Microbiological and Molecular Investigation of Antimicrobial Resistance in Staphylococcus aureus Isolates from Western Romanian Dairy Farms: An Epidemiological Approach.

Antimicrobial therapy is the most frequently used medical intervention for bovine mastitis in the dairy industry. This study aims to monitor the extent of the antimicrobial resistance (AMR) problem in Staphylococcus aureus in the dairy industry in Western Romania. Twenty farms were selected by random sampling in a transverse epidemiological study conducted across four counties in Western Romania and divided into livestock units. This study assessed the association between the resistance genes to phenotypic expression of resistance and susceptibility. Isolates of S. aureus were identified and q-PCR reactions were used to detect antibiotic resistance genes. One hundred and fifty bovine and 20 human samples were positive for S. aureus. Twenty five percent of bovine isolates (30/120) and none(0/30) of the human isolates were methicillin-resistant S. aureus (MRSA). All isolates were susceptible to fosfomycin, ciprofloxacin, netilmicin, and resistant to ampicillin and penicillin. S. aureus isolates regarded as phenotypically resistant (R) were influenced by the origin of the samples (human versus bovine, χ2 = 36.510, p = 0.013), whether they were methicillin-resistant S. aureus (χ2 = 108.891, p < 0.000), the county (χ2 = 103.282, p < 0.000) and farm of isolation (χ2 = 740.841, p < 0.000), but not by the size of the farm (χ2 = 65.036, p = 0.306). The multiple antibiotic resistance index was calculated for each sample as the number regarded as phenotypically resistant (R)/total antibiotics tested (MARI = 0.590 ± 0.023) was significantly higher (p < 0.000) inmethicillin-resistant S. aureus (0.898 ± 0.019) than non-methicillin-resistant S. aureus (0.524 ± 0.024) isolates. For the antibiotics tested, the total penetrance (P%) of the resistance genes was 59%, 83% for blaZ, 56% for cfr, 50% for erm(B), 53% for erm(C), 57% for mecA and 32% for tet(K). Penetrance can be used as a parameter for guidance towards a more accurate targeting of chemotherapy. P% in S. aureus was strongly positively correlated with the multiple antibiotic resistance index (r = +0.878, p < 0.000) with the potential to use the same limit value as an antibiotic management decision criterion. Considering cow mastitis, the penetrance value combined with the multiple antibiotic resistance index suggests that penetrance could serve as a useful parameter for more precise targeting of chemotherapy for S. aureus.

Experimenting with Pig-based Skin Model for Burns. Testing of Mean Literature Findings.

Introduction: Animal models that closely mimic human burn wound healing processes are essential for developing effective burn wound treatments. Pigs are useful animal models for studying burn wound healing. From their extensive literature review, Andrews and Cuttle (2017) reported mean temperature and exposure time values. This study was done to provide initial burn depth for another experiment comparing two burn wound treatments. The secondary goal was to validate a systematic review on porcine burn model standardization. Materials and Methods: Six four-week-old Large White x Landrace gilts were housed in a closed structure for 10 days to acclimatize. The procedures were performed under general anesthesia. A round 2.5 cm copper plate welded to an aluminum rod with a wooden handle caused the injuries. The burning device was used to reach a contact temperature of 110ºC on the pig's skin. The objective was to create a superficial partial thickness (SPT) burn for 10 seconds (Group 10s) and a deep partial thickness (DPT) burn for 20 seconds (Group 20s) using a plate heated at 110ºC. No stabilizer or pressure controller was used. Wounds were conclusively dressed and harvested 24 hours later. The usual hematoxylin-eosin protocol was used to cut and stain 4-micron sections. Results: A significant difference (p 0.01) was observed in dermis involvement, with a mean of 85.61 % (95% CI= 80.62 to 90.61) for group 10s and 123.71% (95% CI= 114.91 to 132.50) for group 20s. An exposure time of 20 seconds increased dermis depth-related total collagen denaturation by almost 50% compared to 10 seconds. Conclusions: In conclusion, our experiment produced DPT burns in 10 seconds and FT burns in 20 seconds without a pressure application device.

Correction: Lungu et al. Molecular Characterisation of Antimicrobial Resistance in E. coli Isolates from Piglets in the West Region of Romania. Antibiotics 2023, 12, 1544.

The authors Bianca Cornelia Lungu and Ioan Hutu did not state contributed equally [...].

Molecular Characterisation of Antimicrobial Resistance in E. coli Isolates from Piglets in the West Region of Romania.

Antibiotics are widely used for prophylaxis and therapy, reducing morbidity and mortality produced by bacterial pathogensin pigs, including infections caused by Escherichia coli. The aim of this study was to characterise antibiotic resistance phenotypes and genotypes in E. coli isolates in pigs in West Romanian grower farms. Differential phenotypic susceptibility profiles and the contribution of resistance genes to phenotypic expression of susceptibility or resistance were evaluated. A total of 76 E. coli isolates were identified and confirmed by the MicroScan Walk Away System. The occurrence of four resistance genes, ampC, blaZ, blaTEM and tetK in strains resistant to 13 antibiotics was assessed. Of the E. coli isolates, 0% showed resistance to meropenem, 3.9% to tigecycline and 10.5% to piperacillin/tazobactam, whereas, in contrast, 100% were resistant to ampicillin and mezlocillin, 76.31% to piperacillin and 59.3% to tetracycline. The prevalence of resistance genes in resistant isolates detected by q-PCR analysis was 97.0% for ampC, 96% for blaZ, 32.9% for blaTEM and 58.8% for tetK. Penetrance (the proportion of individuals carrying a particular variant of a gene that also expresses an associated trait) was 50% for ampC (32% for amoxicillin/clavulanate, 62% for cefazolin, 32% for cefepime, 100% for cefotaxime, 56% for cefuroxime and 99% for ampicillin), 65% for blaZ (32% for amoxicillin/clavulanate and 99% for ampicillin), 51% for blaTEM (81% for piperacillin) and 44% for the tetK gene (83% for tetracycline). The result of phenotypic antibiotic resistance testing may indicate the presence of plasmid-borne resistance, with a diagnostic odds ratio of a positive phenotypic resistance for tetK being 4.52. As a management decision, the maximum penetrance admitted for using a specific antibiotic for E. coli infections in pigs is recommended to be less than 20%.

Unusual Canine Distemper Virus Infection in Captive Raccoons (Procyon lotor).

Canine morbillivirus, also known as canine distemper virus (CDV), is the causative agent of canine distemper (CD), which is a serious contagious disease of canines, large felids, and, occasionally, raccoons. This study included seven raccoons from the Timisoara Zoological Garden, Romania. CDV was detected using RT-qPCR on blood samples, but several other exams were also performed-clinical, bacteriological, immunohistochemistry (IHC) and histopathology, toxicological screening, and necropsy-which confirmed CDV infection. Severe digestive disorders (diarrhea and frequent hematemesis) were observed. The necropsy findings included pseudo membranous gastroenteritis, congestion, and pulmonary edema in two raccoons. Immunohistochemistry showed immunolabeled CDV antigenantibodies on the viral nucleocapsid. Histopathology revealed lymphocyte depletion in mesenteric lymphnodes and intranuclear and intracytoplasmic inclusions in the enterocytes of the small intestine. Based on the RT-qPCR assay, laboratory tests, and the lesions observed, it was established that the raccoons were infected with CDV, which was the cause of death in two cases. The results from the necropsy, histology, and immunohistochemistry in the raccoons are comparable with reported CDV lesions in dogs. In conclusion, several exams may be performed to establish the etiology of possible interspecific viral infection, but only very specific exams can identify aCDV infection. Laboratory analyses must be completed by RT-qPCR assay or IHC to establish infection with uncommon viruses in raccoons with high accuracy.

Interplay between Partial EMT and Cisplatin Resistance as the Drivers for Recurrence in HNSCC.

This study aims to investigate the role of partial epithelial to mesenchymal transition (pEMT)-related proteins in modulating Cisplatin resistance in head and neck squamous cell carcinoma (HNSCC). SCC-25 cells were pre-treated with TGF-beta1 followed by transient Krüppel-like Factor 4 (KLF4)-overexpression and Cisplatin treatment. Cell growth, cell morphological changes and cell migration were assessed using Juli BR live cell video-microscopy. In addition, Ki-67 and Slug immunostaining and follow-up image cytometric analysis of primary and recurrent HNSCC tumors were performed to evaluate the proliferation index (PI) and the EMT-like phenotype. We observed that proliferating and Slug-positive tumor cells expand after therapy in HNSCC. Subsequently, protein analysis revealed the stabilization of Slug, upregulation of Vimentin and phospho-p38 (p-p38) in Cisplatin-resistant SCC-25 cells. Moreover, KLF4-overexpression contributed to Cisplatin sensitivity by reduction of Slug at the protein level. This work strongly suggests that an pEMT-like pathway is activated in recurrent and Cisplatin-resistant HNSCC. Finally, stable KLF4-overexpression might sensitize HNSCC tumor cells for Cisplatin treatment.

Next-Generation Digital Histopathology of the Tumor Microenvironment.

Progress in cancer research is substantially dependent on innovative technologies that permit a concerted analysis of the tumor microenvironment and the cellular phenotypes resulting from somatic mutations and post-translational modifications. In view of a large number of genes, multiplied by differential splicing as well as post-translational protein modifications, the ability to identify and quantify the actual phenotypes of individual cell populations in situ, i.e., in their tissue environment, has become a prerequisite for understanding tumorigenesis and cancer progression. The need for quantitative analyses has led to a renaissance of optical instruments and imaging techniques. With the emergence of precision medicine, automated analysis of a constantly increasing number of cellular markers and their measurement in spatial context have become increasingly necessary to understand the molecular mechanisms that lead to different pathways of disease progression in individual patients. In this review, we summarize the joint effort that academia and industry have undertaken to establish methods and protocols for molecular profiling and immunophenotyping of cancer tissues for next-generation digital histopathology-which is characterized by the use of whole-slide imaging (brightfield, widefield fluorescence, confocal, multispectral, and/or multiplexing technologies) combined with state-of-the-art image cytometry and advanced methods for machine and deep learning.