Osteomyelitis by Microsporum canis and Staphylococcus spp. in cat (Felis catus) - case report.

BACKGROUND: Staphylococcus spp and Microsporum canis are zoonotic microorganisms which can cause infections and systemic diseases. The bone infection is usually caused by invasion of pathogen through the hematologic route. Mixed osteomyelitis caused by bacteria and fungi is rare, and to date, there have been no reports of mixed osteomyelitis with Staphylococcus spp. and Microsporum canis. CASE PRESENTATION: This essay reports an atypical presentation of mixed osteomyelitis (Staphylococcus spp. and Microsporum canis) in a domestic cat. A 15-month-old female Persian cat was presented to a veterinary service; the main complaint was the appearance of a nodule in the mandibular ventral rostral region. A radiographic exam performed on the animal showed proliferative and osteolytic bone lesions. The patient was submitted to a biopsy for histopathological evaluation, along with bacterial and fungal cultures. Results showed mixed osteomyelitis by Staphylococcus spp. and Microsporum canis. Microbial Sensitivity Test was performed to choose a more suitable treatment. Two surgical procedures were executed to resect and curette the lesion, and treatments with anti-inflammatory, antibiotic, and antifungal drugs were established, showing a positive clinical evolution. After 8 months of treatment, the patient's owner moved to a different city, and the animal was seen by other veterinarians, who followed along with the same treatment. However, due to complications and a diminishing quality of life over 4 years of diagnosis, the patient was euthanized. CONCLUSION: Given the above, mixed osteomyelitis is difficult to treat and can cause losses of life quality resulting death, especially in infections where M. canis is the agent causing the disease. Bacterial osteomyelitis is more frequently reported. But the lack of investigation of microorganisms other than bacteria, such as fungal cases, may imply in underdiagnosed cases. Treatment of osteomyelitis can be difficult considering the difficulties in isolating the pathological agent, resistance to the drug used, prolonged treatment time, and cost.

Interference with Bacterial Conjugation and Natural Alternatives to Antibiotics: Bridging a Gap.

Horizontal gene transfer (HGT) in food matrices has been investigated under conditions that favor gene exchange. However, the major challenge lies in determining the specific conditions pertaining to the adapted microbial pairs associated with the food matrix. HGT is primarily responsible for enhancing the microbial repertoire for the evolution and spread of antimicrobial resistance and is a major target for controlling pathogens of public health concern in food ecosystems. In this study, we investigated Salmonella Heidelberg (SH) and Escherichia coli (EC) regarding gene exchange under conditions mimicking the industrial environment, with the coproducts whey (SL) and chicken juice (CJ). The S. Heidelberg strain was characterized by antibiotic susceptibility standards and PCR to detect the blaTEM gene. A concentration of 0.39 mg/mL was determined to evaluate the anti-conjugation activity of nanostructured lipid nanocarriers (NLCs) of essential oils to mitigate ß-lactam resistance gene transfer. The results showed that the addition of these coproducts promoted an increase of more than 3.5 (whey) and 2.5 (chicken juice) orders of magnitude in the conjugation process (p < 0.01), and NLCs of sage essential oil significantly reduced the conjugation frequency (CF) by 74.90, 90.6, and 124.4 times when compared to the transfers in the absence of coproducts and the presence of SL and CJ, respectively. For NLCs from olibanum essential oil, the decrease was 4.46-fold for conjugations without inhibitors and 3.12- and 11.3-fold in the presence of SL and CJ. NLCs associated with sage and olibanum essential oils effectively control the transfer of antibiotic resistance genes and are a promising alternative for use at industrial levels.

New Palladium(II) Complexes Containing Methyl Gallate and Octyl Gallate: Effect against Mycobacterium tuberculosis and Campylobacter jejuni.

This work describes the preparation, characterization and antimicrobial activity of four palladium(II) complexes, namely, [Pd(meg)(1,10-phen)] 1, [Pd(meg)(PPh3)2] 2, [Pd(og)(1,10-phen)] 3 and [Pd(og)(PPh3)2] 4, where meg = methyl gallate, og = octyl gallate, 1,10-phen = 1,10-phenanthroline and PPh3 = triphenylphosphine. As to the chemical structures, spectral and physicochemical studies of 1-4 indicated that methyl or octyl gallate coordinates a palladium(II) ion through two oxygen atoms upon deprotonation. A chelating bidentate phenanthroline or two triphenylphosphine molecules complete the coordination sphere of palladium(II) ion, depending on the complex. The metal complexes were tested against the Mycobacterium tuberculosis H37Rv strain and 2 exhibited high activity (MIC = 3.28 µg/mL). As to the tests with Campylobacter jejuni, complex 1 showed a significant effect in reducing bacterial population (greater than 7 log CFU) in planktonic forms, as well as in the biomass intensity (IBF: 0.87) when compared to peracetic acid (IBF: 1.11) at a concentration of 400 µg/mL. The effect provided by these complexes has specificity according to the target microorganism and represent a promising alternative for the control of microorganisms of public health importance.

Agents of Campylobacteriosis in Different Meat Matrices in Brazil.

We aimed to identify the prevalence of thermophilic species of Campylobacter in meats of different species available on the Brazilian commercial market and to determine the genetic diversity, antimicrobial resistance and virulence potential of the isolates. A total of 906 samples, including chicken, beef and pork carcasses and chicken and beef livers, were purchased in retail outlets, and prevalences of 18.7% (46/246), 3.62% (5/138), 10.14% (14/138), 3.62% (5/138) and 4.47% (11/132), respectively, were identified, evidencing the dissemination of genotypes in the main producing macro-regions. Of all isolates, 62.8% were classified as multidrug resistant (MDR), with resistance to amoxicillin-clavulanate (49.4%), tetracycline (51.8%) and ciprofloxacin (50.6%) and co-resistance to macrolides and fluoroquinolones (37.1%). Multivirulent profiles were identified mainly in isolates from chicken carcasses (84.8%), and the emergence of MDR/virulent strains was determined in pork isolates. All isolates except those from chicken carcasses showed a high potential for biofilm formation (71.4% luxS) and consequent persistence in industrial food processing. For chicken carcasses, the general virulence was higher in C. jejuni (54.3%), followed by C. coli (24%) and Campylobacter spp. (21.7%), and in the other meat matrices, Campylobacter spp. showed a higher prevalence of virulence (57.2%). The high rates of resistance and virulence reinforce the existence of strain selection pressure in the country, in addition to the potential risk of strains isolated not only from chicken carcasses, but also from other meat matrices.

A Ternary Copper (II) Complex with 4-Fluorophenoxyacetic Acid Hydrazide in Combination with Antibiotics Exhibits Positive Synergistic Effect against Salmonella Typhimurium.

Salmonella spp. continues to figure prominently in world epidemiological registries as one of the leading causes of bacterial foodborne disease. We characterised 43 Brazilian lineages of Salmonella Typhimurium (ST) strains, characterized drug resistance patterns, tested copper (II) complex as control options, and proposed effective antimicrobial measures. The minimum inhibitory concentration was evaluated for seven antimicrobials, isolated and combined with the copper (II) complex [Cu(4-FH)(phen)(ClO4)2] (4-FH = 4-fluorophenoxyacetic acid hydrazide and phen = 1,10-phenanthroline), known as DRI-12, in planktonic and sessile ST. In parallel, 42 resistance genes were screened (PCR/microarray). All strains were multidrug resistant (MDR). Resistance to carbapenems and polymyxins (86 and 88%, respectively) have drawn attention to the emergence of the problem in Brazil, and resistance is observed also to CIP and CFT (42 and 67%, respectively), the drugs of choice in treatment. Resistance to beta-lactams was associated with the genes blaTEM/blaCTX-M in 39% of the strains. Lower concentrations of DRI-12 (62.7 mg/L, or 100 µM) controlled planktonic and sessile ST in relation to AMP/SUL/TET and AMP/SUL/TET/COL, respectively. The synergistic effect provided by DRI-12 was significant for COL/CFT and COL/AMP in planktonic and sessile ST, respectively, and represents promising alternatives for the control of MDR ST.

Development of a nonhuman primate challenge model to evaluate CD8+ T cell responses to an adenovirus-based vaccine expressing SIV proteins upon repeat-dose treatment with checkpoint inhibitors.

Targeting immune checkpoint receptors expressed in the T cell synapse induces active and long-lasting antitumor immunity in preclinical tumor models and oncology patients. However, traditional nonhuman primate (NHP) studies in healthy animals have thus far demonstrated little to no pharmacological activity or toxicity for checkpoint inhibitors (CPIs), likely due to a quiescent immune system. We developed a NHP vaccine challenge model in Mauritius cynomolgus monkey (MCMs) that elicits a strong CD8+ T cell response to assess both pharmacology and safety within the same animal. MHC I-genotyped MCMs were immunized with three replication incompetent adenovirus serotype 5 (Adv5) encoding Gag, Nef and Pol simian immunodeficiency virus (SIV) proteins administered 4 weeks apart. Immunized animals received the anti-PD-L1 atezolizumab or an immune checkpoint-targeting bispecific antibody (mAbX) in early development. After a single immunization, Adv5-SIVs induced T-cell activation as assessed by the expression of several co-stimulatory and co-inhibitory molecules, proliferation, and antigen-specific T-cell response as measured by a Nef-dependent interferon-γ ELIspot and tetramer analysis. Administration of atezolizumab increased the number of Ki67+ CD8+ T cells, CD8+ T cells co-expressing TIM3 and LAG3 and the number of CD4+ T cells co-expressing 4-1BB, BTLA, and TIM3 two weeks after vaccination. Both atezolizumab and mAbX extended the cytolytic activity of the SIV antigen-specific CD8+ T cell up to 8 weeks. Taken together, this vaccine challenge model allowed the combined study of pharmacology and safety parameters for a new immunomodulatory protein-based therapeutic targeting CD8+ T cells in an NHP model.

Genotypical Relationship Between Human and Poultry Strains of Campylobacter jejuni.

This study aimed to compare the genotype diversity of C. jejuni isolates. From the total of 64 C. jejuni strains evaluated, 44 were isolated from broiler carcasses (2015-2016) and 20 from hospitalized patients with gastroenteritis caused by the microorganism (2000-2006). The strains were correlated for the presence of flaA, pldA, cadF, ciaB, cdtABC, luxS, dnaJ, cbrA, htrA, pVir, Hcp, cstII, and neuA genes by PCR (polymerase chain reaction) and for phylogenetic proximity by PFGE (pulsed-field gel electrophoresis). Of the total strains studied, 28 (43.7%) presented all the studied genes, except pVir. Among these strains, 25 (89.3%) were of poultry origin. Poultry strains showed a higher prevalence (P < 0.05) of genes linked to adhesion, colonization, invasion, cytotoxicity, biofilm formation, and adaptation to adverse conditions. Additionally, the profile that denotes the presence of all genes identified in the study (P1) was identified in 56.8% of poultry strains and in 15.0% of human strains. Molecular typing analysis identified five pulsotypes, none of which grouped strains from different origins. Although human strains were from hospitalized patients, they presented limited virulence capacity and adaptability to adverse conditions compared to chicken carcasses, besides being different in molecular typing. However, the ability to cause Guillain-Barré Syndrome is equal for both strains. In general, poultry strains, being more recent, are more specialized to adapt to the environment, invade, and cause disease in the human host.

Antibiotic Resistance in the Alternative Lifestyles of Campylobacter jejuni.

Campylobacter jejuni is the main pathogen identified in cases of foodborne gastroenteritis worldwide. Its importance in poultry production and public health is highlighted due to the growing antimicrobial resistance. Our study comparatively investigated the effect of five different classes of antimicrobials on the planktonic and biofilm forms of 35 strains of C. jejuni with high phylogenetic distinction in 30 of them. In the planktonic form, the existence of susceptible strains to colistin (7/35 - 20%) and resistance to meropenem (3/35 - 8.6%) represent a novelty in strains evaluated in Brazil. In biofilms formed with the addition of chicken juice, the number of resistant strains was significantly higher for colistin, erythromycin and meropenem (100%), but the susceptibility to tetracycline was shown as a control strategy for specific cases. High concentrations (1,060 ± 172.1mg/L) of antibiotics were necessary to control the biofilm structure in susceptible strains in the planktonic form, which is consistent with the high biomass produced in these strains. Stainless steel and polyurethane were the most (BFI=2.1) and least (BFI=1.6) favorable surfaces for the production of biomass treated with antimicrobials. It is concluded that the antimicrobial action was detected for all tested drugs in planktonic form. In sessile forms, the biomass production was intensified, except for tetracycline, which showed an antibiofilm effect.

Cross-company evaluation of the human lymphocyte activation assay.

Nonclinical immunotoxicity evaluation is an important component of safety assessment for pharmaceuticals. One in vitro assay that can be applied in a weight of evidence assessment is the human lymphocyte activation (HuLA) assay, an antigen recall assay, similar in many respects to the in vivo T-cell-dependent antibody response (TDAR) in that cooperation of multiple immune cell types are needed to produce responses. This assay uses human cells and is more amenable than the TDAR to compound ranking and mechanistic studies. The HuLA assay requires less time and drug than TDAR assays, uses a relevant antigen (influenza), reflects a human immune response, and applies principles of the 3Rs to non-clinical safety assessment. Peripheral blood mononuclear cells (PBMC) from flu-immunized donors are re-stimulated with flu-vaccine in the presence of test articles, and proliferation is measured. Published data demonstrate the applicability of the HuLA assay, but it has not been evaluated for reproducibility across testing sites. To evaluate assay reproducibility, scientists from a consortium of institutions conducted the assay in parallel, using a common pool of donor PBMC, influenza vaccine, and known immunosuppressant compounds (cyclosporine A and mycophenolic acid). The HuLA assay was highly reproducible in identification of inhibition of antigen-specific responses, and there was significant agreement across testing sites in the half maximal inhibitory concentration (IC50) values. Intra-site variability was the largest contributor to the variability observed within the assay. The HuLA assay was demonstrated to be ideally suited to comparing multiple compounds (i.e. compound ranking or benchmarking) within the same assay. Overall, the data reported herein support the HuLA assay as a useful tool in mechanistic evaluations of antigen-specific immune responses.

Approaching stability challenges for flow cytometry in a regulated bioanalytical environment.

Stability of samples for flow cytometry is a critical parameter since storage period of samples is restricted to only a limited period after collection. For most studies, clinical samples have to be shipped to a testing laboratory, in contrast to preclinical samples, which can be analyzed on-site or off-site. Therefore, evaluating stability is critical to provide flexibility on testing of samples to obtain reliable data. A wide variety of factors contributes to establishing stability from sample collection through acquisition. We provided suggestions for experimental and stability parameters to be taken into consideration when designing a flow cytometry method. The case studies presented represent how certain stability issues were overcome to perform flow cytometry assays in a regulated bioanalytical environment.