In vitro efficacy of lavender oil, otological gel and gentamicin to eradicate biofilm produced by Pseudomonas aeruginosa.

BACKGROUND: Otitis externa (OE) is one of the most frequently diagnosed dermatological diseases in dogs, having a multifactorial aetiology. Among the bacterial agents associated with canine OE, Pseudomonas aeruginosa is of special concern owing to its frequent multidrug resistance profile and ability to form biofilms related to the infection's chronicity and recurrence. OBJECTIVES: The main objective of this study was to evaluate and compare the antibiofilm activity of two innovative antimicrobials-an otological gel containing a synthetic antimicrobial peptide and Lavandula angustifolia essential oil-with gentamicin (a conventional antibiotic) using biofilm-producing P. aeruginosa isolates obtained from dogs with OE. MATERIALS AND METHODS: The biofilm eradication capacity of gentamicin, otological gel and lavender oil was determined against a collection of 12 P. aeruginosa biofilm-producers among 35 clinical isolates obtained from the ear canals of dogs with OE. Also, the antimicrobial activity of the otological gel against P. aeruginosa biofilms was assessed in an in vitro model of dog cerumen. RESULTS: Lavender oil showed the best effectiveness after 30 min of contact, eradicating 58.3% (seven of 12) of the isolates, and gentamicin showed full eradication (12 of 12) after 24 h. The otological gel acted more slowly than the lavender oil; yet at 24 h, the antibiofilm capacity of both compounds was similar, with no significant difference between them. It also was found that triglycerides from synthetic cerumen earwax had antipseudomonal activity and, when combined with the otological gel, led to the full eradication of P. aeruginosa. CONCLUSIONS AND CLINICAL RELEVANCE: The results of this in vitro study indicate that lavender oil and the otological gel may be effective topical treatments for canine OE promoted by P. aeruginosa biofilm-producers, as alternatives to gentamicin.

Osteomyelitis caused by Aspergillus terreus complex in a dog: a case report.

BACKGROUND: 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 different 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 identified 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 profile of the fungal isolate was phenotypically characterized using specific 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 significant 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.

Absence of Synergism between a Dual-AMP Biogel and Antibiotics Used as Therapeutic Agents for Diabetic Foot Infections.

Diabetic foot infections (DFIs) are frequently linked to diabetic-related morbidity and death because of the ineffectiveness of conventional antibiotics against multidrug-resistant bacteria. Pexiganan and nisin A are antimicrobial peptides (AMPs), and their application may complement conventional antibiotics in DFI treatment. A collagen 3D model, previously established to mimic a soft-tissue collagen matrix, was used to evaluate the antibacterial efficacy of a guar gum gel containing pexiganan and nisin alone and combined with three antimicrobials toward the biofilms of Staphylococcus aureus and Pseudomonas aeruginosa isolated from infected foot ulcers. Antimicrobials and bacterial diffusion were confirmed by spot-on-lawn and bacterial growth by bacterial count (cfu/mL). Our main conclusion was that the dual-AMP biogel combined with gentamicin, clindamycin, or vancomycin was not able to significantly reduce bacterial growth or eradicate S. aureus and P. aeruginosa DFI isolates. We further reported an antagonism between dual-AMP and dual-AMP combined with antibiotics against S. aureus.

Public contracting of home respiratory care services through an electronic quasi-market model.

Respiratory diseases are becoming a major challenge in health systems as they cover expanding and diversified pathologies such as chronic obstructive pulmonary disease, chronic bronchitis, emphysema and other types increasing at a fast rate as it is the case of sleep apnoea. Nowadays, most of these pathologies can be treated by home respiratory care services using advanced medical equipment as well as efficient assistance and monitoring services supported by digital technologies. However, the implementation of home respiratory care services implies flexible public contracting of such services to achieve equity conditions of access and to cope with their technological nature, their continuous processes of innovation and their time dependent demand. Unfortunately, traditional procurement processes are not able to meet these challenges explaining their frequent low rates of success and so an alternative procurement approach is proposed in this paper based on an electronic quasi-market model. This new model was successfully applied in Portugal and the evaluated results are also discussed herein.

Liposomes as Antibiotic Delivery Systems: A Promising Nanotechnological Strategy against Antimicrobial Resistance.

Antimicrobial drugs are key tools to prevent and treat bacterial infections. Despite the early success of antibiotics, the current treatment of bacterial infections faces serious challenges due to the emergence and spread of resistant bacteria. Moreover, the decline of research and private investment in new antibiotics further aggravates this antibiotic crisis era. Overcoming the complexity of antimicrobial resistance must go beyond the search of new classes of antibiotics and include the development of alternative solutions. The evolution of nanomedicine has allowed the design of new drug delivery systems with improved therapeutic index for the incorporated compounds. One of the most promising strategies is their association to lipid-based delivery (nano)systems. A drug's encapsulation in liposomes has been demonstrated to increase its accumulation at the infection site, minimizing drug toxicity and protecting the antibiotic from peripheral degradation. In addition, liposomes may be designed to fuse with bacterial cells, holding the potential to overcome antimicrobial resistance and biofilm formation and constituting a promising solution for the treatment of potential fatal multidrug-resistant bacterial infections, such as methicillin resistant Staphylococcus aureus. In this review, we aim to address the applicability of antibiotic encapsulated liposomes as an effective therapeutic strategy for bacterial infections.

A polymicrobial biofilm model for testing the antimicrobial potential of a nisin-biogel for canine periodontal disease control.

BACKGROUND: Periodontal disease (PD) in dogs is prompted by the establishment of a polymicrobial biofilm at the tooth surface and a subsequent host inflammatory response. Several strategies may be used for PD control, including dental hygiene home care procedures, like toothbrushing, special diet and chew toys that reduce dental plaque accumulation, or professional periodontal treatments. Aiming at PD control, a biogel composed by nisin and guar-gum was previously developed. This work aimed to establish an in vitro model mimicking the PD-associated biofilms and to evaluate the nisin-biogel inhibitory activity against this polymicrobial biofilm by determining its Minimum Biofilm Inhibitory (MBIC) and Eradication Concentrations (MBEC). Bacterial species tested included Neisseria zoodegmatis CCUG 52598T, Corynebacterium canis CCUG 58627T, Porphyromonas cangingivalis DSMZ VPB 4874, Peptostreptococcus canis CCUG 57081 and an Enterococcus faecalis isolate belonging to a collection of oral bacteria obtained from dogs with PD. Before establishing the biofilm, coaggregation between species was determined by optical density measurement after 2 and 24 hours. Nisin-biogel MBIC and MBEC values regarding the polymicrobial biofilm were determined using a modified version of the Calgary biofilm pin lid device, after confirming the presence of the five bacterial species by Fluorescent In Situ Hybridization. RESULTS: Only 40% of the bacterial dual suspensions were able to coaggregate at 2 hours, but all species tested exhibited a coaggregation percentage higher than 30% at 24 hours. It was possible to establish a 48 h polymicrobial biofilm model composed by the five bacterial species selected. This model was used to determine nisin-biogel MBIC (26.39 ± 5.89 µg/mL) and MBEC (62.5 ± 27.73 µg/mL) values. CONCLUSIONS: Our results showed that the nisin-biogel can inhibit and eradicate PD multispecies biofilms. As this in vitro model mimics an in vivo periodontal polymicrobial biofilm, our results reinforce the potential of the application of nisin-biogel for canine PD control.

Cyclic fatigue resistance of three rotary file systems in a dynamic model after immersion in sodium hypochlorite.

To evaluate the effect of immersion in 3% sodium hypochlorite solution in the resistance to cyclic fatigue of three nickel-titanium (NiTi) rotary file systems, ProTaper Next (PTN), Hyflex CM (CM), and Hyflex EDM (EDM), in a mechanical model featuring axial movement. Ninety instruments of three different NiTi rotary file systems, PTN (size 25, 0.06 taper), CM (25, 0.06), and EDM (25/~, variable taper), were randomly divided according to a 3 × 3 factorial design and tested under dynamic immersion in a 3% NaOCl solution (1 or 5 min) or without immersion, making a total of 9 groups (n = 10). Files were tested in an artificial root canal with 45° angle and 5 mm radius apical curvature being submitted to back-and-forth movements until fracture. Statistical analysis was performed using two-way factorial ANOVA with Bonferroni post-hoc tests, at a significance level of 5%. Instruments were evaluated for reliability using a Weilbull approach. Regardless of the immersion treatment, PTN had on average 1200 ± 178 cycles to fracture, CM had 1949 ± 362, and EDM had 5573 ± 853, which was a significantly different (P < 0.01). The NaOCl immersion promoted a significant reduction in the mean number of cycles to fracture (P = 0.01), and was reflected in a significant reduction of the characteristic life of the instruments of the CM end EDM groups. Within this study conditions, EDM instruments performed better to cyclic fatigue followed by CM and then PTN. Immersion in NaOCl decreased the resistance to cyclic fatigue of all tested instruments, but affected more those manufactured from CM wire.

Tinea corporis by Microsporum audouinii in a female chimpanzee (Pan troglodytes) from Guinea-Bissau: A case report.

We report a Microsporum audouinii infection in a female juvenile chimpanzee (Pan troglodytes) presenting generalized dermatitis compatible with dermatophytosis. Dermatophyte was identified by macro- and microscopic characterization of skin and scales cultures in Mycosel Agar. The topical treatment applied was effective, having the potential for dermatophytosis treatment in immunocompetent primates.

Evaluation of the humoral immune response induced by vaccination for canine distemper and parvovirus: a pilot study.

BACKGROUND: Canine Distemper Virus (CDV) and Canine Parvovirus (CPV) lead to infections with high mortality rates in dogs. These viruses affect unvaccinated dogs or dogs with incomplete vaccination protocols. Vaccination plays an important role in reducing death rates, preventing clinical cases and controlling the spread of virus However, the efficacy of vaccination might be affected by different factors including vaccine scheduling and the neutralization of the vaccine targets by maternal antibodies. In face of these factors, the main goals of this study are (i) to investigate the antibody responses of puppies undergoing different primary vaccination protocols against CPV and CDV and (ii) to estimate the time until seroreversion in adult dogs unvaccinated for at least 3 years. RESULTS: Antibody protection against CDV and CPV was evaluated in a total of 20 dogs: 5 puppies that initiated immunization at 6 weeks after birth (group A), 8 animals that started vaccination between 8 and 12 weeks of age (group B), and 7 adult dogs that have not been vaccinated for at least 3 years (group C). Blood samples were collected from each animal, with 3 to 4 weeks apart. Antibody responses were measured using indirect ELISA. In the second immunization point, no significant differences were found between the seroconversion of groups A and B for each viral infection (p = 0.81 and 0.20 for CDV and CPV, respectively). In the third immunization, there was evidence for a shorter time to achieve a protective titer against CPV in group B when compared to group A (p = 0.015). Similar evidence was not found for CDV (p-value = 0.41). In Group C, the average time until seroveversion was estimated at 2.86 years and 7.63 years for CDV and CPV, respectively. CONCLUSION: Vaccine response to CDV and CPV is specific in each individual. Effective immune protection in primary vaccination depends mainly on the initial titer of maternal antibodies acquired by the neonate. Other factors such as environmental exposure, immunization schedules and immune system activity influence the duration of immunity in adult dogs. The variability found reinforces the need to determine individual humoral immunity levels in order to assess vaccine efficacy.

Skin asepsis protocols as a preventive measure of surgical site infections in dogs: chlorhexidine-alcohol versus povidone-iodine.

BACKGROUND: Most of surgical site infections (SSI) are caused by commensal and pathogenic agents from the patient's microbiota, which may include antibiotic resistant strains. Pre-surgical asepsis of the skin is one of the preventive measures performed to reduce SSI incidence and also antibiotic resistance dissemination. However, in veterinary medicine there is no agreement on which biocide is the most effective. The aim of this study was to evaluate the effectiveness of two pre-surgical skin asepsis protocols in dogs. A total of 46 animals were randomly assigned for an asepsis protocol with an aqueous solution of 7.5% povidone-iodine or with an alcoholic solution of 2% chlorhexidine. For each dog, two skin swab samples were collected at pre-asepsis and post-asepsis, for bacterial quantification by conventional techniques and isolation of methicillin-resistant species. RESULTS: Most samples collected at the post-asepsis did not present bacterial growth, both for the animals subjected to the povidone-iodine (74%) or to the chlorhexidine (70%) protocols. In only 9% of the cases a significant bacterial logarithmic reduction was not observed, indicating possible resistance to these agents. Also, the logarithmic reduction of the bacterial quantification from pre- and post-asepsis time, was not statistically different for povidone-iodine (6.51 ± 1.94 log10) and chlorhexidine (6.46 ± 2.62 log10) protocol. From the 39% pre-asepsis swabs which showed bacterial growth in MRSA modified chromogenic agar medium, only one isolate was identified as Staphylococcus aureus and one as S. epidermidis. False positives were mainly other staphylococci species, as well as Enterobacteriaceae. CONCLUSIONS: Pre-surgical skin asepsis protocols with povidone-iodine or chlorhexidine showed similar efficacy in the elimination of methicillin resistant bacteria and preventing surgical site infections in dogs undergoing surgery.

Potential of two delivery systems for nisin topical application to dental plaque biofilms in dogs.

BACKGROUND: Periodontal disease (PD) is caused by the development of a microbial biofilm (dental plaque) in the periodontium, affecting approximately 80% of dogs. Several bacterial species present in the canine oral cavity can be implicated in the development of this disease, including Enterococcus spp. To decrease antibiotic administration, a possible control strategy for dog's enterococcal PD may involve the use of the antimicrobial peptide (AMP) nisin. Nisin's inhibitory activity was evaluated against a collection of previously characterized enterococci obtained from the oral cavity of dogs with PD (n = 20), as well as the potential of a guar-gum gel and a veterinary toothpaste as topical delivery systems for this AMP. The Minimum Inhibitory (MIC) and Bactericidal Concentrations (MBC) and the Minimum Biofilm Eradication (MBEC) and Inhibitory Concentrations (MBIC) were determined for nisin and for the supplemented guar-gum gel. For the supplemented veterinary toothpaste an agar-well diffusion assay was used to evaluate its inhibitory potential. RESULTS: Nisin was effective against all isolates. Independently of being or not incorporated in the guar-gum gel, its inhibitory activity on biofilms was higher, with MBIC (12.46 ± 5.16 and 13.60 ± 4.31 µg/mL, respectively) and MBEC values (21.87 ± 11.33 and 42.34 ± 16.61 µg/mL) being lower than MIC (24.61 ± 4.64 and 14.90 ± 4.10 µg/mL) and MBC (63.09 ± 13.22 and 66.63 ± 19.55 µg/mL) values. The supplemented toothpaste was also effective, showing inhibitory activity against 95% of the isolates. CONCLUSIONS: The inhibitory ability of nisin when incorporated in the two delivery systems was maintained or increased, demonstrating the potential of these supplemented vehicles to be applied to PD control in dogs.

Polymicrobial interactions influence the agr copy number in Staphylococcus aureus isolates from diabetic foot ulcers.

Diabetic foot ulcers are a major complication of diabetes and are often colonised by complex bacterial communities, where Staphylococcus aureus is frequently co-present with Pseudomonas aeruginosa. These bacteria interact through quorum sensing, encoded in S. aureus by the accessory gene regulator (agr). Typing and copy number of S. aureus agr were assessed here to give insights on strain variability and possible interspecies influence. As agr is classified in four genetic groups, agr-I, agr-II, agr-III and agr-IV, the agr type of 23 S. aureus diabetic foot ulcers isolates was evaluated by PCR and gene copy number determined by qPCR, including in S. aureus present in polymicrobial infections. agr-I and agr-II were found to be present in 52 and 39% of the isolates, respectively. In two isolates, no agr type was identified, and types III and IV were not detected. Interestingly, agr-II copy number was higher in dual suspensions than in S. aureus single suspension. We conclude that agr type I was the most frequent in clinical centers in Lisbon, and variations in agr-I and agr-II copy numbers were strain specific. Variations in agr copy number in dual suspensions suggests that P. aeruginosa may influence S. aureus agr-II gene regulation, confirming an interaction between these two bacteria. This is a first approach to characterise agr variation in S. aureus from diabetic foot ulcers in vitro.

Antimicrobial resistance and genomic rep-PCR fingerprints of Pseudomonas aeruginosa strains from animals on the background of the global population structure.

BACKGROUND: Pseudomonas aeruginosa is an important human opportunistic pathogen responsible for fatal nosocomial infections worldwide, and has emerged as a relevant animal pathogen. Treatment options are dramatically decreasing, due to antimicrobial resistance and the microorganism's large versatile genome. Antimicrobial resistance profiles, serotype frequency and genomic profile of unrelated P. aeruginosa isolates of veterinary origin (n = 73), including domesticated, farm, zoo and wild animals mainly from Portugal were studied. The genomic profile, determined by DiversiLab system (Rep-PCR-based technique), was compared with the P. aeruginosa global population structure to evaluate their relatedness. RESULTS: Around 40% of the isolates expressed serotypes O6 (20.5%) and O1 (17.8%). A total of 46.6% of isolates was susceptible to all antimicrobials tested. Isolates obtained from most animals were non-multidrug resistant (86.3%), whereas 11% were multidrug resistant, MDR (non-susceptible to at least one agent in ≥ three antimicrobial categories), and 2.7% extensively drug resistant, XDR (non-susceptible to at least one agent in all but two or fewer antimicrobial categories). Resistance percentages were as follows: amikacin (0.0%), aztreonam (41.1%), cefepime (9.6%), ceftazidime (2.7%), ciprofloxacin (15.1%), colistin (0.0%), gentamicin (12.3%), imipenem (1.4%), meropenem (1.4%), piperacillin + tazobactam (12.3%), ticarcillin (16.4%), ticarcillin + clavulanic acid (17.8%), and tobramycin (1.4%). Animal isolates form a population with a non-clonal epidemic structure indistinguishable from the global P. aeruginosa population structure, where no specific 'animal clonal lineage' was detected. CONCLUSIONS: Serotypes O6 and O1 were the most frequent. Serotype frequency and antimicrobial resistance patterns found in P. aeruginosa from animals were as expected for this species. This study confirms earlier results that P. aeruginosa has a non-clonal population structure, and shows that P. aeruginosa population from animals is homogeneously scattered and indistinguishable from the global population structure.

Susceptibility patterns of Staphylococcus aureus biofilms in diabetic foot infections.

BACKGROUND: Foot infections are a major cause of morbidity in people with diabetes and the most common cause of diabetes-related hospitalization and lower extremity amputation. Staphylococcus aureus is by far the most frequent species isolated from these infections. In particular, methicillin-resistant S. aureus (MRSA) has emerged as a major clinical and epidemiological problem in hospitals. MRSA strains have the ability to be resistant to most ß-lactam antibiotics, but also to a wide range of other antimicrobials, making infections difficult to manage and very costly to treat. To date, there are two fifth-generation cephalosporins generally efficacious against MRSA, ceftaroline and ceftobripole, sharing a similar spectrum. Biofilm formation is one of the most important virulence traits of S. aureus. Biofilm growth plays an important role during infection by providing defence against several antagonistic mechanisms. In this study, we analysed the antimicrobial susceptibility patterns of biofilm-producing S. aureus strains isolated from diabetic foot infections. The antibiotic minimum inhibitory concentration (MIC) was determined for ten antimicrobial compounds, along with the minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC), followed by PCR identification of genetic determinants of biofilm production and antimicrobial resistance. RESULTS: Results demonstrate that very high concentrations of the most used antibiotics in treating diabetic foot infections (DFI) are required to inhibit S. aureus biofilms in vitro, which may explain why monotherapy with these agents frequently fails to eradicate biofilm infections. In fact, biofilms were resistant to antibiotics at concentrations 10-1000 times greater than the ones required to kill free-living or planktonic cells. The only antibiotics able to inhibit biofilm eradication on 50 % of isolates were ceftaroline and gentamicin. CONCLUSIONS: The results suggest that the antibiotic susceptibility patterns cannot be applied to biofilm established infections. Selection of antimicrobial therapy is a critical step in DFI and should aim at overcoming biofilm disease in order to optimize the outcomes of this complex pathology.

Molecular typing, virulence traits and antimicrobial resistance of diabetic foot staphylococci.

BACKGROUND: Diabetes mellitus is a major chronic disease that continues to increase significantly. One of the most important and costly complications of diabetes are foot infections that may be colonized by pathogenic and antimicrobial resistant bacteria, harboring several virulence factors, that could impair its successful treatment. Staphylococcus aureus is one of the most prevalent isolate in diabetic foot infections, together with aerobes and anaerobes. METHODS: In this study, conducted in the Lisbon area, staphylococci isolated (n = 53) from diabetic foot ulcers were identified, genotyped and screened for virulence and antimicrobial resistance traits. Genetic relationship amongst isolates was evaluated by pulsed-field-gel-electrophoresis with further multilocus sequence typing of the identified pulsotypes. PCR was applied for detection of 12 virulence genes and e-test technique was performed to determine minimal inhibitory concentration of ten antibiotics. RESULTS: Among the 53 isolates included in this study, 41 Staphylococcus aureus were identified. Staphylococcal isolates were positive for intercellular adhesins icaA and icaD, negative for biofilm associated protein bap and pantone-valentine leucocidin pvl. S. aureus quorum sensing genes agrI and agrII were identified and only one isolate was positive for toxic shock syndrome toxin tst. 36 % of staphylococci tested were multiresistant and higher rates of resistance were obtained for ciprofloxacin and erythromycin. Clonality analysis revealed high genomic diversity and numerous S. aureus sequence types, both community- and hospital-acquired, belonging mostly to clonal complexes CC5 and C22, widely diffused in Portugal nowadays. CONCLUSIONS: This study shows that diabetic foot ulcer staphylococci are genomically diverse, present resistance to medically important antibiotics and harbour virulence determinants. These properties suggest staphylococci can contribute to persistence and severity of these infections, leading to treatment failure and to the possibility of transmitting these features to other microorganisms sharing the same niche. In this context, diabetic patients may become a transmission vehicle for microorganisms' clones between community and clinical environments.

New insight into dolphin morbillivirus phylogeny and epidemiology in the northeast Atlantic: opportunistic study in cetaceans stranded along the Portuguese and Galician coasts.

BACKGROUND: Screening Atlantic cetacean populations for Cetacean Morbillivirus (CeMV) is essential to understand the epidemiology of the disease. In Europe, Portugal and Spain have the highest cetacean stranding rates, mostly due to the vast extension of coastline. Morbillivirus infection has been associated with high morbidity and mortality in cetaceans, especially in outbreaks reported in the Mediterranean Sea. However, scarce information is available regarding this disease in cetaceans from the North-East Atlantic populations. The presence of CeMV genomic RNA was investigated by reverse transcription-quantitative PCR in samples from 279 specimens stranded along the Portuguese and Galician coastlines collected between 2004 and 2015. RESULTS: A total of sixteen animals (n = 16/279, 5.7 %) were positive. The highest prevalence of DMV was registered in striped dolphins (Stenella coeruleoalba) (n = 14/69; 20.3 %), slightly higher in those collected in Galicia (n = 8/33; 24.2 %) than in Portugal (n = 6/36; 16.7 %). CONCLUSIONS: Phylogenetic analysis revealed that, despite the low genetic distances between samples, the high posterior probability (PP) values obtained strongly support the separation of the Portuguese and Galician sequences in an independent branch, separately from samples from the Mediterranean and the Canary Islands. Furthermore, evidence suggests an endemic rather than an epidemic situation in the striped dolphin populations from Portugal and Galicia, since no outbreaks have been detected and positive samples have been detected annually since 2007, indicating that this virus is actively circulating in these populations and reaching prevalence values as high as 24 % among the Galician samples tested.

Characterization of multidrug-resistant diabetic foot ulcer enterococci.

INTRODUCTION: Diabetes mellitus is a highly prevalent chronic progressive disease with complications that include diabetic-foot ulcers. METHODS: Enterococci isolated from diabetic-foot infections were identified, evaluated by macro-restriction analysis, and screened for virulence traits and antimicrobial resistance. RESULTS: All isolates were considered multidrug-resistant, cytolysin and gelatinase producers, and the majority also demonstrated the ability to produce biofilms. CONCLUSIONS: These results indicate the importance of enterococci in diabetic-foot infection development and persistence, especially regarding their biofilm-forming ability and resistance to clinically relevant antibiotics.

Phenotypic and Molecular Characterization of Salmonella 1,4,[5],12:i:- R-Type ASSuT Isolates from Humans, Animals, and Environment in Portugal, 2006-2011.

The increase in prevalence of Salmonella 1,4,[5],12:i:- related infections over the last few years has been considered a public health issue in many European countries, especially as this serovar may be associated with tetraresistance to ampicillin, streptomycin, sulfonamides, and tetracyclines (R-type ASSuT). Salmonella 1,4,[5],12:i:- isolates (n = 187) obtained by the Portuguese National Laboratory from different sources, including human clinical cases (n = 170), veterinary (n = 10), environmental (n = 6), and food samples (n = 1), were collected from 15 districts between 2006 and 2011. All isolates were serotyped using the slide agglutination method and results were confirmed by multiplex PCR for the monophasic variant. From the confirmed Salmonella 1,4,[5],12:i:-, R-type ASSuT isolates were selected by disc diffusion and minimal inhibitory concentration (MIC) determination for further characterization by pulsed-field gel electrophoresis restriction with XbaI, virulence genes determination by PCR, additional antimicrobial resistance profiling by disc diffusion, and epidemiological distribution evaluation. Out of the 187 serotyped isolates, 133 were confirmed as Salmonella 1,4,[5],12:i:- with a R-type ASSuT occurrence of 61.7%. Distribution among Portuguese districts showed a higher percentage of reported cases in coastal areas, in particular, in Porto (24.8%), Setúbal (13.5%), and Aveiro (12.8%), probably due to the higher population density. Clonality analysis revealed a high diversity of pulsotypes with the majority of human salmonellosis cases being attributed to sporadic events. All isolates harbored 14 out of the 18 virulence genes evaluated and 87.8% of the isolates showed all the resistance genes frequently associated with the European clone, blaTEM+sul2+straA-straB+tetB+. This study shows that Salmonella 1,4,[5],12:i:- resistant isolates are widely distributed in Portugal. This may be related to a selective advantage offered by R-type ASSuT profile, the presence of multiple virulent features, including the ability to form biofilms, which along with a high diversity of pulsotypes may be responsible for the dissemination through the country.

First report of Corynebacterium pseudotuberculosis from caseous lymphadenitis lesions in Black Alentejano pig (Sus scrofa domesticus).

BACKGROUND: Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis, a common disease in small ruminant populations throughout the world and responsible for a significant economic impact for producers. CASE PRESENTATION: To our knowledge, this is the first characterization of C. pseudotuberculosis from caseous lymphadenitis lesions in Black Alentejano pig (Sus scrofa domesticus). In this study, phenotypic and genotypic identification methods allocated the swine isolates in C. pseudotuberculosis biovar ovis. The vast majority of the isolates were able to produce phospholipase D and were susceptible to most of the antimicrobial compounds tested. Macrorestriction patterns obtained by Pulsed Field Gel Electrophoresis (PFGE) grouped the C. pseudotuberculosis in two clusters with a high similarity index, which reveals their clonal relatedness. Furthermore, swine isolates were compared with C. pseudotuberculosis from caprines and PFGE patterns also showed high similarity, suggesting the prevalence of dominant clones and a potential cross-dissemination between these two animal hosts. CONCLUSIONS: This work represents the first report of Corynebacterium pseudotuberculosis from caseous lymphadenitis lesions in Black Alentejano pig and alerts for the importance of the establishment of suitable control and sanitary management practices to control the infection and avoid further dissemination of this important pathogen to other animal hosts.

Molecular and serological surveillance of canine enteric viruses in stray dogs from Vila do Maio, Cape Verde.

BACKGROUND: Infections caused by canine parvovirus, canine distemper virus and canine coronavirus are an important cause of mortality and morbidity in dogs worldwide. Prior to this study, no information was available concerning the incidence and prevalence of these viruses in Cape Verde archipelago. RESULTS: To provide information regarding the health status of the canine population in Vila do Maio, Maio Island, Cape Verde, 53 rectal swabs were collected from 53 stray dogs during 2010 and 93 rectal swabs and 88 blood samples were collected from 125 stray dogs in 2011. All rectal swabs (2010 n = 53; 2011 n = 93) were analysed for the presence of canine parvovirus, canine distemper virus and canine coronavirus nucleic acids by quantitative PCR methods. Specific antibodies against canine distemper virus and canine parvovirus were also assessed (2011 n = 88).From the 2010 sampling, 43.3% (23/53) were positive for canine parvovirus DNA, 11.3% (6/53) for canine distemper virus RNA and 1.9% (1/53) for canine coronavirus RNA. In 2011, the prevalence values for canine parvovirus and canine coronavirus were quite similar to those from the previous year, respectively 44.1% (41/93), and 1.1% (1/93), but canine distemper virus was not detected in any of the samples analysed (0%, 0/93). Antibodies against canine parvovirus were detected in 71.6% (63/88) blood samples and the seroprevalence found for canine distemper virus was 51.1% (45/88). CONCLUSIONS: This study discloses the data obtained in a molecular and serological epidemiological surveillance carried out in urban populations of stray and domestic animals. Virus transmission and spreading occurs easily in large dog populations leading to high mortality rates particularly in unvaccinated susceptible animals. In addition, these animals can act as disease reservoirs for wild animal populations by occasional contact. Identification of susceptible wildlife of Maio Island is of upmost importance to evaluate the risk of pathogen spill over from domestic to wild animals in Cape Verde and to evaluate the associated threat to the wild susceptible species.