Study of Antimicrobial Resistance, Biofilm Formation, and Motility of Pseudomonas aeruginosa Derived from Urine Samples.

Pseudomonas aeruginosa causes urinary tract infections associated with catheters by forming biofilms on the surface of indwelling catheters. Therefore, controlling the spread of the bacteria is crucial to preventing its transmission in hospitals and the environment. Thus, our objective was to determine the antibiotic susceptibility profiles of twenty-five P. aeruginosa isolates from UTIs at the Medical Center of Trás-os-Montes and Alto Douro (CHTMAD). Biofilm formation and motility are also virulence factors studied in this work. Out of the twenty-five P. aeruginosa isolates, 16% exhibited multidrug resistance, being resistant to at least three classes of antibiotics. However, the isolates showed a high prevalence of susceptibility to amikacin and tobramycin. Resistance to carbapenem antibiotics, essential for treating infections when other antibiotics fail, was low in this study, Notably, 92% of the isolates demonstrated intermediate sensitivity to ciprofloxacin, raising concerns about its efficacy in controlling the disease. Genotypic analysis revealed the presence of various ß-lactamase genes, with class B metallo-ß-lactamases (MBLs) being the most common. The blaNDM, blaSPM, and blaVIM-VIM2 genes were detected in 16%, 60%, and 12% of the strains, respectively. The presence of these genes highlights the emerging threat of MBL-mediated resistance. Additionally, virulence gene analysis showed varying prevalence rates among the strains. The exoU gene, associated with cytotoxicity, was found in only one isolate, while other genes such as exoS, exoA, exoY, and exoT had a high prevalence. The toxA and lasB genes were present in all isolates, whereas the lasA gene was absent. The presence of various virulence genes suggests the potential of these strains to cause severe infections. This pathogen demonstrated proficiency in producing biofilms, as 92% of the isolates were found to be capable of doing so. Currently, antibiotic resistance is one of the most serious public health problems, as options become inadequate with the continued emergence and spread of multidrug-resistant strains, combined with the high rate of biofilm production and the ease of dissemination. In conclusion, this study provides insights into the antibiotic resistance and virulence profiles of P. aeruginosa strains isolated from human urine infections, highlighting the need for continued surveillance and appropriate therapeutic approaches.

Antimicrobial Resistance and Clonal Lineages of Escherichia coli from Food-Producing Animals.

Escherichia coli are one of the most important pathogenic bacteria readily found in the livestock and widely studied as an indicator that carries drug-resistant genes between humans, animals, and the environment. The use of antimicrobials in the food chain, particularly in food-producing animals, is recognized as a significant contributor to the development and spread of antimicrobial resistance (AMR) and resistance genes can be transferred from the farm through the food-chain. The objective of this review is to highlight the background of the antimicrobials use in food-producing animals, more specifically, to study clonal lineages and the resistance profiles observed in E. coli, as well as in extended spectrum beta-lactamases (ESBL) producing E. coli, in a set of food-production animals with greater relevance in food consumption, such as pigs, poultry, cattle, fish farming and rabbits. Regarding the prevalence of ESBL-producing E. coli among farm animals, high-to-moderate prevalence was observed, and the highest resistance rates to tetracycline and ampicillin was detected in different farms in all geographic regions. Worldwide pandemic clones and high-risk zoonotic E. coli clones have been identified in most food-producing animals, and some of these clones are already disseminated in different niches, such as the environment and humans. A better understanding of the epidemiology of E. coli and ESBL-producing E. coli in livestock is urgently needed. Animal production is one of the major causes of the antibiotic resistance problem worldwide and a One Health approach is needed.

Staphylococcus aureus and MRSA in Livestock: Antimicrobial Resistance and Genetic Lineages.

Animal production is associated with the frequent use of antimicrobial agents for growth promotion and for the prevention, treatment, and control of animal diseases, thus maintaining animal health and productivity. Staphylococcus aureus, in particular methicillin-resistant S. aureus (MRSA), can cause a variety of infections from superficial skin and soft tissue infections to life-threatening septicaemia. S. aureus represents a serious public health problem in hospital and community settings, as well as an economic and animal welfare problem. Livestock-associated MRSA (LA-MRSA) was first described associated with the sequence (ST) 398 that was grouped within the clonal complex (CC) 398. Initially, LA-MRSA strains were restricted to CC398, but over the years it has become clear that its diversity is much greater and that it is constantly changing, a trend increasingly associated with multidrug resistance. Therefore, in this review, we aimed to describe the main clonal lineages associated with different production animals, such as swine, cattle, rabbits, and poultry, as well as verify the multidrug resistance associated with each animal species and clonal lineage. Overall, S. aureus ST398 still remains the most common clone among livestock and was reported in rabbits, goats, cattle, pigs, and birds, often together with spa-type t011. Nevertheless, a wide diversity of clonal lineages was reported worldwide in livestock.

Influence of Environmental Factors on Biofilm Formation of Staphylococci Isolated from Wastewater and Surface Water.

The presence of biofilms can negatively affect several different areas, such as the food industry, environment, and biomedical sectors. Conditions under which bacteria grow and develop, such as temperature, nutrients, and pH, among others, can largely influence biofilm production. Staphylococcus species survive in the natural environment due to their tolerance to a wide range of temperatures, dryness, dehydration, and low water activity. Therefore, we aimed to evaluate the influence of external environmental factors on the formation of biofilm of staphylococci isolated from hospital wastewater and surface waters. We investigated the biofilm formation of methicillin-resistant and -susceptible S. aureus (MRSA and MSSA) and coagulase-negative staphylococci (CoNS) under various temperatures, pH values, salt concentrations, glucose concentrations, and under anaerobic and aerobic conditions. CoNS had the ability to produce more biofilm biomass than MSSA and MRSA. All environmental factors studied influenced the biofilm formation of staphylococci isolates after 24 h of incubation. Higher biofilm formation was achieved at 4% of NaCl and 0.5% of glucose for MSSA and CoNS, and 1% of NaCl and 1.5% of glucose for MRSA isolates. Biofilm formation of isolates was greater at 25 °C and 37 °C than at 10 °C and 4 °C. pH values between 6 and 8 led to more robust biofilm formation than pH levels of 9 and 5. Although staphylococci are facultative anaerobes, biofilm formation was higher in the presence of oxygen. The results demonstrated that multiple environmental factors affect staphylococci biofilm formation. Different conditions affect differently the biofilm formation of MRSA, MSSA, and CoNS strains.

MRSA in Humans, Pets and Livestock in Portugal: Where We Came from and Where We Are Going.

Over the years, molecular typing of methicillin-resistant S. aureus (MRSA) has allowed for the identification of endemic MRSA strains and pathogenic strains. After reaching a peak of predominance in a given geographic region, MRSA strains are usually replaced by a new strain. This process is called clonal replacement and is observed worldwide. The worldwide spread of hospital-associated MRSA (HA-MRSA), community-associated MRSA (CA-MRSA) and livestock-associated MRSA (LA-MRSA) clones over the last few decades has allowed this microorganism to be currently considered a pandemic. In Portugal, most HA-MRSA infections are associated with EMRSA-15 (S22-IV), New York/Japan (ST5-II) and Iberian (ST247-I) clones. Regarding the strains found in the community, many of them are frequently associated with the hospital environment, namely the Pediatric, Brazilian and Iberian clones. On the other hand, a strain that is typically found in animals, MRSA clonal complex (CC) 398, has been described in humans as colonizing and causing infections. The ST398 clone is found across all animal species, particularly in farm animals where the economic impact of LA-MRSA infections can have disastrous consequences for industries. In contrast, the EMRSA-15 clone seems to be more related to companion animals. The objective of this review is to better understand the MRSA epidemiology because it is, undoubtedly, an important public health concern that requires more attention, in order to achieve an effective response in all sectors.

Antimicrobial Resistance and Molecular Epidemiology of Staphylococcus aureus from Hunters and Hunting Dogs.

Several studies have showed that a dog-to-human transmission of Staphylococcus aureus occurs. Hunting dogs do not have as much contact with their owners as dogs that live in the same household as the owners; however, these dogs have contact with their owners during hunting activities as well as when hunting game; therefore, we aimed to isolate S. aureus from hunters and their hunting dogs to investigate a possible S. aureus transmission. Nose and mouth samples were collected from 30 hunters and their 78 hunting dogs for staphylococcal isolation. The species identification was performed using MALDI-TOF. The antimicrobial susceptibility profiles were accessed using the Kirby-Bauer method and respective antimicrobial resistance genes were investigated by PCR. Multilocus sequence typing (MLST) and spa- and agr-typing was performed in all S. aureus isolates. S. aureus were detected in 10 (30%) human samples and in 11 (15.4%) dog samples of which 11 and 5 were methicillin-resistant S. aureus (MRSA). Other staphylococci were identified, particularly, S. pseudintermedius. Most S. aureus isolates were resistant to penicillin, erythromycin, and tetracycline. Evidence of a possible transmission of S. aureus between human and dogs was detected in three hunters and their dogs. S. aureus isolates were ascribed to 10 STs and 9 spa-types. A moderate colonization of S. aureus in hunting dogs and their owners was detected in this study. A few dog-to-dog and dog-to-human possible transmissions were identified.

Nocturnal Birds of Prey as Carriers of Staphylococcus aureus and Other Staphylococci: Diversity, Antimicrobial Resistance and Clonal Lineages.

Owls are nocturnal predators that inhabit urbanized and farmlands. They are in direct contact with other animals, both livestock and small wild rodents that they mostly feed on. Staphylococci can be both commensal and pathogenic bacteria that are widespread across the various ecological niches. We aimed to isolate staphylococci from owls and to characterize their antimicrobial resistance, virulence factors and genetic lineages. Swab samples were collected from the throat and cloaca of 114 owls admitted to two rehabilitation centers in Portugal. The identification of staphylococci species was performed by MALDI-TOF. Staphylococci antimicrobial resistance and virulence genes were investigated by means of the disk diffusion method and PCR. Staphylococcus aureus isolates were characterized by MLST, agr and spa-typing. Of the tested animals, 66 isolates were recovered, including 10 different species of staphylococci, of which 25 were coagulase-positive (CoPS) and 41 were coagulase-negative (CoNS). Twenty-three S. aureus were isolated, of which one mecC-MRSA was identified. The isolates were mainly resistant to penicillin, aminoglycosides, clindamycin and tetracycline. mecC-MRSA belonged to ST1245 and spa-type t843 and the remaining S. aureus were ascribed to 12 STs and 15 spa types. A high diversity of clonal lineages was identified among the S. aureus isolated from wild owls. Owls feed mainly on small rodents often exposed to waste and anthropogenic sources, which may explain the moderate prevalence of S. aureus in these animals.

High Frequency of the EMRSA-15 Clone (ST22-MRSA-IV) in Hospital Wastewater.

Hospital wastewaters often carry multidrug-resistant bacteria and priority pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA). Pathogens and antibiotic resistance genes present in wastewaters may reach the natural environment facilitating their spread. Thus, we aimed to isolate MRSA from wastewater of 3 hospitals located in the north of Portugal and to characterize the isolates regarding the antimicrobial resistance and genetic lineages. A total of 96 wastewater samples were collected over six months. The water was filtered, and the filtration membrane was immersed in BHI broth supplemented with 6.5% of NaCl and incubated. The inoculum was streaked in ORSAB agar plates for MRSA isolation. The isolates susceptibility testing was performed against 14 antimicrobial agents. The presence of resistance and virulence genes was accessed by PCR. Molecular typing was performed in all isolates. From the 96 samples, 28 (29.2%) were MRSA-positive. Most isolates had a multidrug-resistant profile and carried the mecA, blaZ, aac(6')-Ie-aph(2″)-Ia, aph(3')-IIIa, ermA, ermB, ermC, tetL, tetM, dfrA dfrG and catpC221 genes. Most of the isolates were ascribed to the immune evasion cluster (IEC) type B. The isolates belonged to ST22-IV, ST8-IV and ST105-II and spa-types t747, t1302, t19963, t6966, t020, t008 and tOur study shows that MRSA can be found over time in hospital wastewater. The wastewater treatment processes can reduce the MRSA load. The great majority of the isolates belonged to ST22 and spa-type t747 which suggests the fitness of these genetic lineages in hospital effluents.

Genomic and Metabolic Characteristics of the Pathogenicity in Pseudomonas aeruginosa.

In recent years, the effectiveness of antimicrobials in the treatment of Pseudomonas aeruginosa infections has gradually decreased. This pathogen can be observed in several clinical cases, such as pneumonia, urinary tract infections, sepsis, in immunocompromised hosts, such as neutropenic cancer, burns, and AIDS patients. Furthermore, Pseudomonas aeruginosa causes diseases in both livestock and pets. The highly flexible and versatile genome of P. aeruginosa allows it to have a high rate of pathogenicity. The numerous secreted virulence factors, resulting from its numerous secretion systems, the multi-resistance to different classes of antibiotics, and the ability to produce biofilms are pathogenicity factors that cause numerous problems in the fight against P. aeruginosa infections and that must be better understood for an effective treatment. Infections by P. aeruginosa represent, therefore, a major health problem and, as resistance genes can be disseminated between the microbiotas associated with humans, animals, and the environment, this issue needs be addressed on the basis of an One Health approach. This review intends to bring together and describe in detail the molecular and metabolic pathways in P. aeruginosa's pathogenesis, to contribute for the development of a more targeted therapy against this pathogen.

Topical Application of Ozonated Oils for the Treatment of MRSA Skin Infection in an Animal Model of Infected Ulcer.

Diabetic foot ulcers are a common cause of morbidity in diabetic patients. One of the main pathogens found in these ulcers is methicillin-resistant Staphylococcus aureus (MRSA). MRSA often carries resistance to several classes of antibiotics and their infections are becoming harder to treat. Therefore, new alternatives are urgently needed. Thus, this study aimed to investigate the capacity of topical ozonated oil application on the treatment of early-stage skin infected with MRSA in an animal model. Ozonated oil was prepared from a mixture of oils subjected to a gas stream of O2/O3 mixture. Sixteen Wistar rats were inoculated by an intradermic injection of MRSA suspension, producing an abscess lesion. After 3 days, the skin epidermis was removed to open the wound. Group 1 received an application of oil mixture without ozone treatment and Group 2 received an application of ozonated oil. After the treatment period, skin was collected, colony-forming units (CFU) of bacteria were quantified and the histological analysis of the skin was carried out. Skin samples from the control 1 and 2 had a bacterial load was of 1.1 × 105 and 5.7 × 103 CFU/mL, respectively. Group 2 showed better wound healing from mild to moderate epidermal regeneration. Topical application of ozonated vegetable oil in MRSA-infected skin in rats showed a small reduction of the bacterial load and better wound healing.

Anti-biofilm activity of dalbavancin against methicillin-resistant Staphylococcus aureus (MRSA) isolated from human bone infection.

The presence of methicillin-resistant Staphylococcus aureus (MRSA) in bone infections difficults its treatment and is a sign of concern. The aim of this study was to evaluate in vitro activity of dalbavancin on pre-established adhered cells and 24 h old biofilms of MRSA strains isolated from a human bone infection. Thirty-three MRSA were isolated from osteomyelitis episodes. The antimicrobial susceptibility of these strains was assessed by the Kirby-Bauer disc diffusion method and the presence of resistance genes was screened by PCR. MRSA planktonic minimum inhibitory concentration and minimum bactericidal concentration were assessed. Minimum biofilm eradication concentration (MBEC) was performed by the microtiter biofilm formation assay. All 33 MRSA strains were classified as multidrug-resistant strains and susceptible to dalbavancin. Dalbavancin inhibited the growth of 54.6% and 52% of strains at the concentrations of 0.05 µg/mL and 1 µg/mL, respectively. The MBEC values up to 0.4 µg/mL demonstrated that dalbavancin was active against most strains in pre-established adhered cells and 24 h old biofilms. The current results show that dalbavancin is active against adhered cells and biofilms in vitro, suggesting that this antimicrobial agent may be an option for the treatment of bone infections caused by MRSA.

Survey of the Knowledge and Use of Antibiotics among Medical and Veterinary Health Professionals and Students in Portugal.

Antimicrobial resistance (AMR) is an urgent and complex problem worldwide, exacerbated by the frequently inappropriate use of antibiotics. The purpose of this study was to survey the levels of knowledge and awareness about antibiotic use and stewardship, among human and veterinary health professionals or students in Portugal, and the associations between antibiotic knowledge factors and socio-professional groups. In cross-sectional survey design, a total of 449 online structured questionnaires were completed in 2018-2019. The statistical analysis was performed dividing the respondents into four groups, A (undergraduate students), B (PhD students and researchers), C (lecturers), and D (technicians and other occupation). Among all respondents, 17% (n = 75) revealed some gap in knowledge about antibiotic resistance and the antibiotics that should be administered for different infection types (bacterial, viral, or fungal). Of the 159 pet owners among the respondents, only half had administered antibiotics to their animal and 64% (n = 102) knew that veterinary prescription is mandatory when administering antibiotics to animals. All groups statistically agreed that the AMR is a major public health problem and the antibiotics should be administrated for bacterial infections and used until the whole pack has been finished (p = 0.00). As expected, only groups B and C demonstrated a higher level of knowledge to recognize the antibiotic name and their active ingredient than undergraduate students (p = 0.00). About the antibiotic use on pets, only group B was statistically significant to no used antibiotics on their pets (p = 0.00). However, groups A, C, and D were statistically significant for the knowledge about the mandatory veterinarian prescription and groups C and D were significantly statistics for fully aware of the transmission of bacteria between animals and humans. In conclusion, in matters related to AMR, the behavior, education, and training of the general public and health professionals, including those who prescribe antibiotics for humans and animals, need to be improved.

Regeneration of critical-sized defects, in a goat model, using a dextrin-based hydrogel associated with granular synthetic bone substitute.

The development of injectable bone substitutes (IBS) have obtained great importance in the bone regeneration field, as a strategy to reach hardly accessible defects using minimally invasive techniques and able to fit to irregular topographies. In this scenario, the association of injectable hydrogels and bone graft granules is emerging as a well-established trend. Particularly, in situ forming hydrogels have arisen as a new IBS generation. An in situ forming and injectable dextrin-based hydrogel (HG) was developed, aiming to act as a carrier of granular bone substitutes and bioactive agents. In this work, the HG was associated to a granular bone substitute (Bonelike®) and implanted in goat critical-sized calvarial defects (14 mm) for 3, 6 and 12 weeks. The results showed that HG improved the handling properties of the Bonelike® granules and did not affect its osteoconductive features, neither impairing the bone regeneration process. Human multipotent mesenchymal stromal cells from the umbilical cord, extracellular matrix hydrolysates and the pro-angiogenic peptide LLKKK18 were also combined with the IBS. These bioactive agents did not enhance the new bone formation significantly under the conditions tested, according to micro-computed tomography and histological analysis.

High Efficacy of Ozonated Oils on the Removal of Biofilms Produced by Methicillin-Resistant Staphylococcus aureus (MRSA) from Infected Diabetic Foot Ulcers.

Ozone has a high wound healing capacity and antibacterial properties and can be used as a complementary treatment in infections. Methicillin-resistant S. aureus (MRSA) is the most common pathogen found in infected diabetic foot ulcers. Most of MRSA are resistant to several classes of antibiotics and, therefore, there is a need for new, effective, and well-tolerated agents. Thus, we aimed evaluate the antimicrobial and antibiofilm potentials of ozonated vegetable oils against MRSA strains isolated from diabetic foot ulcers. Six ozonated oils were produced with concentrations of ozone ranging from 0.53 to 17 mg of ozone/g of oil. The peroxide values were determined for each oil. Ozonated oils content on fatty acid was determined by gas chromatography equipped with a flame ionization detector. The antimicrobial susceptibility testing was performed by the Kirby-Bauer disk diffusion method and the effect of ozonated oils on biofilm formation ability and on established biofilms was investigated. In general, the content in identified unsaturated fatty acid in oils decreased with the increase of ozonation time and, consequently, the peroxide value increased. Most bacterial strains were inhibited by ozonated oil at a concentration of 4.24 mg/g. Ozonated oils had moderate to high ability to remove adhered cells and showed a high capacity to eradicate 24 h old biofilms. Our results show promising use of ozonated oils on the treatment of infections, in particular those caused by multidrug-resistant MRSA strains.

Genetic Characterization of Methicillin-Resistant Staphylococcus aureus Isolates from Human Bloodstream Infections: Detection of MLSB Resistance.

In this study we aimed to characterize antimicrobial resistance in methicillin-resistant Staphylococcus aureus (MRSA) isolated from bloodstream infections as well as the associated genetic lineages of the isolates. Sixteen MRSA isolates were recovered from bacteremia samples from inpatients between 2016 and 2019. The antimicrobial susceptibility of these isolates was tested by the Kirby-Bauer disk diffusion method against 14 antimicrobial agents. To determine the macrolide-lincosamide-streptogramin B (MLSB) resistance phenotype of the isolates, erythromycin-resistant isolates were assessed by double-disk diffusion (D-test). The resistance and virulence genes were screened by polymerase chain reaction (PCR). All isolates were characterized by multilocus sequence typing (MLST), spa typing, staphylococcal chromosomal cassette mec (SCCmec) typing, and accessory gene regulator (agr) typing. Isolates showed resistance to cefoxitin, penicillin, ciprofloxacin, erythromycin, fusidic acid, clindamycin, and aminoglycosides, confirmed by the presence of the blaZ, ermA, ermC, mphC, msrA/B, aac(6')-Ie-aph(2'')-Ia, and ant(4')-Ia genes. Three isolates were Panton-Valentine-leukocidin-positive. Most strains (n = 12) presented an inducible MLSB phenotype. The isolates were ascribed to eight spa-types (t747, t002, t020, t1084, t008, t10682, t18526, and t1370) and four MLSTs (ST22, ST5, ST105, and ST8). Overall, most (n = 12) MRSA isolates had a multidrug-resistance profile with inducible MLSB phenotypes and belonged to epidemic MRSA clones.

Therapeutic potential of dalbavancin in a rat model of methicillin-resistant Staphylococcus aureus (MRSA)-osteomyelitis.

Bacterial osteomyelitis is a major clinical challenge in human and veterinary patients. This infection is an infrequent but feared complication of orthopedic surgery and is mainly caused by methicillin-resistant Staphylococcus aureus (MRSA). The aim of this study was to evaluate the efficacy of dalbavancin (dosed for either 7 or 14 days) in an MRSA-osteomyelitis tibial bone model. A total of 39 rats were included in the study. All animals received an inoculum of a clinical strain of MRSA (106 colony-forming units [CFU]) injected into the proximal tibia under general anesthesia. Dalbavancin was injected intraperitoneally for 7 or 14 days in 13 animals each; the remaining 13 animals received saline solution. After treatment, the animals were sacrificed. Infected tibiae were recovered for histological evaluation and microbiological analysis (MRSA count per gram of bone). Rats that received dalbavancin showed a statistically significant reduction of MRSA counts compared with the control group: median 0 CFU/g bone (14 days of dalbavancin) vs. 70 CFU/g bone (7 days of dalbavancin) and 1600 CFU/g bone (control). Histological evaluation showed typical signs of osteomyelitis in the control group, whereas there were no signs of bone infection in 92% of the rats that received 14 days of dalbavancin. According to this model, dalbavancin seems to have good efficacy for treating serious Gram-positive bone infections, including those caused by MRSA.

Efficacy of dalbavancin against MRSA biofilms in a rat model of orthopaedic implant-associated infection.

OBJECTIVES: The aim of this study was to evaluate the efficacy of dalbavancin against MRSA biofilm-related infection in orthopaedic implants in vivo. METHODS: One MRSA strain isolated from human osteomyelitis was used to promote biofilm formation on the surface of screws. The implants were inserted in the proximal tibia under general anaesthesia. Thirty-nine Wistar rats were divided into three groups [control group (no treatment), Group 1 (7 days of treatment) and Group 2 (14 days of treatment)]; both treatment groups were administered dalbavancin intraperitoneally and euthanized after treatment. cfu of bacteria present in both the tibia and the implant were quantified. The infection severity was assessed by histopathology and scored from 0 (no infection) to 4 (severe infection). RESULTS: The high number of cfu/g and cfu/mL present in the control group indicated a well-established infection. There was a significant reduction in cfu in rats treated with dalbavancin both in the tibia (2.8 × 105 cfu/g) and the implant (1.1 × 106 cfu/mL) in Group 1 (1.8 × 103 cfu/g and 2.4 × 105 cfu/mL, respectively) and in Group 2 (8.2 cfu/g and 8.2 × 103 cfu/mL, respectively). Most animals from the control group presented an infection scored as 3 (severe). At the end of the experiment, most rats from Groups 1 and 2 presented an infection scored as 2 (moderate) and 0 (no infection), respectively. CONCLUSIONS: Although there was a marked decrease in cfu number, signs of biofilm-induced infection prevailed after 14 days of treatment. Further studies should be carried out to evaluate the potential of dalbavancin in the treatment of bone and orthopaedic implant-associated MRSA infections.

Diversity of methicillin-resistant staphylococci among wild Lepus granatensis: first detection of mecA-MRSA in hares.

MRSA in humans, pets and livestock have been widely investigated, nevertheless, there is still little information of MRSA in wild animals. Therefore, this study aimed to investigate the occurrence and antimicrobial resistance profiles of methicillin-resistant staphylococci (MRS) in wild Iberian hares and to characterize their genetic lineages. Samples from 83 wild hares (Lepus granatensis) were collected during the hunting season. Isolation of MRS was accomplish using Oxacillin Resistant Screening Agar medium with 2 mg/L of oxacillin. The susceptibility of the isolates was tested by the Kirby-Bauer disc diffusion method. The presence of resistance and virulence genes was studied by PCR. S. aureus strains were further characterized by multilocus sequence typing, agr, spa and SCCmec typing. From the 83 samples, 12 (14.45%) coagulase-negative staphylococci and 3 (3.6%) MRSA strains were isolated. Nine coagulase-negative isolates showed resistance to at least one antibiotic. One MRSA isolate showed a multidrug-resistant profile with resistances to ß-lactams, aminoglycosides, macrolides and lincosamides. All MRSA strains were ascribed to ST2855, t1190 and SCCmec type III. The frequency of MRSA strains in wild hares was low, nevertheless, the presence of MRSA in game animals is considered a public health problem and may represent a route of transmission between animals and humans.

In vivo systemic toxicity assessment of an oxidized dextrin-based hydrogel and its effectiveness as a carrier and stabilizer of granular synthetic bone substitutes.

The worldwide incidence of bone disorders is raising, mainly due to aging population. The lack of effective treatments is pushing the development of synthetic bone substitutes (SBSs). Most ceramic-based SBSs commercially available display limited handling properties. Attempting to solve these issues and achieve wider acceptance by the clinicians, granular ceramics have been associated with hydrogels (HGs) to produce injectable/moldable SBSs. Dextrin, a low-molecular-weight carbohydrate, was used to develop a fully resorbable and injectable HG. It was first oxidized with sodium periodate and then cross-linked with adipic acid dihydrazide. The in vivo biocompatibility and safety of the dextrin-based HG was assessed by subacute systemic toxicity and skin sensitization tests, using rodent models. The results showed that the HG did not induce any systemic toxic effect, skin reaction, or genotoxicity, neither impaired the bone repair/regeneration process. Then, the HG was successfully combined with granular bone substitute, registered as Bonelike (250-500 µm) to obtain a moldable/injectable SBS, which was implanted in tibial fractures in goats for 3 and 6 weeks. The obtained results showed that HG allowed the stabilization of the granules into the defect, ensuring effective handling, and molding properties of the formulation, as well as an efficient cohesion of the granules. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1678-1689, 2019.

Blepharitis due to Cryptococcus neoformans in a cat from northern Portugal.

Case summary We report a clinical case of blepharitis due to Cryptococcus neoformans yeasts in a 2-year-old stray cat from northern Portugal (Vila Real) without concurrent naso-ocular signs. Ophthalmological examination revealed mucopurulent discharge from an open wound in the right upper and lower lids. Slit-lamp biomicroscopy showed a normal anterior segment, and intraocular pressure was within the normal reference interval. No fundoscopic alterations were detected in either eye by direct and indirect ophthalmoscopic examination. Cytological examination of an appositional smear showed numerous polymorphic neutrophils and macrophages, together with spherical yeast cells compatible with Cryptococcus species. Molecular analysis by means of PCR and restriction fragment length polymorphism identified C neoformans genotype VNI. The cat was treated with itraconazole, and amoxicillin and clavulanic acid, combined with a commercial ear ointment and an imidacloprid/moxidectin spot-on application for bilateral parasitic otitis caused by Otodectes cynotis. One month after treatment, the clinical signs were completely resolved. Localised cutaneous lesions, as in the present case, probably result from contamination of cat-scratch injuries with viable encapsulated yeasts. Relevance and novel information This is, to the best of our knowledge, the first clinical report of feline blepharitis due to C neoformans without concurrent naso-ocular signs. The current findings, together with those from recent reports of the infection in domestic animals, should alert the veterinary community both in Portugal and in Europe to this underdiagnosed disease.