Teresa Semedo-Lemsaddek

Transcriptional analysis of virulence-related genes in enterococci from distinct origins.

Aims:  The role of enterococci in food fermentation and as probiotics counteracts with their increasing importance as human pathogens. Over the years, several virulence factors have been described, mainly in clinical strains but also in food isolates. However, differential expression of such traits may modulate the pathogenic potential of the harbouring enterococci. To further unravel such differential response, this study aims to identify environmental cues responsible for triggering the expression of virulence‐related genes.

Enterococci from artisanal dairy products show high levels of adaptability

Enterococci are ubiquitous organisms able to promote both health (fermented food/probiotics) and illness (human/animal infections). Disturbingly, several enterococcal species commonly found in artisanal cheeses, such as Enterococcus faecalis and E. faecium, are being increasingly established as causes of infection, posing a problem for food safety. In this study enterococci from ewes milk and cheese were compared to clinical and reference strains by growth in media simulating environmental colonization and infection sites: 2YT, BHI, skim milk, urine and rabbit serum at different pHs, NaCl concentrations and temperatures. Growth curves were obtained with Microbiology Workstation Bioscreen C and used to calculate relative indexes--RIs--(based on absorbance, lag phase and specific growth rate) for each strain and environmental condition. Similar or higher RIs were obtained for food strains growing in infection-related environments when compared to clinical ones, revealing their ability to adapt and grow in these conditions. A dendrogram built using Pearsons correlation coefficient and a PCA analysis clustered the strains regardless of their origin or species allocation, suggesting a strain-specific mode of growth and a high environmental adaptability of enterococcal strains. These evidences turn essential the evaluation of strains to be used as starters or probiotics.

In vitro antimicrobial activity of royleanone derivatives against Gram-positive bacterial pathogens.

Infections caused by multiresistant bacterial pathogens are a significant problem worldwide, turning the search for natural compounds to act as alternatives to antibiotics of major importance. The aim of the present study was to investigate the in vitro antimicrobial activity of 7α‐acetoxy‐6β‐hydroxyroyleanone (1), isolated from Plectranthus grandidentatus (Lamiaceae), and 11 additional royleanone abietane derivatives of 1 (2–12) against important Gram‐positive human bacterial pathogens. Results showed that the aromatic and alkylic esters 2, 3 and 5 are more active than 1 against Enterococcus and Staphylococcus (minimum inhibitory concentration (MIC) values ranging from 0.98 to 62.50 µg/mL). Moreover, 7α‐acetoxy‐6β‐hydroxy‐12‐O‐(4‐chloro)benzoylroyleanone (2) gave rise to a new antibacterial‐prototype (MIC values of 3.91–15.63 µg/mL against Staphylococcus and of 0.98–3.91 µg/mL against Enterococcus). The results showed that the compounds under analysis also present antimicrobial activity against resistant bacteria. The hydrophobic extra‐interactions with bacterial targets seem to play an important role on the activity of royleanones derivatives. Copyright

Characterization of Enterococci from Food and Food-Related Settings

Enterococcus species are ubiquitous in nature, exist at high levels in food, and can cause severe diseases in humans. Thus, surveillance of enterococci harboring antibiotic resistance and virulence factors in food and food-related environments is needed. In the present study, 89 samples from food and food processing surfaces were collected in a cheese factory, a swine slaughterhouse, and a supermarket, and 132 Enterococcus isolates were recovered. Most isolates were identified as E. faecalis, which is considered the most pathogenic member of this genus. Safety analysis covering antibiotic resistance revealed that all isolates were resistant to sulfamethoxazole-trimethoprim and sensitive to amoxicillin-clavulanate, ampicillin, ciprofloxacin, gentamicin, levofloxacin, linezolid, nitrofurantoin, or teicoplanin. More than half of the isolates were resistant to quinupristin-dalfopristin, tetracycline, and bacitracin, and less than half were resistant to the other antibiotics evaluated. Regarding virulence factors, 52% the isolates were beta-hemolytic, 39% were gelatinase producers, and 45% contained the gelE gene. For the remaining genes evaluated, efaAfs was detected in more than half of the isolates, and agg, esp, and efaAfm were found in less than half of the isolates. The present investigation revealed that food-related enterococci obtained from very different settings have multidrug resistance and virulence factors, highlighting the importance of effective surveillance networks to avoid the spread of putative pathogenic enterococci.

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

BackgroundCorynebacterium 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 presentationTo 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.ConclusionsThis 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.

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.

Occurrence of Putative Pathogenicity Islands in Enterococci from Distinct Species and of Differing Origins

ABSTRACT Enterococci isolated from ewes milk and cheese, clinical isolates of human and veterinary origins, and reference strains obtained from culture collections were screened for the occurrence of putative pathogenicity island (PAIs). Results obtained after PCR amplification and hybridization point toward PAI dissemination among enterococci of diverse origins (food/clinical) and species (Enterococcus faecalis/non-E. faecalis).

Virulence traits and antibiotic resistance among enterococci isolated from dogs with periodontal disease

Periodontal disease - PD - is one of the most widespread diseases in dogs, but the role of this odontogenic infection in the dissemination of pathogenic bacteria present in the oral mucosa to other animals or pet owners is understudied. Trying to unveil the putative pathogenicity of enterococci present in the gums of dogs diagnosed with PD, thirty-two animals were investigated during routine visits to a private veterinary clinic. Seventy-one enterococci were recovered and characterized regarding species, genomic variability, virulence traits, antimicrobial resistance and biofilm-forming ability. Isolates were mainly identified as Enterococcus faecalis, with the large majority (95%) being able to produce biofilm. Regarding antibiotic resistance, all dog-enterococci were susceptible to ampicillin, amoxicillin/clavulanate, gentamicin-120, imipenem and vancomycin; while distinct levels of resistance were observed for chloramphenicol (10%), erythromycin (20%), streptomycin-300 (35%) and tetracycline (95%). For virulence traits incidence levels of 35% were observed for β-hemolysis and 25% for cylA, 25% for gelatinase and 35% for gelE; 85% harbor efaAfs and ebpABC; while ace, agg and esp are present respectively in 50, 30 and 10% of the dog-enterococci; efaAfm and acm were detected in all the Enterococcus faecium. Overall, the widespread prevalence of PD in dogs, associated with the close contact between companion animals, other animals and humans, may act as source for the dissemination of opportunistic pathogenic bacteria. Hence, aforementioned data on virulence and resistance features, emphasizes the need for active surveillance measures, such as the diagnose of PD in companion animals during routine visits to the veterinary clinic.

Enterococcal Infective Endocarditis following Periodontal Disease in Dogs

In humans, one of the major factors associated with infective endocarditis (IE) is the concurrent presence of periodontal disease (PD). However, in veterinary medicine, the relevance of PD in the evolution of dogs’ endocarditis remains poorly understood. In order to try to establish a correlation between mouth-associated Enterococcus spp. and infective endocarditis in dogs, the present study evaluated the presence and diversity of enterococci in the gum and heart of dogs with PD. Samples were collected during necropsy of 32 dogs with PD and visually diagnosed with IE, which died of natural causes or euthanasia. Enterococci were isolated, identified and further characterized by Pulsed-Field Gel Electrophoresis (PFGE); susceptibility to antimicrobial agents and pathogenicity potential was also evaluated. In seven sampled animals, PFGE-patterns, resistance and virulence profiles were found to be identical between mouth and heart enterococci obtained from the same dog, allowing the establishment of an association between enterococcal periodontal disease and endocarditis in dogs. These findings represent a crucial step towards understanding the pathogenesis of PD-driven IE, and constitute a major progress in veterinary medicine.

Putative probiotic lactic acid bacteria isolated from sauerkraut fermentations

Probiotics are live microorganisms which confer health benefits to the host, and may be isolated from several sources, such as vegetable foodstuffs. Sauerkraut is a cabbage product resulting from fermentation by a lactic acid bacteria microbial succession, and is a potential source for probiotics. The aim of the present study was the isolation and characterization of probiotic microorganisms from sauerkraut fermentations. Four distinct fermentations were performed, from which lactic acid bacteria were recovered. Overall, 114 isolates were obtained, phenotypically and genotypically characterized, identified to the genus level and evaluated regarding safety and probiotic potential. Representative bacteria were selected for further analysis, 52% being Lactobacillus spp. and 33% belonging to Leuconostoc spp. genus. One isolate revealed to be β-hemolytic, 42% possessed potentially mobile antimicrobial resistance, 88% were resistant to bile and 20% to low pH. The six most promising candidates were further characterized and presented antimicrobial activity against Listeria monocytogenes, three being resistant to lower pH values. Thus, global analysis of data gathered during this study highlighted the identification of three Lactobacillus strains with putative probiotic potential, suggesting the applicability of sauerkraut fermentations as a source for probiotic isolation. Due to their origin these strains should be suited for future application in the food industry, namely vegetable products such as sauerkraut itself.