Extraction and Quantification of Azelaic Acid from Different Wheat Samples (Triticum durum Desf.) and Evaluation of Their Antimicrobial and Antioxidant Activities.

Azelaic Acid (AzA) is a 9-carbon atom dicarboxylic acid, with numerous pharmacological uses in dermatology. Its effectiveness in papulopustular rosacea and acne vulgaris, among other dermatological disorders such as keratinization and hyper-pigmentation, is thought to be related to its anti-inflammatory and antimicrobial properties. It is a by-product of Pityrosporum fungal mycelia metabolism but also it is found in different cereals such as barley, wheat, and rye. Diverse topical formulations of AzA exist in commerce, and it is mainly produced via chemical synthesis. In this study we describe the extraction of AzA from whole grains and whole-grain flour (Triticum durum Desf.) through green methods. Seventeen different extracts were prepared and analyzed for their AzA content by HPLC-MS methods and then screened for their antioxidant activity using spectrophotometric assays (ABTS, DPPH, and Folin-Ciocalteu). Minimum-inhibitory-concentration (MIC) assays against several bacterial and fungal pathogens were performed, to validate their antimicrobial activity. The obtained results indicate that whole grain extracts provide a wider spectrum of activity than the flour matrix; in particular, the Naviglio® extract showed higher AzA content, while the hydroalcoholic ultrasound-assisted extract provided better antimicrobial and antioxidant activity. The data analysis was performed using principal component analysis (PCA), as an unsupervised-pattern-recognition technique, to extract useful analytical and biological information.

Metabolite Profiling and Bioactivities of Leaves, Stems, and Flowers of Rumex usambarensis (Dammer) Dammer, a Traditional African Medicinal Plant.

The comprehensive identification of secondary metabolites represents a fundamental step for the assessment of bioactivities and pharmacological properties of traditional herbal drugs. Rumex usambarensis (Dammer) Dammer has been described as a multipurpose remedy in different African traditional pharmacopoeias, but its phytochemical profile has not been properly investigated. Herein we report a high throughput metabolomic screening, based on ultra-high performance liquid chromatography-travelling wave ion mobility spectrometry quadrupole time-of-flight (UHPLC-TWINS-QTOF), which was performed for the first time on different R. usambarensis plant parts. By applying high-resolution mass spectrometry-based metabolomics and chemometric analysis, a complete discrimination of different aerial parts was obtained, with the annotation of 153 significant metabolites in leaves, stems, and flowers, suggesting an easy authentication and discrimination route. Phytochemical data were correlated to antimicrobial and antioxidant properties. Flavonoids, benzopyranes, chromones, and xanthones derivatives, along with a richer phytocomplex, might be responsible for the stronger bioactivities obtained from flowers.

Seleno Containing Compounds as Potent and Selective Antifungal Agents.

Fungal promoted infections are becoming a severe health global emergency due to drug-resistant phenomena and zoonosis. This work investigated compounds bearing acyl-/selenoureido moieties and primary/secondary sulfonamide groups as novel antifungal agents acting through organism-directed selenium toxicity and inhibition of the newly emergent therapeutic target, the Carbonic Anhydrases (CAs; EC 4.2.1.1). Reported data clearly indicate that seleno-containing scaffolds with respect to the standard-of-care drugs showed appreciable antifungal activity, which was suppressed when the chalcogen was replaced with its cognate isosteric elements sulfur and oxygen. In addition, such compounds showed excellent selectivity against Malassezia pachydermatis over its related genus strains Malassezia furfur and Malassezia globosa. Safe cytotoxicity profiles on bovine kidney cells (MDBK) and human HaCat cells, as well as the shallow hemolytic activity on defibrinated sheep blood, allowed us to consider these compounds as up-and-coming novel antifungals.

Inhibitors of O-Acetylserine Sulfhydrylase with a Cyclopropane-Carboxylic Acid Scaffold Are Effective Colistin Adjuvants in Gram Negative Bacteria.

Antibacterial adjuvants are of great significance, since they allow one to downscale the therapeutic dose of conventional antibiotics and reduce the insurgence of antibacterial resistance. Herein, we report that O-acetylserine sulfhydrylase (OASS) inhibitors could be used as colistin adjuvants to treat infections caused by critical pathogens spreading worldwide, Escherichia coli, Salmonella enterica serovar Typhimurium, and Klebsiella pneumoniae. Starting from a hit compound endowed with a nanomolar dissociation constant, we have rationally designed and synthesized a series of derivatives to be tested against S. Typhimurium OASS isoenzymes, StOASS-A and StOASS-B. All acidic derivatives have shown good activities in the nanomolar range against both OASS isoforms in vitro. Minimal Inhibitory Concentrations (MICs) were then evaluated, as well as compounds' toxicity. The compounds endowed with good activity in vitro and low cytotoxicity have been challenged as a potential colistin adjuvant against pathogenic bacteria in vitro and the fractional inhibitory concentration (FIC) index has been calculated to define additive or synergistic effects. Finally, the target engagement inside the S. Typhimurium cells was confirmed by using a mutant strain in which the OASS enzymes were inactivated. Our results provide a robust proof of principle supporting OASS as a potential nonessential antibacterial target to develop a new class of adjuvants.

Survey on Carbapenem-Resistant Bacteria in Pigs at Slaughter and Comparison with Human Clinical Isolates in Italy.

This study is focused on resistance to carbapenems and third-generation cephalosporins in Gram-negative microorganisms isolated from swine, whose transmission to humans via pork consumption cannot be excluded. In addition, the common carriage of carbapenem-resistant (CR) bacteria between humans and pigs was evaluated. Sampling involved 300 faecal samples collected from slaughtered pigs and 300 urine samples collected from 187 hospitalised patients in Parma Province (Italy). In swine, MIC testing confirmed resistance to meropenem for isolates of Pseudomonas aeruginosa and Pseudomonas oryzihabitans and resistance to cefotaxime and ceftazidime for Escherichia coli, Ewingella americana, Enterobacter agglomerans, and Citrobacter freundii. For Acinetobacter lwoffii, Aeromonas hydrofila, Burkolderia cepacia, Corynebacterium indologenes, Flavobacterium odoratum, and Stenotrophomonas maltophilia, no EUCAST MIC breakpoints were available. However, ESBL genes (blaCTXM-1, blaCTX-M-2, blaTEM-1, and blaSHV) and AmpC genes (blaCIT, blaACC, and blaEBC) were found in 38 and 16 isolates, respectively. P. aeruginosa was the only CR species shared by pigs (4/300 pigs; 1.3%) and patients (2/187; 1.1%). P. aeruginosa ST938 carrying blaPAO and blaOXA396 was detected in one pig as well as an 83-year-old patient. Although no direct epidemiological link was demonstrable, SNP calling and cgMLST showed a genetic relationship of the isolates (86 SNPs and 661 allele difference), thus suggesting possible circulation of CR bacteria between swine and humans.

Leptospira Seroprevalence in Colombian Dairy Herds.

Leptospirosis in cattle has important economic effects on the infected farms. Moreover, livestock farming is considered a major occupational risk factor for the transmission of Leptospira infection to humans. A survey was performed to determine the overall and within-herd seroprevalence and mapping of different Leptospira serovars in dairy cattle from farms located in some municipalities of the Colombian department of Boyacá. Nine hundred and fifty-nine animals, from 20 unvaccinated and one vaccinated herd, were included in the study. Anti-Leptospira serum antibodies were detected by the microscopic agglutination test (MAT). Only one herd was seronegative. Overall seroprevalence to at least one serovar of Leptospira was 24.1% for unvaccinated animals and 62.3% for animals from the vaccinated herd. A very high within-herd seroprevalence (>60%) was present in 20% of the unvaccinated herds. The presence in the vaccinated herd of 20/398 animals showing high titers, between 1000 and 4000, to at least one serovar of Leptospira suggest that some animals could have been infected. Moreover, due to the presence of seronegative animals, a failure of vaccination immunity or the presence of unvaccinated animals in the vaccinated herd cannot be excluded. In all farms, domestic animals other than cattle were present. Considering the farming practices occurring on dairy farms in the study area, higher hygienic standards and stricter biosecurity measures are suggested.

Antimicrobial activity of a standardized medical honey on bacterial isolates from infected skin lesions of non-traditional companion animals.

In recent years, due to the growing phenomenon of antimicrobial resistance, the search for alternative strategies to antibiotic treatments is increasing and a considerable interest for the use of medical honey in clinical practice has emerged. Honey has been used for the treatment of skin lesions, in both humans and animals. However, knowledge concerning the use of medical honey in non­traditional companion animals is scarce. The aim of this study was to assess the antibacterial activity of a standardized medical honey (Revamil, BFactory) against bacterial strains isolated from skin lesions of non­traditional companion animals. The minimum bactericidal concentration (MBC) of Revamil honey against seventeen clinical isolates and three reference strains was established.The medical honey showed antimicrobial activity against both Gram­positive and Gram­negative bacteria. Growth was inhibited for all the strains at concentrations of medical honey ranging from 10 to 40%. Pseudomonas oryzihabitans and Alcaligenes faecalis showed the lowest MBC (10%). The reference strain Staphylococcus aureus ATCC25923 showed a higher sensitivity to 20% honey compare to the corresponding clinical isolate (P = 0.001). The observed results suggest that Revamil could represent an effective therapeutic aid, useful for the reduction of antibiotic use, in case of pathological skin infections in non­traditional companion animals.

Development of novel cocrystal-based active food packaging by a Quality by Design approach.

A way to reduce food waste is related to the increase of the shelf-life of food as a result of improving the package type. An innovative active food packaging material based on cocrystallization of microbiologically active compounds present in essential oils i.e. carvacrol, thymol and cinnamaldehyde was developed following the Quality by Design principles. The selected active components were used to produce antimicrobial plastic films with solidified active ingredients on their surface characterized by antimicrobial properties against four bacterial strains involved in fruit and vegetable spoilage. The developed packaging prototypes exhibited good antimicrobial activity in vitro providing inhibition percentage of 69 (±15)% by contact and inhibition diameters of 32 (±6) mm in the gas phase, along with a prolonged release of the active components. Finally, the prolonged shelf-life of grape samples up to 7 days at room temperature was demonstrated.

OBP-functionalized/hybrid superparamagnetic nanoparticles for Candida albicans treatment.

Infections caused by the opportunistic yeast Candida albicans are one of the major life threats for hospitalized and immunocompromised patients, as a result of antibiotic and long-term antifungal treatment abuse. Odorant binding proteins can be considered interesting candidates to develop systems able to reduce the proliferation and virulence of this yeast, because of their intrinsic antimicrobial properties and complexation capabilities toward farnesol, the major quorum sensing molecule of Candida albicans. In the present study, a hybrid system characterized by a superparamagnetic iron oxide core functionalized with bovine odorant binding protein (bOBP) was successfully developed. The nanoparticles were designed to be suitable for magnetic protein delivery to inflamed areas of the body. The inorganic superparamagnetic core was characterized by an average diameter of 6.5 ± 1.1 nm and a spherical shape. Nanoparticles were functionalized by using 11-phosphonoundecanoic acid as spacer and linked to bOBP via amide bonds, resulting in a concentration level of 26.0 ± 1.2 mg bOBP/g SPIONs. Finally, both the biocompatibility of the developed hybrid system and the fungistatic activity against Candida albicans by submicromolar OBP levels were demonstrated by in vitro experiments.

Discovery of Substituted (2-Aminooxazol-4-yl)Isoxazole-3-carboxylic Acids as Inhibitors of Bacterial Serine Acetyltransferase in the Quest for Novel Potential Antibacterial Adjuvants.

Many bacteria and actinomycetales use L-cysteine biosynthesis to increase their tolerance to antibacterial treatment and establish a long-lasting infection. In turn, this might lead to the onset of antimicrobial resistance that currently represents one of the most menacing threats to public health worldwide. The biosynthetic machinery required to synthesise L-cysteine is absent in mammals; therefore, its exploitation as a drug target is particularly promising. In this article, we report a series of inhibitors of Salmonella thyphimurium serine acetyltransferase (SAT), the enzyme that catalyzes the rate-limiting step of L-cysteine biosynthesis. The development of such inhibitors started with the virtual screening of an in-house library of compounds that led to the selection of seven structurally unrelated hit derivatives. A set of molecules structurally related to hit compound 5, coming either from the original library or from medicinal chemistry efforts, were tested to determine a preliminary structure-activity relationship and, especially, to improve the inhibitory potency of the derivatives, that was indeed ameliorated by several folds compared to hit compound 5 Despite these progresses, at this stage, the most promising compound failed to interfere with bacterial growth when tested on a Gram-negative model organism, anticipating the need for further research efforts.

Investigational Studies on a Hit Compound Cyclopropane-Carboxylic Acid Derivative Targeting O-Acetylserine Sulfhydrylase as a Colistin Adjuvant.

Antibacterial adjuvants are of great significance, since they allow the therapeutic dose of conventional antibiotics to be lowered and reduce the insurgence of antibiotic resistance. Herein, we report that an O-acetylserine sulfhydrylase (OASS) inhibitor can be used as a colistin adjuvant to treat infections caused by Gram-positive and Gram-negative pathogens. A compound that binds OASS with a nM dissociation constant was tested as an adjuvant of colistin against six critical pathogens responsible for infections spreading worldwide, Escherichia coli, Salmonella enterica serovar Typhimurium, Klebisiella pneumoniae, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Staphylococcus pseudintermedius. The compound showed promising synergistic or additive activities against all of them. Knockout experiments confirmed the intracellular target engagement supporting the proposed mechanism of action. Moreover, compound toxicity was evaluated by means of its hemolytic activity against sheep defibrinated blood cells, showing a good safety profile. The 3D structure of the compound in complex with OASS was determined at 1.2 Å resolution by macromolecular crystallography, providing for the first time structural insights about the nature of the interaction between the enzyme and this class of competitive inhibitors. Our results provide a robust proof of principle supporting OASS as a potential nonessential antibacterial target to develop a new class of adjuvants and the structural basis for further structure-activity relationship studies.

Wild Micromammals as Bioindicators of Antibiotic Resistance in Ecopathology in Northern Italy.

Antimicrobial resistance (AMR) is an increasing threat to human health and an important issue also in the natural environment. For this study, an ecopathological approach was applied to the monitoring of the antimicrobial resistance in the province of Parma, Northern Italy. Fourteen monitoring sites and seventy-four faecal samples from four species of wild micromammals (Apodemus sylvaticus, Microtus savii, Mus domesticus and Suncus etruscus) were collected. Samples were subjected to bacteriological examination and antimicrobial susceptibility testing. Antibiotics belonging to 13 different antibiotic classes were tested. Collected data showed a prevalence of multi-drug resistant (MDR) strains of 55.13% and significant differences in the prevalence of MDR strains among the different micromammal species, while sex, age and anthropization level did not significantly affected MDR strains prevalence. Moreover, a high prevalence of bacterial strains resistant to colistin (95%), gentamicin (87%) and amikacin (83%) was observed. To our knowledge, this is the first report on antibiotic resistance in wild micromammals in the province of Parma.

The prevalence of Pseudomonas aeruginosa and multidrug resistant Pseudomonas aeruginosa in healthy captive ophidian.

BACKGROUND: Snakes are globally considered as pet animals, and millions of ophidians are bred in captivity. Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium that can act as an opportunistic pathogen of man and animals and is frequently present in the oral and cloacal microbiota of healthy ophidians. It can cause severe clinical diseases and often shows antibiotic resistance. The aim of this study was to evaluate the prevalence and antibiotic resistance profiles of P. aeruginosa isolated from the cloacal microbiota of a large population sample of healthy captive ophidians and to evaluate the statistical associations with farming conditions. METHODS: A total of 419 cloacal swabs were collected from snakes belonging to the Boidae (n = 45), Colubridae (n = 48) and Pythonidae (n = 326) families and inoculated onto complete culture media. Food, water and bedding samples were also analyzed. The antimicrobial susceptibility of P. aeruginosa isolates was evaluated through the Kirby-Bauer agar diffusion test. Statistical analyses were performed with the chi-square test. RESULTS: The prevalence of P. aeruginosa was 59.9%, and 35.5% of these strains were multidrug resistant (MDR). The prevalence of MDR P. aeruginosa was significantly higher in adult samples than in young samples, and widespread resistance to Cephalosporins, Polymyxins and Sulfonamides was observed. Statistically significant differences in the prevalence of P. aeruginosa were observed depending on the farm size and snake family. Feeding thawed prey was associated with a higher P. aeruginosa and MDR P. aeruginosa prevalence. Moreover, snakes fed home-raised prey had a significantly higher MDR P. aeruginosa prevalence than snakes fed commercially available feed. Less frequent terrarium cleaning was associated with a higher MDR P. aeruginosa prevalence. On the other hand, snake reproductive status was not significantly associated with P. aeruginosa or MDR P. aeruginosa prevalence. All food, water and bedding samples were negative for P. aeruginosa presence. DISCUSSION: The overall P. aeruginosa prevalence found in this study was lower than that found by other authors, but a high proportion of the isolates were MDR. This study highlighted the presence of constitutive (such as age and taxonomic family) and managerial (farm size, cleaning cycle frequency and food type) factors associated with P. aeruginosa and/or MDR P. aeruginosa prevalence. Good breeding management and proper antibiotic treatment of P. aeruginosa infections could help reduce the presence of P. aeruginosa and MDR P. aeruginosa in the gut microbiota of snakes and consequently reduce the risk to public health.

Vertebrate odorant binding proteins as antimicrobial humoral components of innate immunity for pathogenic microorganisms.

The bacterium Pseudomonas aeruginosa (PA) and the yeast Candida albicans (CA) are pathogens that cohabit the mucosa of the respiratory tracts of animals and humans. Their virulence is largely determined by chemical communication driven by quorum sensing systems (QS), and the cross perception of their quorum sensing molecules (QSM) can modulate the prevalence of one microorganism over the other. Aiming to investigate whether some of the protein components dissolved in the mucus layering the respiratory mucosa might interfere with virulence and cross-communication of these, and eventually other microorganisms, ligand binding assays were carried out to test the scavenging potential of the bovine and porcine forms of the Lipocalin odorant binding protein (OBP) for several QSMs (farnesol, and acylhomoserine lactones), and for pyocyanin, a toxin produced by PA. In addition, the direct antimicrobial activity of the OBPs was tested by time kill assay (TKA) against CA, PA and other bacteria and yeasts. The positivity of all the ligand binding assays and the antimicrobial activity determined for CA, and for some of the other microorganisms tested, let hypothesize that vertebrate OBPs might behave as humoral components of innate immunity, active against pathogenic bacteria and fungi. In addition, TKAs with mutants of bovine OBP with structural properties different from those of the native form, and with OBP forms tagged with histidines at the amino terminal, provided information about the mechanisms responsible of their antimicrobial activity and suggested possible applications of the OBPs as alternative or co-adjuvants to antibiotic therapeutic treatments.

Microbial Isolates from Vegetable Foreign Bodies Inhaled by Dogs.

Grass-seed inhalation is a common problem in canine patients, in particular during summer months, migrating in upper and lower respiratory tract. Grass awns can harbor bacteria and fungi, causing grass seeds foreign body-related disease (GSFBD). Aim of this study was to investigate the aerobic microbial flora isolated from grass awns extracted from 41 dogs with GSFBD and the antibiotic susceptibility of the isolated bacterial strains. Fifty-four grass awns were localized with diagnostic imaging tests and removed by endoscopy from respiratory tract. The most frequent localizations were in the left nostril and the right hemithorax. Only one grass awn was extracted from each patient except in 7 that had more than one. Bacteriological and mycological cultures, strains identification, and antibiotic susceptibility tests were performed. One or more bacterial strains were isolated from all grass awns. Fungal strains were isolated only in 4 cases. Staphylococcus sp. was the most frequent isolate in the upper respiratory tract (36.8%), while E. coli (24.4%) was the most frequent isolate in the lower tract. Fluoroquinolones and Doxycycline were the most effective antibiotics, while resistance was observed against Gentamicin (>93%), Cefapirin, and Clindamycin (>80%). These data are relevant in relation to the use of these antibiotics in both animals and humans, for the risk of transmission of antibiotic resistant bacteria or resistance genes.