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- Periodontal disease in dogs : an experimental approach towards prevention using antimicrobial peptidesPublication . Cunha, Eva; Oliveira, Maria Manuela Castilho Monteiro de; Veiga, Ana Salomé Rocha do NascimentoABSTRACT - Periodontal disease (PD) is one of the most widespread inflammatory diseases in dogs. This disease is initiated by a polymicrobial biofilm (dental plaque) in the teeth surface, along with a subsequent local inflammatory response leading to periodontium damage and systemic consequences. Enterococci are opportunistic bacteria that may be found in the plaque biofilm. Their ability to act as reservoirs of resistant determinants and their previously linkage to PD-systemic consequences, makes enterococci an interesting bacterial model for antimicrobial studies. Several strategies can be used for PD control, being the removal and inhibition of dental plaque the keystone for PD prevention. Antimicrobial peptides (AMPs) are promising compounds for the management of bacterial infections with low rate of resistance incidence. Nisin is an AMP with antimicrobial activity against Gram-positive and some Gram-negative bacteria, including periodontal pathogens. Considering PD high prevalence, its consequences, and the urgent need to control antimicrobial resistant (AMR) strains and develop new antimicrobial strategies, the main goal of this project was to evaluate the potential of the AMP nisin A for PD control in dogs. First, the antimicrobial activity of nisin and of nisin incorporated in two delivery systems (guar gum biogel and a veterinary toothpaste) were evaluated against a previously characterized collection of canine oral enterococci (n=20). Nisin and nisin-biogel were able to inhibit and eradicate all isolates tested in both planktonic and biofilm forms. Supplemented toothpaste was able to inhibit 95% of the isolates tested. As such, nisin-biogel was selected for the following assays, being observed that it also presented inhibition and eradication abilities against an in vitro multispecies biofilm. Then, nisin-biogel antimicrobial ability was evaluated in the presence of canine saliva and over a 24-month storage, as well as its safety towards eukaryotic cells. Saliva hampered nisin-biogel activity, but it maintained its antimicrobial activity over the storage period at freezing, cooling and room temperatures. In addition, nisin-biogel showed no cytotoxicity at concentrations up to 200 μg/mL. Later, nisin-biogel influence on the canine oral microbiome was evaluated by next generation sequencing, revealing that one dental application of nisin-biogel promoted a reduction in bacterial diversity. Still, a diversity recovery was detected after three applications, along with a reduction in some PD-related bacterial species. Finally, nisin influence in AMR development was evaluated by determination of the mutant section window, mutants AMR profiles and horizontal gene transfer (HGT). It was observed that nisin selective pressure may induce changes in the bacteria’s AMR profile, but not influencing vanA HGT between enterococci and staphylococci. Together, these results show that nisin-biogel seems to be an appropriate approach for PD control in dogs, being important to test this product in an in vivo controlled clinical trial.
- Osteomyelitis caused by Aspergillus terreus complex in a dog: a case reportPublication . Abreu, Raquel; Martinho, António; Noiva, Rute; Pissarra, Hugo; Cota, João; Cunha, Eva; Tavares, Luis; Oliveira, ManuelaBackground In dogs, the most frequently reported mycosis associated with Aspergillus spp. are respiratory infections. Systemic aspergillosis is uncommon, with reported cases been associated with several Aspergillus species. Aspergillus terreus species complex are ubiquitous organisms, unfrequently associated with local or systemic disease in animals and humans, and treatment of osteomyelitis caused by this species is usually unfavorable. Case presentation This case report describes the case of a 5-year-old dog, referred to the Veterinary Hospital of the Faculty of Veterinary Medicine of the University of Lisbon, Portugal, with a history of lameness of the right thoracic limb. Radiographs and CT scan revealed two diferent lesions on right humerus and radio, which were biopsied. The samples collected were submitted to cytological and histopathological evaluation and bacterial and mycological culture. Environmental samples, including of the surgery room and of the biopsy needle were also evaluated for the presence of fungi. Regarding biopsy samples, bacterial culture was negative, but mycological analysis originated a pure culture of a fungal species later identifed as Aspergillus terreus by Sanger sequencing. Results were compatible with histopathologic examination, which revealed periosteal reaction and invasion of hyphae elements. Also, mycological analysis of both environmental samples evaluated were negative. The virulence profle of the fungal isolate was phenotypically characterized using specifc media, allowing to reveal its ability to produce several enzymes involved in its pathogenicity, namely lipase, hemolysin and DNAse, corresponding to a Virulence Index (V. Index.) of 0.43. The patient was submitted to itraconazole therapy for 8 weeks. After 3 weeks, the patient showed signifcant clinical improvement, and after 6 weeks no radiographic signs were observed. Conclusions Antifungal therapy with itraconazole can contribute to the remission of canine infections promoted by Aspergillus terreus complex with a relevant V. Index
- Antibiotic resistance and virulence profiles of Gram-Negative bacteria isolated from loggerhead sea turtles (Caretta caretta) of the Island of Maio, Cape VerdePublication . Fernandes, Matilde; Grilo, Miguel; Cunha, Eva; Carneiro, Carla; Tavares, Luis; Patino-Martinez, Juan; Oliveira, ManuelaPrevious studies revealed high levels of antimicrobial resistance (AMR) in loggerhead sea turtles (Caretta caretta), describing this species as prime reservoir of antimicrobial-resistant bacteria. This study aimed to characterise, for the first time, the AMR and virulence profiles of Gram-negative bacteria isolated from 33 nesting loggerhead turtles of the island of Maio, Cape Verde. Cloacal, oral, and egg content swab samples (n = 99) were collected and analysed using conventional bacteriological techniques. Shewanella putrefaciens, Morganella morganii, and Vibrio alginolyticus were isolated from the samples under study. The isolates obtained from this loggerhead subpopulation (North-East Atlantic) revealed lower levels of AMR, compared with the results of studies performed in other subpopulations (e.g., Mediterranean). However, the detection of resistance to carbapenems and multiple antimicrobial resistance indices higher than 0.20, raises concern about the potential association of these animals to points of high antimicrobial exposure. Furthermore, virulence phenotypic characterisation revealed that the isolates presented complex virulence profiles, including the ability to produce biofilms. Finally, due to their pathogenic potential, and considering the evidence of illegal consumption of turtle-related products on the island of Maio, the identified bacteria may represent a significant threat to public health.
- Antimicrobial resistance and virulence profiles of Enterobacterales isolated from two-finger and three-finger sloths (Choloepus hoffmanni and Bradypus variegatus) of Costa RicaPublication . Fernandes, Matilde; Carneiro, Carla; Villada Rosales, Ana Maria; Grilo, Miguel; Ramiro, Yolanda; Cunha, Eva; Nunes, Telmo; Tavares, Luis; Sandi, Janet; Oliveira, ManuelaBackground: Wildlife has been recently recognized as an environmental reservoir for antimicrobial resistance (AMR). However, less information on this topic is available in animals released back into the wild after rehabilitation in wildlife facilities, compared with studies performed exclusively in captive or free-ranging wildlife. This study aimed to evaluate the potential influence of captivity and/or treatment while in captivity of wild sloths on the AMR and virulence profiles of sloths’ Enterobacterales. Methods: Oral and rectal swab samples were collected from 39 two-finger (Choloepus hoffmanni) and three-finger sloths (Bradypus variegatus) of Costa Rica (n = 78) and analyzed using conventional bacteriological techniques. A generalized linear mixed model was applied to estimate the isolates’ multiple antimicrobial resistance and virulence indices as a function of animal status. Results: A considerable level of resistance was detected, especially for Citrobacter youngae and Escherichia coli, with 17.5% of isolates classified as multidrug-resistant. Virulence indices of isolates from rehabilitated sloths were significantly higher than the ones from sloths being hand-reared for shorter periods. Conclusions: To our knowledge, this is the first description of sloths’ antimicrobial resistant Enterobacterales, suggesting that sloths’ rehabilitation and consequent exposure to humans, may promote the selection of bacteria with higher virulence. Ultimately, these bacteria may represent a threat to human and animal health due to their zoonotic potential and AMR and virulence profiles.
- Evaluation of a biocide used in the Biological Isolation and Containment Unit of a Veterinary Teaching HospitalPublication . GERALDES, CATARINA; Verdial, Cláudia; Cunha, Eva; Almeida, Virgilio; Tavares, Luis; Oliveira, Manuela; Gil, SolangeHospital-acquired infections (HAIs) are a rising problem worldwide, and the best way of coping with them is through infection tracking and surveillance systems, combined with prevention strategies, namely efficient disinfection protocols, that employ various biocides. However, increasing reports about reductions in biocide susceptibility and the development of cross-resistance to antimicrobials emphasize the need for identifying the factors influencing biocide efficiency. In this study, 29 bacterial isolates (n = 3 E. coli, n = 2 Pseudomonas spp., n = 23 Enterococcus spp., and n = 1 Staphylococcus pseudintermedius), obtained from environmental samples collected from the Biological Isolation and Containment Unit (BICU), of the Veterinary Teaching Hospital of the Faculty of Veterinary Medicine, University of Lisbon, were tested in order to determine their antimicrobial susceptibility to various antibiotics. Thirteen of these isolates were further selected in order to determine their antimicrobial susceptibility to Virkon™ S, with and without the presence of organic matter. Afterward, seven of these isolates were incubated in the presence of sub-lethal concentrations of this formulation and, subsequently, new susceptibility profiles were determined. Fourteen of the 29 isolates (48.3%) were classified as multidrug resistant, all previously identified as enterococci. Concerning Virkon™ S’s susceptibility, the Minimal Bactericidal Concentration (MBC) of this biocide regarding all isolates was at least eight times lower than the concentration regularly used, when no organic matter was present. However, when organic matter was added, MBC values rose up to 23 times. After exposure to sub-lethal concentrations of Virkon™ S, four enterococci presented a phenotypical change regarding antimicrobial susceptibility towards gentamicin. Virkon™ S also resulted in higher MBC values, up to 1.5 times, in the presence of low concentrations of organic matter, but no rise in these values was observed in assays without interfering substance. Virkon™ S seemed to be an efficient formulation in eliminating all bacteria isolates isolated from the BICU. However, organic matter could represent a hindrance to this ability, which emphasizes the importance of sanitization before disinfection procedures. The changes seen in antimicrobial susceptibility could be explained by a general stress-induced response promoted by the sub-lethal levels of Virkon™ S. Additionally, when no organic matter was present, a decrease in susceptibility to this biocide seemed to be non-existent.
- Analyzing the safety of the parasiticide fungus Mucor circinelloides: first insights on its virulence profile and interactions with the avian gut microbial communityPublication . Lozano, João; Cunha, Eva; Almeida, Cristina; Nunes, Mónica; Dias, Ricardo; Vicente, Eduardo; Sebastião, Daniela; Henriques, Sérgio; Madeira de Carvalho, Luís; Paz-Silva, Adolfo; Oliveira, ManuelaParasiticide fungi are considered an accurate, sustainable, and safe solution for the biocontrol of animal gastrointestinal (GI) parasites. This research provides an initial characterization of the virulence of the native parasiticide fungus Mucor circinelloides (FMV-FR1) and an assessment of its impact on birds’ gut microbes. The genome of this fungus was sequenced to identify the genes coding for virulence factors. Also, this fungus was checked for the phenotypic expression of proteinase, lecithinase, DNase, gelatinase, hemolysin, and biofilm production. Finally, an in vivo trial was developed based on feeding M. circinelloides spores to laying hens and peacocks three times a week. Bird feces were collected for 3 months, with total genomic DNA being extracted and subjected to long-read 16S and 25S-28S sequencing. Genes coding for an iron permease (FTR1), iron receptors (FOB1 and FOB2), ADP-ribosylation factors (ARFs) (ARF2 and ARF6), and a GTPase (CDC42) were identified in this M. circinelloides genome. Also, this fungus was positive only for lecithinase activity. The field trial revealed a fecal microbiome dominated by Firmicutes and Proteobacteria in laying hens, and Firmicutes and Bacteroidetes in peacocks, whereas the fecal mycobiome of both bird species was mainly composed of Ascomycetes and Basidiomycetes fungi. Bacterial and fungal alpha-diversities did not differ between sampling time points after M. circinelloides administrations (P = 0.62 and P = 0.15, respectively). Although findings from this research suggest the lack of virulence of this M. circinelloides parasiticide isolate, more complementary in vitro and in vivo research is needed to conclude about the safety of its administration to birds, aiming at controlling their GI parasites.
- Avaliação dos efeitos secundários dos fármacos quimioterápicos em animais de companhia : estudo retrospetivoPublication . Cunha, Eva; Vilhena, Hugo Corte Real; São Braz, Berta Maria Fernandes FerreiraA quimioterapia é uma modalidade terapêutica usada em oncologia, cujo objetivo passa pela total erradicação das células tumorais ou pela cura, através da administração de fármacos quimioterápicos ou anti-neoplásicos. Estes atuam, na sua grande maioria, em células com elevado índice mitótico, atingindo não apenas as células neoplásicas como também as células normais, o que conduz aos chamados efeitos secundários. Este estudo teve como objetivo a avaliação e caracterização dos efeitos secundários dos fármacos quimioterápicos, na prática clínica de animais de companhia consistindo num estudo retrospetivo de casos. O desenho experimental incluiu todos os cães e gatos, machos e fêmeas, submetidos a pelo menos uma sessão de tratamento com fármacos quimioterápicos, independentemente do tipo de neoplasia, no Hospital Veterinário do Baixo Vouga e Policlínica Veterinária de Aveiro (desde 1 de Março de 2008 e de 2010, respetivamente, até dia 31 de Janeiro de 2014). Os efeitos secundários em análise foram a toxicidade hematopoiética, gastrointestinal, dermatológica e pulmonar, reações alérgicas/anafiláticas, cistite hemorrágica estéril, necrose perivascular/extravasamento, cardiotoxicidade, nefrotoxicidade, hepatotoxicidade, neurotoxicidade e síndrome de lise tumoral aguda. A análise estatística foi realizada com recurso ao programa Microsoft Excel 2010® e software R®. Foram avaliadas 266 sessões de quimioterapia realizadas em 42 animais, verificando-se que 71,43% destes exibiram toxicidade. Verificou-se uma clara predominância da toxicidade gastrointestinal e hematopoiética na amostra e nas sessões quimioterápicas avaliadas. Também a toxicidade dermatológica, reações anafiláticas/alérgicas, cistite hemorrágica estéril, necrose perivascular/extravasamento e cardiotoxicidade, foram identificados durante o estudo. Pela análise estatística foi possível identificar uma associação entre a toxicidade gastrointestinal e os fármacos em geral, e a epirrubicina em particular; as reações alérgicas/anafiláticas e a cardiotoxicidade com a doxorrubicina; e a cistite hemorrágica estéril com a ciclofosfamida e com a quimioterapia metronómica (ciclofosfamida e meloxicam).
- Commonality of multidrug-resistant klebsiella pneumoniae ST348 isolates in horses and humans in PortugalPublication . Trigo Da Roza, Filipa; Couto, Natacha; Carneiro, Carla; Cunha, Eva; Rosa, Teresa; Magalhães, Mariana; Tavares, Luis; Novais, Ângela; Peixe, Luísa; Lamas, Luís P.; Oliveira, ManuelaABSTRACT - Multidrug-resistant (MDR) Klebsiella pneumoniae is considered a major global concern by the World Health Organization. Evidence is growing on the importance of circulation of MDR bacterial populations between animals and humans. Horses have been shown to carry commensal isolates of this bacterial species and can act as human MDR bacteria reservoirs. In this study, we characterized an extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae sequence type (ST) 348 isolate from a horse, an ST reported for the first time in an animal, using next-generation sequencing. We compared it with six other MDR K. pneumoniae ST348 human isolates previously identified in health-care facilities in Portugal using a core genome multi-locus sequence typing approach to evaluate a possible genetic link. The horse isolate was resistant to most of the antimicrobials tested, including 3rd generation cephalosporins, fluoroquinolones, and aminoglycosides, and presented several antimicrobial resistance genes, including blaESBL. Twenty-one allele differences were found between the horse isolate and the most similar human isolate, suggesting a recent common ancestor. Other similarities were observed regarding the content on antimicrobial resistance genes, plasmid incompatibility groups, and capsular and somatic antigens. This study illustrates the relevance of the dissemination of MDR strains, and enhances that identification of these types of bacterial strains in both human and veterinary settings is of significant relevance in order to understand and implement combined control strategies for MDR bacteria in animals and humans.
- Influence of the dental topical application of a nisin-biogel in the oral microbiome of dogs: a pilot studyPublication . Cunha, Eva; Valente, Sara; Nascimento, Mariana; Pereira, Marcelo; Tavares, Luis; Dias, Ricardo; Oliveira, ManuelaPeriodontal disease (PD) is one of the most widespread inflammatory diseases in dogs. This disease is initiated by a polymicrobial biofilm in the teeth surface (dental plaque), leading to a local inflammatory response, with gingivitis and/or several degrees of periodontitis. For instance, the prevention of bacterial dental plaque formation and its removal are essential steps in PD control. Recent research revealed that the antimicrobial peptide nisin incorporated in the delivery system guar gum (biogel) can inhibit and eradicate bacteria from canine dental plaque, being a promising compound for prevention of PD onset in dogs. However, no information is available regarding its effect on the dog's oral microbiome. In this pilot study, the influence of the nisin-biogel on the diversity of canine oral microbiome was evaluated using next generation sequencing (NGS), aiming to access the viability of nisin-biogel to be used in long-term experiment in dogs. Composite toothbrushing samples of the supragingival plaque from two dogs were collected at three timepoints: T1-before any application of the nisin-biogel to the animals' teeth surface; T2-one hour after one application of the nisin-biogel; and T3-one hour after a total of three applications of the nisin-biogel, each 48 hours. After that, microbial profiling was performed by NGS of the V3V4 16s rRNA region. After only one application of the nisin-biogel to the oral cavity of dogs, a statistically significant reduction in microbial diversity was observed (T2) as well as a reduction of some bacterial species potentially related with distinct stages of PD, when compared with samples collected before any application (T1). However, after a total of three nisin-biogel applications (T3), a recovery of the microbial diversity was detected. In conclusion, the nisin-biogel may influence the canine oral microbiome. A reduction in some bacterial species potentially related with distinct stages of PD was observed. This pilot study will help to design a controlled in vivo clinical trial to evaluate nisin-biogel effect on dental plaque progression and canine periodontal indices evolution in a long-term application period.
- Diabetic foot infections : application of a nisin-biogel to complement the activity of conventional antibiotics and antiseptics against Staphylococcus aureus biofilmsPublication . Santos, Raquel; Ruza, Diana; Cunha, Eva; Tavares, Luis; Oliveira, ManuelaABSTRACT - Background Diabetic foot infections (DFIs) are a frequent complication of Diabetes mellitus and a major cause of nontraumatic limb amputations. The Gram-positive bacterium Staphylococcus aureus, known for its resilient biofilms and antibiotic resistant profile, is the most frequent DFI pathogen. It is urgent to develop innovative treatments for these infections, being the antimicrobial peptide (AMP) nisin a potential candidate. We have previously proposed the use of a guar gum biogel as a delivery system for nisin. Here, we evaluated the potential of the nisin-biogel to enhance the efficacy of conventional antibiotics and antiseptics against DFIs S. aureus clinical isolates. Methods A collection of 23 S. aureus strains isolated from DFI patients, including multidrug- and methicillin-resistant strains, was used. The antimicrobial activity of the nisin-biogel was tested alone and in different combinations with the antiseptic chlorhexidine and the antibiotics clindamycin, gentamicin and vancomycin. Isolates' in vitro susceptibility to the different protocols was assessed using broth microdilution methods in order to determine their ability to inhibit and/or eradicate established S. aureus biofilms. Antimicrobials were added to the 96-well plates every 8 h to simulate a typical DFI treatment protocol. Statistical analysis was conducted using RCBD ANOVA in SPSS. Results The nisin-biogel showed a high antibacterial activity against biofilms formed by DFI S. aureus. The combined protocol using nisin-biogel and chlorhexidine presented the highest efficacy in biofilm formation inhibition, significantly higher (p<0.05) than the ones presented by the antibiotics-based protocols tested. Regarding biofilm eradication, there were no significant differences (p>0.05) between the activity of the combination nisin-biogel plus chlorhexidine and the conventional antibiotic-based protocols. Conclusions Results provide a valuable contribution for the development of complementary strategies to conventional antibiotics protocols. A combined protocol including chlorhexidine and nisinbiogel could be potentially applied in medical centres, contributing for the reduction of antibiotic administration, selection pressure on DFI pathogens and resistance strains dissemination.