Advantages and disadvantages of non-starter lactic acid bacteria from traditional fermented foods: Potential use as starters or probiotics.

Traditional fermented foods are a significant source of starter and/or non-starter lactic acid bacteria (nsLAB). Moreover, these microorganisms are also known for their role as probiotics. The potential of nsLAB is huge; however, there are still challenges to be overcome with respect to characterization and application. In the present review, the most important steps that autochthonous lactic acid bacteria isolated from fermented foods need to overcome, to qualify as novel starter cultures, or as probiotics, in food technology and biotechnology, are considered. These different characterization steps include precise identification, detection of health-promoting properties, and safety evaluation. Each of these features is strain specific and needs to be accurately determined. This review highlights the advantages and disadvantages of nsLAB, isolated from traditional fermented foods, discussing safety aspects and sensory impact.

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.

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.

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-6ß-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-6ß-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.

Dynamics of bovine intramammary infections due to coagulase-negative staphylococci on four farms.

The objectives of this study were to compare the impact of different coagulase-negative species (CNS) on udder health measured in terms of individual quarter milk somatic cell count (SCC) and duration of intramammary infection, and to get some insight into most likely routes of infection for different CNS species. This longitudinal observational study was performed on four farms that were sampled at 4-week intervals for a total of 12 visits each. Quarters infected with CNS were followed through time with milk samples being submitted for bacteriological culture and SCC determination. PCR amplification of the internal transcribed spacer region and sequencing of the sodA and rpoB genes were used for species allocation. Pulsed-field gel electrophoresis (PFGE) was performed to assess strain identity. The percentage of quarters affected per farm varied between 6 and 35%, with the most frequently isolated CNS species being Staphylococcus epidermidis, followed by Staph. simulans, Staph. chromogenes and Staph. haemolyticus. It was possible to follow 111 intramammary infections due to CNS through time. Duration of infection had a mean of 188 d and was not significantly different between CNS species. Geometric mean quarter SCC overall was 132 000 cells/ml and was also not significantly different between CNS species. Despite the possibility of a different epidemiology of infection, the impact in terms of udder health seems to be similar for different CNS species.

Antimicrobial Drug Resistance in Poultry Production: Current Status and Innovative Strategies for Bacterial Control.

The world population's significant increase has promoted a higher consumption of poultry products, which must meet the specified demand while maintaining their quality and safety. It is well known that conventional antimicrobials (antibiotics) have been used in livestock production, including poultry, as a preventive measure against or for the treatment of infectious bacterial diseases. Unfortunately, the use and misuse of these compounds has led to the development and dissemination of antimicrobial drug resistance, which is currently a serious public health concern. Multidrug-resistant bacteria are on the rise, being responsible for serious infections in humans and animals; hence, the goal of this review is to discuss the consequences of antimicrobial drug resistance in poultry production, focusing on the current status of this agroeconomic sector. Novel bacterial control strategies under investigation for application in this industry are also described. These innovative approaches include antimicrobial peptides, bacteriophages, probiotics and nanoparticles. Challenges related to the application of these methods are also discussed.

Dissemination of Enterococcal Genetic Lineages: A One Health Perspective.

Enterococcus spp. are commensals of the gastrointestinal tracts of humans and animals and colonize a variety of niches such as water, soil, and food. Over the last three decades, enterococci have evolved as opportunistic pathogens, being considered ESKAPE pathogens responsible for hospital-associated infections. Enterococci's ubiquitous nature, excellent adaptative capacity, and ability to acquire virulence and resistance genes make them excellent sentinel proxies for assessing the presence/spread of pathogenic and virulent clones and hazardous determinants across settings of the human-animal-environment triad, allowing for a more comprehensive analysis of the One Health continuum. This review provides an overview of enterococcal fitness and pathogenic traits; the most common clonal complexes identified in clinical, veterinary, food, and environmental sources; as well as the dissemination of pathogenic genomic traits (virulome, resistome, and mobilome) found in high-risk clones worldwide, across the One Health continuum.

Antimicrobial and Antibiofilm Potential of Thymus vulgaris and Cymbopogon flexuosus Essential Oils against Pure and Mixed Cultures of Foodborne Bacteria.

The spread of pathogenic and food spoilage microorganisms through the food chain still faces major mitigation challenges, despite modern advances. Although multiple cleaning and disinfection procedures are available for microbial load reduction in food-related settings, microbes can still remain on surfaces, equipment, or machinery, especially if they have the ability to form biofilms. The present study assessed the biofilm-forming properties of pure and mixed cultures of foodborne and spoilage bacteria (Listeria monocytogenes, Enterococcus faecalis, Aeromonas hydrophila, Brochothrix thermosphacta), using polystyrene and stainless steel contact surfaces. Subsequently, the antimicrobial and antibiofilm properties of Thymus vulgaris and Cymbopogon flexuosus essential oils-EOs-were evaluated against these bacteria. Moreover, in silico prediction of the absorption and toxicity values of the EOs' major constituents was also performed, perceiving the putative application in food-related settings. Overall, biofilm formation was observed for all microbes under study, at different temperatures and both contact surfaces. In polystyrene, at 25 °C, when comparing pure with mixed cultures, the combination Listeria-Aeromonas achieved the highest biofilm biomass. Moreover, at 4 °C, increased biofilm formation was detected in stainless steel. Regarding thyme, this EO showed promising antimicrobial features (especially against A. hydrophila, with a MIC of 0.60 µg/µL) and antibiofilm abilities (MBEC of 110.79 µg/µL against L. monocytogenes, a major concern in food settings). As for lemongrass EO, the highest antimicrobial activity, with a MIC of 0.49 µg/µL, was also observed against L. monocytogenes. Overall, despite promising results, the in situ effectiveness of these essential oils, alone or in combination with other antimicrobial compounds, should be further explored.

Lactiplantibacillus plantarum from Unexplored Tunisian Ecological Niches: Antimicrobial Potential, Probiotic and Food Applications.

The continued exploration of the diversity of lactic acid bacteria in little-studied ecological niches represents a fundamental activity to understand the diffusion and biotechnological significance of this heterogeneous class of prokaryotes. In this study, Lactiplantibacillus plantarum (Lpb. plantarum) strains were isolated from Tunisian vegetable sources, including fermented olive and fermented pepper, and from dead locust intestines, which were subsequently evaluated for their antimicrobial activity against foodborne pathogenic bacteria, including Escherichia coli O157:H7 CECT 4267 and Listeria monocytogenes CECT 4031, as well as against some fungi, including Penicillium expansum, Aspergilus niger, and Botrytis cinerea. In addition, their resistance to oro-gastro-intestinal transit, aggregation capabilities, biofilm production capacity, adhesion to human enterocyte-like cells, and cytotoxicity to colorectal adenocarcinoma cell line were determined. Further, adhesion to tomatoes and the biocontrol potential of this model food matrix were analyzed. It was found that all the strains were able to inhibit the indicator growth, mostly through organic acid production. Furthermore, these strains showed promising probiotic traits, including in vitro tolerance to oro-gastrointestinal conditions, and adhesion to abiotic surfaces and Caco-2 cells. Moreover, all tested Lpb. plantarum strains were able to adhere to tomatoes with similar rates (4.0-6.0 LogCFU/g tomato). The co-culture of LAB strains with pathogens on tomatoes showed that Lpb. plantarum could be a good candidate to control pathogen growth. Nonetheless, further studies are needed to guarantee their use as probiotic strains for biocontrol on food matrices.

Colicins and Microcins Produced by Enterobacteriaceae: Characterization, Mode of Action, and Putative Applications.

Enterobacteriaceae are widely present in many environments related to humans, including the human body and the food that they consume, from both plant or animal origin. Hence, they are considered relevant members of the gastrointestinal tract microbiota. On the other hand, these bacteria are also recognized as putative pathogens, able to impair human health and, in food, they are considered indicators for the microbiological quality and hygiene status of a production process. Nevertheless, beneficial properties have also been associated with Enterobacteriaceae, such as the ability to synthesize peptides and proteins, which can have a role in the structure of microbial communities. Among these antimicrobial molecules, those with higher molecular mass are called colicins, while those with lower molecular mass are named microcins. In recent years, some studies show an emphasis on molecules that can help control the development of pathogens. However, not enough data are available on this subject, especially related to microcins. Hence, this review gathers and summarizes current knowledge on colicins and microcins, potential usage in the treatment of pathogen-associated diseases and cancer, as well as putative applications in food biotechnology.

Antimicrobial Resistance and Clonal Lineages of Staphylococcus aureus from Cattle, Their Handlers, and Their Surroundings: A Cross-Sectional Study from the One Health Perspective.

Staphylococcus aureus have been progressively identified in farm animals and in humans with direct contact with these animals showing that S. aureus may be a major zoonotic pathogen. Therefore, we aimed to isolate S. aureus from cows, their handlers, and their immediate surroundings, and to investigate the antimicrobial resistance and genetic lineages of the isolates. Mouth and nose swabs of 244 healthy cows (195 Maronesa, 11 Holstein-Friesians, and 28 crossbreeds), 82 farm workers, 53 water and 63 soil samples were collected. Identification of species was carried out by MALDI-TOF MS Biotyper. The presence of antimicrobial resistance genes and virulence factors was assessed based on gene search by PCR. All isolates were typed by multilocus sequence typing and spa-typing. From 442 samples, 33 (13.9%), 24 (29.3%), 1 (2%), and 1 (2%) S. aureus were recovered from cows, farm workers, water, and soil samples, respectively. Most of the isolates showed resistance only to penicillin. S. aureus isolates were ascribed to 17 sequence types (STs) and 26 spa-types. Some clonal lineages were common to both cows and farm workers such as ST30-t9413, ST72-t148, and ST45-t350. Through a One Health approach, this study revealed that there is a great diversity of clonal lineages of S. aureus in cows and their handlers. Furthermore, some S. aureus lineages are common to cows and handlers, which may suggest a possible transmission.

Serbian Traditional Goat Cheese: Physico-Chemical, Sensory, Hygienic and Safety Characteristics.

This research project aimed to investigate the physico-chemical, sensory, hygienic and safety characteristics of raw goat milk, whey, brine and traditional goat cheese during the ripening period of 28 days. Physico-chemical parameters included the determination of dry matter, fat, ash, protein, pH, water activity and NaCl content. The presence of Enterobacteriaceae and fungi was estimated on milk and cheese samples, and a sensory panel evaluated the products' features and acceptability during ripening. The results show that the cheese under study belongs to the acid full-fat cheese group. A consumer panel attributed high scores to the goat cheese, until the 21st day of ripening. After this period, the overall features altered significantly, including augmented bitterness, odor intensification and the development of molds on the surface. The presence of fungi, associated with Enterobacteriaceae, suggests that the hygiene of the production processes needs to be improved. Regarding microbial safety, the detection of putative pathogens and antibiotic resistances recommend an active surveillance of traditional foods to avoid foodborne infections and/or the dissemination of resistant microorganisms along the food chain.

Resistance and virulence distribution in enterococci isolated from broilers reared in two farming systems.

BACKGROUND: The impact of enterococci in human health has been growing for the last decades, mainly due to their resistance to several antimicrobial agents. Human consumption of contaminated meat, especially poultry, has been identified as a possible route of transmission. The aim of the present study was to evaluate and compare the antimicrobial resistance profiles and virulence genes of enterococci isolated from Portuguese conventional and free-range broiler farms. RESULTS: Antibiotic susceptibility testing showed high frequencies of resistance to tetracycline in both farming systems. Resistance to erythromycin and gentamicin were detected in about half of the isolates. Resistance to penicillin was the less frequently observed and no vancomycin resistant isolates were identified. The majority of the enterococcal isolates, from either farming systems, were resistant to more than one antibiotic, and no statistical associations were found, except for penicillin resistance which associated with the genetic clusters. No differences were found between farming systems regarding the prevalence of tet(M), erm(B), aac (6')-Ie-aph (2″)-Ia and pbp5 genes, nevertheless pbp5 prevalence was associated with the different genetic clusters. Hemolytic activity was identified in 26.47% of all isolates and gelatinase activity in 50%. The gelE gene was identified in the majority of the isolates, whereas esp and agg genes were rarely detected. The cylA determinant was not detected in any of the isolates. CONCLUSIONS: Overall, results suggest that similar resistance patterns and virulence genes can be found in both farming systems, though enterococci in free-range conditions should be less prone to acquire further resistance genes.

Antimicrobial Effects of Essential Oils on Oral Microbiota Biofilms: The Toothbrush In Vitro Model.

The present investigation intended to evaluate the bacteriostatic and bactericidal abilities of clove, oregano and thyme essential oils against oral bacteria in planktonic and biofilm states. Furthermore, aiming to mimic everyday conditions, a toothbrush in vitro model was developed. Determination of the minimum inhibitory concentration, minimum bactericidal concentration, minimum biofilm inhibitory concentration and minimum biofilm eradication concentration were achieved using the microdilution procedure. To simulate the toothbrush environment, nylon fibers were inoculated with oral bacteria, which, after incubation to allow biofilm development, were submitted to contact with the essential oils under study. Thyme and oregano essential oils revealed promising antimicrobial effects, both in growth inhibition and the destruction of cells in planktonic and biofilm states, while clove essential oil showed a weaker potential. Regarding the toothbrush in vitro model, observation of the nylon fibers under a magnifying glass proved the essential oil anti-biofilm properties. Considering the effects observed using the in vitro toothbrush model, a realistic approximation to oral biofilm establishment in an everyday use object, a putative application of essential oils as toothbrush sanitizers to help prevent the establishment of bacterial biofilm can be verified.

Occurrence of putative pathogenicity islands in enterococci from distinct species and of differing origins.

Enterococci isolated from ewe's 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).

Antimicrobial activity of essential oils against foodborne multidrug-resistant enterococci and aeromonads in planktonic and biofilm state.

The selection and use of natural compounds with antimicrobial activity against foodborne pathogens is of major importance. The present study evaluated the antimicrobial activity of commercial essential oils against multidrug-resistant Enterococcus spp. and Aeromonas spp. Cymbopogon flexuosus and Thymus vulgaris essential oils presented the highest inhibitory zones against both bacterial groups ( p < 0.05). Subsequent determination of the minimum inhibitory concentrations showed values between 0.47 and 1.9 mg/ml for Aeromonas spp. and from 1.9 to 15 mg/ml for Enterococcus spp. The antimicrobial effect of C. flexuosus and T. vulgaris essential oils was also assessed against biofilms. Bacteria in biofilm state were subjected to 30 min or 1 h of exposure to each essential oil and eradication ability estimated by colony counting. Both essential oils exhibited antimicrobial activity against preformed Aeromonas biofilms, but were unable to successfully eradicate biofilms produced by enterococci, in the conditions under investigation. Nonetheless, the presumptive use of essential oils in the food industry should be considered in further investigations.

What is the Main Processing Factor Influencing Staphylococcus Species Diversity in Different Manufacturing Units?

The microbiota of traditional dry-cured sausages and industrial environment was assessed to characterize the diversity of coagulase-negative staphylococci (CNS), and establish potential relationships with hygiene level or technological characteristics. Eight processing units from South Portugal were audited according to a checklist of requirements. Environmental and products' samples at different production stages were evaluated regarding hygiene and safety criteria. CNS were recovered, characterized, and their potential use as starters evaluated. Low genetic diversity was observed for Staphylococcus xylosus, whereas Staphylococcus equorum showed diverse genetic profiles. Staphylococcus xylosus predominated in products with a long period of cold smoking, Staphylococcus saprophyticus in products with a long period of hot smoking, Staphylococcus epidermidis in products with a short period of cold smoking, and S. equorum in nonsmoked products. Most S. xylosus were resistant to tetracycline, whereas S. equorum were susceptible. Antibioresistance restricted the selection of starters due to safety recommendations. PRACTICAL APPLICATION: The present manuscript highlighted a few staphylococci strains that could potentially be used as starter cultures in fermented meat products. These selected strains do not show resistance to antimicrobials, exhibit adequate technological features, and are well adapted to the industrial environments of meat processing industries using different processing technologies. Therefore, the selected strains ready to be used in the manufacturing of traditional fermented meat products to ensure safety, standardize product properties, and shorten ripening.

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 ß-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.