Manuela Pintado

Biodiversity and characterization of Staphylococcus species isolated from a small manufacturing dairy plant in Portugal

The level and the diversity of the staphylococcal community occurring in the environment and dairy products of a small manufacturing dairy plant were investigated. Species identification was performed using different molecular methods, viz. Multiplex-PCR, amplified ribosomal DNA restriction analysis (ARDRA), and sodA gene sequencing. The main species encountered corresponded to Staphylococcus equorum (41 isolates, 39.0%), S. saprophyticus (28 isolates, 26.7%) and S. epidermidis (15 isolates, 14.3%). Additionally, low incidence of enterotoxin genes was obtained, with only 9 strains (8.6%) being positive for one or more toxin genes. With regard to antimicrobial resistance, 57.1% of the isolates showed at least resistance against one antibiotic, and 28.6% were multi-resistant, which might accomplish resistance for up to 6 antibiotics simultaneously. These results provided evidence that the presence of Staphylococcus species in dairy environment are mostly represented by S. equorum and S. saprophyticus, and illustrate that carrying antimicrobial resistance genes has become reasonably widespread in cheese and dairy environment.

Study of antimicrobial activity and atomic force microscopy imaging of the action mechanism of cashew tree gum.

The aim of this work was to evaluate the antimicrobial potential of two grades of cashew tree gum (crude and purified) against eight microorganisms and to analyze the mechanism of cashew tree gum antimicrobial action via atomic force microscopy (AFM) imaging. The results indicated strong antimicrobial properties of pure cashew tree gum against all tested microorganisms, except for Candida albicans and Lactobacillus acidophilus. On the other hand crude cashew gum showed antimicrobial activity only against Gram-positive bacteria (MRSA, MSSA, Listeria innocua and Enterococcus faecium). Atomic force microscopy imaging showed that pure cashew tree gum lead to bacterial cell collapse. In conclusion cashew tree gum presented relevant antimicrobial activity against most of the studied bacteria, and the purification of the cashew gum affected its antimicrobial spectrum.

Chitosan mouthwash: Toxicity and in vivo validation

A previous study showed that a chitosan mouthwash would be a valid alternative to current mouthwashes as it demonstrated, in vitro, significantly higher antibiofilm activity than two commercial mouthwashes. As such, the aim of this work was to verify the safety of the developed product and to validate, in vivo, the biological activity ascertained in vitro. Chitosan mouthwash safety was evaluated through Ames, MTT and V79 chromosomal aberration assay while antimicrobial activity was evaluated through in vivo assays. The results showed that the chitosan mouthwash was safe, presenting lower cytotoxicity than a commercial mouthwash, and that it effectively reduced viable counts of Streptococcus spp. and Enterococcus spp. by ca. 5.5 log of CFU. Furthermore, in direct comparison with a commercial mouthwash the chitosan mouthwash possessed significantly higher antimicrobial activity. The conjunction of these results proves that the chitosan mouthwash is a safe, effective, natural alternative to the existent chemical mouthwashes.

A novel direct contact method for the assessment of the antimicrobial activity of dental cements

Dental cements are a crucial part of the odontological treatment, however, due to the hazardous nature and reduced biological efficiency of some of the used materials, newer and safer alternatives are needed, particularly so those possessing higher antimicrobial activity than their traditional counterparts. The evaluation of the antimicrobial properties of solid and semi-solid antimicrobials, such as dental cements and gels, is challenging, particularly due to the low sensitivity of the current methods. Thus, the main aim of this study was the evaluation of the antimicrobial activity of a novel chitosan containing dental cement while simultaneous assessing/validating a new, more efficient, method for the evaluation of the antimicrobial activity of solid and gel like materials. The results obtained showed that the proposed method exhibited a higher sensitivity than the standard 96 well microtiter assay and allowed the determination of bactericidal activity. Additionally, it is interesting to note that the chitosan containing cement, which presented higher antimicrobial activity than the traditional zinc oxide/eugenol mix, was capable of inducing a viable count reduction above 5 log of CFU for all of the studied microorganisms.

Anti-biofilm potential of phenolic acids: the influence of environmental pH and intrinsic physico-chemical properties

Abstract Phenolic acids are a particular group of small phenolic compounds which have exhibited some anti-biofilm activity, although the link between their activity and their intrinsic pH is not clear. Therefore, the present work examined the anti-biofilm activity (inhibition of biomass and metabolic activity) of phenolic acids in relation to the environmental pH, as well as other physico-chemical properties. The results indicate that, while Escherichia coli was not inhibited by the phenolic acids, both methicillin resistant Staphylococcus aureus and methicillin resistant Staphylococcus epidermidis were susceptible to the action of all phenolic acids, with the pH playing a relevant role in the activity: a neutral pH favored MRSE inhibition, while acidic conditions favored MRSA inhibition. Some links between molecular polarity and size were associated only with their potential as metabolic inhibitors, with the overall interactions hinting at a membrane-based mechanism for MRSA and a cytoplasmic effect for MRSE.

Chitosan as an effective inhibitor of multidrug resistant Acinetobacter baumannii

Over the last two decades worldwide levels of antibiotic resistance have risen leading to the appearance of multidrug resistant microorganisms. Acinetobacter baumannii is a known skin pathogen which has emerged as a major cause of nosocomial outbreaks due to its capacity to colonize indwelling medical devices and natural antibiotic resistance. With chitosan being an effective antimicrobial agent against antibiotic resistant microorganisms, the aim of this work was to access its potential as an alternative to traditional antimicrobials in the management of A. baumannii growth. What the results showed was that both chitosan MWs tested were active upon A. baumanniis planktonic and sessile growth. For planktonic growth MICs and MBCs were obtained at relatively low concentrations (0.5-2mg/mL) while for sessile growth chitosan proved to be an effective inhibitor of A. baumanniis adhesion and biofilm formation. Considering these results chitosan shows a high potential for control of A. baumannii infections.

Chapter 8 – Environmental Footprint of Emerging Technologies, Regulatory and Legislative Issues

Abstract Consumers are more demanding, better educated in terms of food quality and nutritional aspects, and forcing producers along with regulatory agencies to search for alternative processing technologies. Some of these technologies like high pressure, pulsed electrical fields, supercritical CO 2 , ultrasound, ozone, or plasma treatment are at industrially use, pilot scale, or even at the edge of application, but the most successful ones at the moment, with already a wide variety of commercial products, are high hydrostatic pressure and pulsed electrical fields. These technologies offer better products, both “natural” in terms of fresh-like flavor and ingredients and safe with extended shelf-life. This tendency leads to the need for a global regulation system that ensures quality of food regardless of country of origin and, at the same time, without compromising safety. In addition to this system, there should be governing bodies that regulate and monitor the enforcement of these food-processing regulations, to avoid regulations that often only apply to countries belonging to specific organizations. Together with food legislation concerns, sustainable food manufacturing and related efficient energy use have also became priorities of the food industries. While conventional preservation processes (e.g., canning, freezing, drying) mainly use thermal energy, more complex processes use mechanical, electromagnetic, electrical, and other forms of energy, which can reduce the energy consumption. Indeed the efficient use of resources in food industry is a critical element for the future generations for sustainable food processing, but the impact of energy requirements in emerging technologies has not been a matter of intense discussion. The objective of this chapter is to provide a concise overview of environmental footprint of emerging technologies, namely high-pressure processing and pulsed electric fields, and their current related food legislation status in various countries.