in Gram-negative isolates and norB in Staphylococci isolates. This research underscores the potential for the development of an eco-friendly antimicrobial alternative in the form of green synthesized silver nanoparticles to combat drug resistance offering potential antibiofilm and efflux pump inhibitory activities.

Unravelling the complex mechanisms of multidrug resistance in bovine mastitis pathogens: Insights into antimicrobial resistance genes, biofilm dynamics, and efflux systems.

Mastitis remains a paramount economic threat to dairy livestock, with antibiotic resistance severely compromising treatment efficacy. This study provides an in-depth investigation into the multidrug resistance (MDR) mechanisms in bacterial isolates from bovine mastitis, emphasizing the roles of antimicrobial resistance genes (ARGs), biofilm formation, and active efflux systems. A total of 162 Staphylococci, eight Escherichia coli, and seven Klebsiella spp. isolates were obtained from 215 milk samples of clinical and subclinical mastitis cases. Antibiotic susceptibility testing identified Twenty Staphylococci (12.35 %), six E. coli (75 %) and seven Klebsiella (100 %) identified as MDR displaying significant resistance to ß-lactams and tetracyclines The Multiple Antibiotic Resistance (MAR) index of these isolates ranged from 0.375 to 1.0, highlighting extensive resistance. Notably, 29 of the 33 MDR isolates produced biofilms on Congo red agar, while all exhibited biofilm formation in the Microtitre Plate assay. Critical ARGs (blaZ, blaTEM, blaCTX-M, tetM, tetA, tetB, tetC, strA/B, aadA) and efflux pump genes (acrB, acrE, acrF, emrB, norB) regulating active efflux were identified. This pioneering study elucidates the synergistic contribution of ARGs, biofilm production, and efflux pump activity to MDR in bovine mastitis pathogens. To our knowledge, this comprehensive study is the first of its kind, offering novel insights into the complex resistance mechanisms. The findings underscore the imperative need for advanced antibiotic stewardship and strategic interventions in dairy farming to curb the rise of antibiotic-resistant infections, thereby protecting both animal and public health.

Diagnostic efficacy of urinary neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 for early detection of acute kidney injury in dogs with leptospirosis or babesiosis.

This study evaluates the diagnostic efficacy of urinary biomarkers, Neutrophil Gelatinase-Associated Lipocalin (uNGAL), and Kidney Injury Molecule-1 (uKIM-1), in identifying Acute Kidney Injury (AKI) in dogs affected with leptospirosis or babesiosis. Acute kidney injury was diagnosed based on the increase in serum creatinine levels above 0.3 mg/dL within 48 h and dogs were categorized according to AKI grades based on International Renal Interest Society guidelines. Traditional biomarkers (serum creatinine and blood urea nitrogen) and novel biomarkers like urinary NGAL and urinary KIM-1 levels were measured and compared to concentrations obtained in control dogs. Statistical analysis assessed significant differences (P < 0.01) across AKI grades, specifically noting elevated urinary NGAL and KIM-1 in IRIS grade I AKI (P < 0.001). The study highlights the diagnostic significance of urinary NGAL and KIM-1 as early indicators of renal damage, particularly valuable in non-azotemic AKI cases, offering promising markers for early AKI diagnosis in veterinary clinical settings. These biomarkers demonstrate clinical utility and underscore their potential for improving AKI management in veterinary medicine. Further validation studies involving larger cohorts and diverse etiologies of AKI are needed to confirm the diagnostic accuracy and clinical utility of urinary NGAL and KIM-1 in veterinary practice.

A novel loop-mediated isothermal amplification (LAMP) assay to diagnose feline panleukopenia.

Protoparvovirus carnivoran1, known commonly as feline panleukopenia virus (FPV) is a highly contagious and environmentally stable parvovirus of domestic as well as wild felids. A rapid and robust diagnostic tool will aid in implementing prompt treatment and control measures. A loop-mediated isothermal amplification (LAMP) as a point-of-care diagnostic tool for diagnosing feline panleukopenia was standardised using faecal samples of cats. The assay will reduce the cost and time required to diagnose feline panleukopenia. A set of two outer primers (F3 and B3) and two inner primers (FIP and BIP) were designed to target the viral polypeptide (VP2) gene of FPV. Optimisation of the LAMP reaction was done at 60 °C for one hour after an initial denaturation at 95 °C for five minutes. Visualisation of the result based on the addition of SYBR Green 1 dye offered an easy and reliable diagnosis. The detection limit of the standardised LAMP assay was as low as 1.25 ng/µl of the target DNA. Species specificity of the LAMP primers revealed no amplification of the non-target DNA of any other species except that of the canine parvovirus DNA template. DNA extracted from 100 PCR-positive and 20 PCR-negative faecal samples were subjected to the standardised assay and compared with PCR. Analysis of the results revealed that the LAMP assay was 100% sensitive and 90% specific compared to PCR. The LAMP assay could be a reliable tool for the point-of-care diagnosis of feline panleukopenia in limited resource settings.