Aerobic Vaginal Microflora in Gestational and Non-Gestational Bitches (Canis lupus familiaris).

The vaginal tract comprises commensal microorganisms, which play an essential role in the health of the reproductive tract. Any dysbiosis in the vaginal microenvironment may lead to severe urinary tract infections or even infertility. This study aimed to evaluate the aerobic bacterial flora isolated from vaginal samples from 100 lactating bitches in the antepartum period (n = 3), postpartum period (n = 80), and with Lactatio sine graviditate (n = 17). Before vaginal swabs, all the bitches went through a gynecology consult, along with milk and blood sampling. Standard microbiological techniques were used for bacterial isolation. Among the 100 vaginal samples analyzed, 82% had a positive microbiological outcome, while 18% were negative. The microbiologic profile listed 17 different genera. The main isolated bacterial families were Micrococcaceae, Staphylococcaceae, Morganellaceae, Bacillaceae, and Rhizobiaceae. At the same time, strains like Agrobacterium radiobacter, Ochrobactrum anthropi, Chromobacterium violaceum, Burkholderia mallei, Bacillus pumllus, or Staphylococcus xylosus were isolated for the first time from the vaginal secretion of lactating bitches. The microbiological data demonstrates that lactating bitches' vaginal discharge is heterogeneous and may be affected by coitus, sampling season, age, and reproductive status.

Colon Drug Delivery Systems Based on Swellable and Microbially Degradable High-Methoxyl Pectin: Coating Process and In Vitro Performance.

Oral colon delivery systems based on a dual targeting strategy, harnessing time- and microbiota-dependent release mechanisms, were designed in the form of a drug-containing core, a swellable/biodegradable polysaccharide inner layer and a gastroresistant outer film. High-methoxyl pectin was employed as the functional coating polymer and was applied by spray-coating or powder-layering. Stratification of pectin powder required the use of low-viscosity hydroxypropyl methylcellulose in water solution as the binder. These coatings exhibited rough surfaces and higher thicknesses than the spray-coated ones. Using a finer powder fraction improved the process outcome, coating quality and inherent barrier properties in aqueous fluids. Pulsatile release profiles and reproducible lag phases of the pursued duration were obtained from systems manufactured by both techniques. This performance was confirmed by double-coated systems, provided with a Kollicoat® MAE outer film that yielded resistance in the acidic stage of the test. Moreover, HM pectin-based coatings manufactured by powder-layering, tested in the presence of bacteria from a Crohn's disease patient, showed earlier release, supporting the role of microbial degradation as a triggering mechanism at the target site. The overall results highlighted viable coating options and in vitro release characteristics, sparking new interest in naturally occurring pectin as a coating agent for oral colon delivery.

Antibacterial Potential of Essential Oils and Silver Nanoparticles against Multidrug-Resistant Staphylococcus pseudintermedius Isolates.

Staphylococcus pseudintermedius is an emergent zoonotic agent associated with multidrug resistance (MDR). This work aimed to describe the antibacterial activity of four essential oils (EOs) and silver nanoparticles (AgNPs) against 15 S. pseudintermedius strains isolated from pyoderma. The four EOs, namely Rosmarinus officinalis (RO), Juniperus communis (GI), Citrus sinensis (AR), and Abies alba (AB), and AgNPs were used alone and in combination to determine the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). All strains were MDR and methicillin-resistant. Among the antibiotic cohort, only rifampicin, doxycycline, and amikacin were effective. EOs' chemical analysis revealed 124 compounds belonging to various chemical classes. Of them, 35 were found in AR, 75 in AB, 77 in GI, and 57 in RO. The monoterpenic fraction prevailed over the sesquiterpenic in all EOs. When EOs were tested alone, AB showed the lowest MIC followed by GI, AR, and RO (with values ranging from 1:128 to 1:2048). MBC increased in the following order: AB, AR, GI, and RO (with values ranging from 1:512 to 1:2048). MIC and MBC values for AgNPs were 10.74 mg/L ± 4.23 and 261.05 mg/L ± 172.74. In conclusion, EOs and AgNPs could limit the use of antibiotics or improve the efficacy of conventional therapies.

Guar gum as a microbially degradable component for an oral colon delivery system based on a combination strategy: formulation and in vitro evaluation.

Oral colon delivery has widely been pursued exploiting naturally occurring polysaccharides degraded by the resident microbiota. However, their hydrophilicity may hinder the targeting performance. The aim of the present study was to manufacture and evaluate a double-coated delivery system leveraging intestinal microbiota, pH, and transit time for reliable colonic release. This system comprised a tablet core, a hydroxypropyl methylcellulose (HPMC) inner layer and an outer coating based on Eudragit® S and guar gum. The tablets were loaded with paracetamol, selected as a tracer drug because of the well-known analytical profile and lack of major effects on bacterial viability. The HPMC and Eudragit® S layers were applied by film-coating. Tested for in vitro release, the double-coated systems showed gastroresistance in 0.1 N HCl followed by lag phases of consistent duration in phosphate buffer pH 7.4, imparted by the HPMC layer and synergistically extended by the Eudragit® S/guar gum one. In simulated colonic fluid with fecal bacteria from an inflammatory bowel disease patient, release was faster than in the presence of ß-mannanase and in control culture medium. The bacteria-containing fluid was obtained by an experimental procedure making multiple tests possible from a single sampling and processing run. Thus, the study conducted proved the feasibility of the delivery system and ability of guar gum to trigger release in the presence of colon bacteria without impairing the barrier properties of the coating. Finally, it allowed an advantageous simulated colonic fluid preparation procedure to be set up, reducing the time, costs, and complexity of testing and enhancing replicability.

Innovative Elastomers with Antimicrobial Activity May Decrease Infection Risks during Milking.

Contagious pathogens are very costly to dairy herds, and they may have zoonotic and reverse-zoonotic potentials and may contribute to the spread of antimicrobial resistance. One of the most important risk factors for spreading these infections is milking, when liner contamination may transfer the pathogens from infected to healthy cows. There is no effective protocol to prevent the transmission of infection without the segregation of infected cows. Recently, the availability of elastomers with patented antimicrobial components in their formulations has allowed the exploration of alternative methods to reduce the risk of infection. Two different types of elastomers (rubber and silicone) and nine different formulations were challenged with three major mastitis pathogens (S. aureus, S. agalactiae, and E. coli). The results that were obtained in this study were interesting and unexpected. Indeed, to our knowledge, this is the first study to show that basic rubber materials have intrinsic antimicrobial activity. Silicone elastomers did not exhibit the same levels of bactericidal activity, although they did exhibit some antibacterial capacity. A significant decrease in bacterial survival curves was observed for all the formulations tested when antimicrobial components were added. The different results observed for the various products are likely due to the different formulations and diverse manufacturing processes. The availability of these new materials that significantly reduce the bacterial load on the liner surface may reduce the risk of spreading intramammary infections during milking. This would be an important step forward in achieving global sustainability of dairy herds, consistent with the objectives of One Health, by reducing the risks of zoonotic diseases and antimicrobial treatments.

Epidemiology of Antimicrobial Resistance Genes in Staphylococcus aureus Isolates from a Public Database from a One Health Perspective-Sample Origin and Geographical Distribution of Isolates.

Staphylococcus aureus are commensal bacteria that are found in food, water, and a variety of settings in addition to being present on the skin and mucosae of both humans and animals. They are regarded as a significant pathogen as well, with a high morbidity that can cause a variety of illnesses. The Centers for Disease Control and Prevention (CDC) has listed them among the most virulent and resistant to antibiotics bacterial pathogens, along with Escherichia coli, Staphylococcus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterococcus faecalis, and Enterococcus faecium. Additionally, S. aureus is a part of the global threat posed by the existence of antimicrobial resistance (AMR). Using 26,430 S. aureus isolates from a global public database (NPDIB; NCBI Pathogen Detection Isolate Browser), epidemiological research was conducted. The results corroborate the evidence of notable variations in isolate distribution and ARG (Antimicrobial Resistance Gene) clusters between isolate sources and geographic origins. Furthermore, a link between the isolates from human and animal populations is suggested by the ARG cluster patterns. This result and the widespread dissemination of the pathogens among animal and human populations highlight how crucial it is to learn more about the epidemiology of these antibiotic-resistance-related infections using a One Health approach.

Epidemiology of Antimicrobial Resistance Genes in Staphyloccocus aureus Isolates from a Public Database in a One Health Perspective-Sample Characteristics and Isolates' Sources.

Staphylococcus aureus is considered one of the most widespread bacterial pathogens for both animals and humans, being the causative agent of various diseases like food poisoning, respiratory tract infections, nosocomial bacteremia, and surgical site and cardiovascular infections in humans, as well as clinical and subclinical mastitis, dermatitis, and suppurative infections in animals. Thanks to their genetic flexibility, several virulent and drug-resistant strains have evolved mainly due to horizontal gene transfer and insurgence of point mutations. Infections caused by the colonization of such strains are particularly problematic due to frequently occurring antibiotic resistance, particulary methicillin-resistant S. aureus (MRSA), and are characterized by increased mortality, morbidity, and hospitalization rates compared to those caused by methicillin-sensitive S. aureus (MSSA). S. aureus infections in humans and animals are a prime example of a disease that may be managed by a One Health strategy. In fact, S. aureus is a significant target for control efforts due to its zoonotic potential, the frequency of its illnesses in both humans and animals, and the threat posed by S. aureus antibiotic resistance globally. The results of an epidemiological analysis on a worldwide public database (NCBI Pathogen Detection Isolate Browser; NPDIB) of 35,026 S. aureus isolates were described. We considered the diffusion of antibiotic resistance genes (ARGs), in both human and animal setting, and the results may be considered alarming. The result of this study allowed us to identify the presence of clusters with specific ARG patterns, and that these clusters are associated with different sources of isolation (e.g., human, non-human).

Early Milk Total and Differential Cell Counts as a Diagnostic Tool to Improve Antimicrobial Therapy Protocols.

Mastitis is a major cause of antimicrobial treatments either during lactation or at drying off. From a One Health perspective, there should be a balance between the risk of IMI that may impair cow health and welfare and the reduction of antimicrobial usage to decrease antimicrobial resistance, as may happen when applying selective dry-cow therapy. This reduction may be achieved by an early and accurate diagnosis followed by prudent and rationale therapeutical protocols. This study aims to assess the accuracy of PLCC (neutrophils + lymphocyte count/mL) in identifying cows at risk of having IMI due to major pathogens (S.aureus, Str.agalactiae, Str.uberis, and Str.dysgalactiae), and to simulate the impact of this early diagnosis on the potential number of treatments using a decision-tree model. The results of this study showed that PLCC had an overall accuracy of 77.6%. The results of the decision-tree model based on data from the 12 participating herds, with an overall prevalence of major pathogens of 1.5%, showed a potential decrease in the number of treatments of about 30% (from 3.4% to 2.5%) when PLCC in early lactation (days 5-16) was used to identify cows at risk for major pathogens compared with using SCC at the first milk test (days 17-43). The study confirmed that it is possible to improve animal health and reduce the risk of antimicrobial use through early IMI detection based on PLCC and applying a rationale and prudent antimicrobial protocol.

Epidemiology of Antimicrobial Resistance Genes in Streptococcus agalactiae Sequences from a Public Database in a One Health Perspective.

Streptococcus agalactiae is a well-known pathogen in humans and food-producing animals. Therefore, this bacterium is a paradigmatic example of a pathogen to be controlled by a One Health approach. Indeed, the zoonotic and reverse-zoonotic potential of the bacteria, the prevalence of Group B Streptococci (GBS) diseases in both human and animal domains, and the threatening global situation on GBS antibiotic resistance make these bacteria an important target for control programs. An epidemiological analysis using a public database containing sequences of S. agalactiae from all over the world was conducted to evaluate the frequency and evolution of antibiotic resistance genes in those isolates. The database we considered (NCBI pathogen detection isolate browser-NPDIB) is maintained on a voluntary basis. Therefore, it does not follow strict epidemiological criteria. However, it may be considered representative of the bacterial population related to human diseases. The results showed that the number of reported sequences increased largely in the last four years, and about 50% are of European origin. The frequency data and the cluster analysis showed that the AMR genes increased in frequency in recent years and suggest the importance of verifying the application of prudent protocols for antimicrobials in areas with an increasing frequency of GBS infections both in human and veterinary medicine.

Climate Change and Effects on Molds and Mycotoxins.

Earth's climate is undergoing adverse global changes as an unequivocal result of anthropogenic activity. The occurring environmental changes are slowly shaping the balance between plant growth and related fungal diseases. Climate (temperature, available water, and light quality/quantity; as well as extreme drought, desertification, and fluctuations of humid/dry cycles) represents the most important agroecosystem factor influencing the life cycle stages of fungi and their ability to colonize crops, survive, and produce toxins. The ability of mycotoxigenic fungi to respond to Climate Change (CC) may induce a shift in their geographical distribution and in the pattern of mycotoxin occurrence. The present review examines the available evidence on the impact of CC factors on growth and mycotoxin production by the key mycotoxigenic fungi belonging to the genera Aspergillus, Penicillium, and Fusarium, which include several species producing mycotoxins of the greatest concern worldwide: aflatoxins (AFs), ochratoxins, and fumonisins (FUMs).

A Journey into Animal Models of Human Osteomyelitis: A Review.

Osteomyelitis is an infection of the bone characterized by progressive inflammatory destruction and apposition of new bone that can spread via the hematogenous route (hematogenous osteomyelitis (HO)), contiguous spread (contiguous osteomyelitis (CO)), and direct inoculation (osteomyelitis associated with peripheral vascular insufficiency (PVI)). Given the significant financial burden posed by osteomyelitis patient management, the development of new preventive and treatment methods is warranted. To achieve this objective, implementing animal models (AMs) of infection such as rats, mice, rabbits, avians, dogs, sheep, goats, and pigs might be of the essence. This review provides a literature analysis of the AMs developed and used to study osteomyelitis. Historical relevance and clinical applicability were taken into account to choose the best AMs, and some study methods are briefly described. Furthermore, the most significant strengths and limitations of each species as AM are discussed, as no single model incorporates all features of osteomyelitis. HO's clinical manifestation results in extreme variability between patients due to multiple variables (e.g., age, sex, route of infection, anatomical location, and concomitant diseases) that could alter clinical studies. However, these variables can be controlled and tested through different animal models.

Enzymatic continuous-flow preparation of nature-inspired phenolic esters as antiradical and antimicrobial agents.

A collection of nature-inspired lipophilic phenolic esters have been prepared by an enzymatic synthesis under flow conditions, using the immobilized lipase B from Candida antarctica (Novozyme 435®) as a catalyst in cyclopentyl methyl ether (CPME), a non-conventional and green solvent. Their antimicrobial activity against four selected bacterial strains together with their efficiency as radical scavengers were evaluated. The obtained compounds were characterized by enhanced lipophilicity in comparison with the parent non-esterified compounds, which increased the possibility of their use as additives in the food industry.

Biocatalyzed Synthesis of Vanillamides and Evaluation of Their Antimicrobial Activity.

A series of vanillamides were easily synthesized, exploiting an acyltransferase from Mycobacterium smegmatis (MsAcT). After their evaluation as antimicrobial agents against a panel of Gram-positive and Gram-negative bacteria, three compounds were demonstrated to be 9-fold more effective toward Pseudomonas aeruginosa than the vanillic acid precursor. Taking into consideration the scarce permeability of the Gram-negative bacteria cell envelope when compared to Gram-positive strains or yeasts, these molecules can be considered the basis for the generation of new nature-inspired antimicrobials. To increase the process productivity and avoid any problem related to the poor water solubility of the starting material, a tailored flow biocatalyzed strategy in pure toluene was set up. While a robust immobilization protocol exploiting glyoxyl-agarose was employed to increase the stability of MsAcT, in-line work-up procedures were added downstream the process to enhance the system automation and reduce the overall costs.

Bacteriological and pathological investigations on the preputial glands of one-year-old C57BL/6NCrl mice maintained in individually ventilated cages.

Spontaneous infections of the preputial glands represent overlooked health problems in mice that could raise welfare concerns and potentially confound scientific experiments. Agents involved in preputial gland infections have rarely been investigated, with opportunistic pathogens of laboratory animals usually detected in inflamed preputial glands. The aim of this study was to investigate the prevalence of bacterial infection in the preputial glands and the relationship between haematological and pathological changes and infection status. We analysed 40 preputial glands from 20 one-year-old C57BL/6NCrl male mice by using bacteriology, haematology and pathology. Bacteria were isolated from 16/20 (80%) mice, for a total of 32/40 (80%) examined preputial glands. Enterobacter cloacae, Pasteurella spp., Klebsiella spp. and Staphylococcus aureus were identified in 35%, 17.5%, 15% and 12.5% of the examined glands, respectively. Preputial gland inflammation was identified in 29/40 (72.5%) glands and was classified as chronic interstitial adenitis in 27 cases and suppurative adenitis in the remaining two glands. No haematological changes were found in mice with infected glands. Histologically, the presence of intralesional bacteria, intraluminal necrotic material, intraluminal keratin accumulation, interstitial inflammatory cell infiltrate and granulocytes (intraluminal and/or interstitial), along with total inflammatory score and total histopathological score, were significantly increased in infected glands and correlated with the bacterial load. Most severe inflammatory changes were identified after S. aureus infection, while ductal hyperkeratosis was significantly increased in glands infected with Klebsiella spp. In conclusion, preputial gland infection was a common event in one-year-old C57BL/6NCrl mice, and bacterial load correlated with pathological findings, while systemic effects were not highlighted by haematology.

Aerobic Isolates from Gestational and Non-Gestational Lactating Bitches (Canis lupus familiaris).

Mastitis is a complex and well-defined mammary gland pathology, and an emergency in bitches. In dogs, its prevalence is about 1% of all reported diseases and about 5.3% of all reproductive pathologies. Lactating bitches are naturally prone to developing mastitis since puppies can easily overstimulate the epidermal layer of nipples during feeding, facilitating bacterial colonization of the glands. This study aimed to describe the aerobic bacterial flora isolated from milk samples derived from a cohort of patients (n = 87) diagnosed with clinical mastitis (n = 29), subclinical mastitis (n = 17) and healthy mammary glands (n = 46). All of the patients underwent a gynecology consultation to diagnose mammary gland afflictions; physical examination results were coupled with traditional hematological findings. The milk samples were plated on specific microbiological media for bacterial isolation. Among the 162 milk samples analyzed, 93.2% (151/162) had a positive microbiological result, while 6.8% (11/162) were sterile. The bacteriological profile of the milk samples showed 47 different species. The most common bacterial families detected in healthy bitches and bitches with subclinical and clinical mastitis were the Staphylococcaceae, Enterobacteriaceae and Enterococcaceae families. The results indicated that half of the isolated bacteria are novel findings in dogs and that some of them are normal components of human milk.

Efficacy of a Semi Automated Commercial Closed System for Autologous Leukocyte- and Platelet-Rich Plasma (l-prp) Production in Dogs: A Preliminary Study.

BACKGROUND: To characterize the cellular composition (platelets, erythrocytes, and leukocytes) and determine platelet-derived growth factor isoform BB (PDGF-BB) concentration in canine leukocyte- and platelet rich plasma (L-PRP) produced using a commercial semi-automated closed system. METHODS: Twenty milliliters of citrated whole blood were obtained from 30 healthy un-sedated canine blood donors and processed using a semi-automated completely closed commercial system (CPUNT 20, Eltek group, Casale Monferrato, Alessandria, Italy) according to the manufacturer's instructions. Erythrocyte, leukocyte, and platelet counts were determined in both whole blood (WB) and resultant L-PRP. The PDGF-BB concentration was evaluated after bovine thrombin activation of 10 L-PRP samples. RESULTS: This commercial system produced on average 2.3 ± 0.7 mL of L-PRP containing a high concentration of platelets (767,633 ± 291,001 µL, p < 0.001), with a 4.4 fold increase in platelet count, lower concentration of erythrocytes (528,600 ± 222,773 µL, p < 0.001) and similar concentration of leukocytes (8422 ± 6346 µL, p = 0.9918) compared with WB. L-PRP had an average of 3442 ± 2061 pg/mL of PDGF-BB after thrombin activation. Neutrophils, lymphocytes and monocytes average percent content in L-PRP was 14.8 ± 13.2, 71.7 ± 18.5 and 10.7 ± 6.4, respectively. CONCLUSION: Sterile canine L-PRP prepared using this semi-automated closed system is easy to obtain, produces a significant increase in platelet count compared to WB and contains a detectable concentration of PDGF-BB after activation. Additional in vitro and in vivo studies are needed to assess inflammatory markers concentration and the therapeutic efficacy of this L-PRP in dogs.

In Vitro Efficacy of Essential Oils from Melaleuca Alternifolia and Rosmarinus Officinalis, Manuka Honey-based Gel, and Propolis as Antibacterial Agents Against Canine Staphylococcus Pseudintermedius Strains.

Essential oils (EOs) and honeybee products (e.g., honey and propolis) are natural mixtures of different volatile compounds that are frequently used in traditional medicine and for pathogen eradication. The aim of this study was to evaluate the antibacterial properties of tea tree (Melaleuca alternifolia) EO (TTEO), Rosmarinus officinalis EO (ROEO), manuka-based gel, and propolis against 23 strains of Staphylococcus pseudintermedius (SP) isolated from canine pyoderma. Antimicrobial resistance screening was assessed using a panel of nine antimicrobial agents coupled with a PCR approach. An aromatogram was done for both EOs, using the disk diffusion method. The minimum inhibitory concentration (MIC) was determined for all the compounds. Among the 23 SP strains, 14 (60.9%) were multidrug-resistant (MDR), 11 strains (47.8%) were methicillin-resistant (MRSP), and 9 (39.1%) were non-MDR. The mean diameter of the inhibition zone for Melaleuca and Rosmarinus were 24.5 ± 8.8 mm and 15.2 ± 8.9 mm, respectively, resulting as statistically different (p = 0.0006). MIC values of TTEO and ROEO were similar (7.6 ± 3.2% and 8.9 ± 2.1%, respectively) and no statistical significances were found. Honeybee products showed lower MIC compared to those of EOs, 0.22 ± 0.1% for Manuka and 0.8 ± 0.5% for propolis. These findings reveal a significant antibacterial effect for all the tested products.

Diagnostic evaluation of a point-of-care test for culture and microbial susceptibility testing in canine dermatological infections in clinical practice.

BACKGROUND AND AIM: Empirical antimicrobial therapy is frequently given in superficial bacterial folliculitis (SBF) and otitis externa (OE) in dogs, especially for the initial clinical presentation. Culture and subsequent antimicrobial susceptibility testing (AST) are generally limited to chronic cases with poor response to initial therapy. Several factors contribute to the failure to implement the use of AST in veterinary practice, i.e., long laboratory turnaround time or special requirements for sample shipping. Point-of-care (PoC) testing might reduce laboratory turnaround time and costs and the risk of emergence of multidrug-resistant pathogens. This study evaluated the Speed Biogram™ PoC test in canine SBF and OE compared with conventional methods for culture and AST. MATERIALS AND METHODS: Thirty-four canine samples were analyzed: eleven from SBF, seven from bacterial OE, four from mixed OE, six from Malassezia spp. OE, and six negative controls. Sensitivity (Se) and specificity (Sp) of the PoC test and the agreement between the PoC test and conventional methods were evaluated. RESULTS: Se and Sp of PoC test in discriminating between healthy and unhealthy subjects were 100% (95% confidence interval [CI] 87.66-100.00) and 100% (95% CI 54.1-100.0), respectively. For bacterial identification, the k value was 0.532. Se and Sp of PoC tests for AST were 81.73% (95% CI 72.95-88.63) and 93.10% (95% CI 88.86-96.98), respectively with a total good agreement between tests (mean k=0.714), but major (8/27) and very major (19/27) errors were observed in 55% of bacterial conventional culture-positive samples. CONCLUSION: PoC test can identify dogs with SBF and OE, but AST is not sufficiently accurate. The lack of susceptibility testing for methicillin makes this test inappropriate for use in small animal practice.