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It is generally assumed that French fries are likely to have weak in vitro mutagenic activity, but most studies thereof have only assessed gene mutations. In this article, the genotoxicity of 10 extracts of French fries was assessed using the in vitro micronucleus test (following the principles of the OECD 487 guidelines). Each sample was obtained from a different mass catering company in Navarra (Spain). This assay, together with the Ames test, is recommended in the basic in vitro phase included in the European Food Safety Authority Opinion on Genotoxicity Testing Strategies Applicable to Food and Feed Safety Assessment. Eight of 10 samples from mass catering companies induced chromosomal aberrations in the in vitro micronucleus test. Moreover, French fries deep-fried in the laboratory for different periods of time (0, 3, 5, 10, 20, 30 min) were assessed using the in vitro micronucleus test. Genotoxicity was observed in all time periods from 3 min on. The biological relevance of these results must be further explored.

The current work describes the capability of casein-chitosan microparticles to encapsulate Lactobacillus plantarum (CECT 220 and WCFS1 strains) and evaluates their ability to target the distal areas of the gut and to stimulate the immune system. Microparticles were prepared by complex coacervation, between sodium caseinate and chitosan in an aqueous suspension of the bacteria, and dried by spray-drying. In order to increase the survival rate of the loaded bacteria, microparticles were cross-linked with one of the following cross-linkers: tripolyphosphate, calcium salts or vanillin. Overall, microparticles displayed a mean size of about 7.5 μm with a bacteria loading of about 11 Log CFU/g, when cross-linked with vanillin (MP-LP-V). For conventional microparticles, the payload was 10.12 Log CFU/g. The storage stability study at 25 ◦C/60% RH, MP-LP-V offered the highest degree of protection without signif- icant modification of the payload in 260 days. Compared with control (aqueous suspension of bacteria), MP-LP-V also displayed a significantly higher degree of protection against probiotic inactivation in simulated gastric and intestinal fluids. In vivo results evidenced that microparticles, orally administered to rats, were able to reach the distal ileum and colon in about 4 h post-administration. Additionally, the effect of the daily administration of 107 CFU/mouse of MP-LP-V, for 3 weeks, induced an immunomodulatory effect characterized by an important enhancement of Th1 and Th17 responses. In conclusion, these microparticles seem to be a promising strategy for increasing survival and efficacy of probiotics, allowing the formulation of cost-effective and more stable and effective probiotic-based nutraceuticals.

This paper reports the use of the Layer-by-Layer self-assembly (LbL) as an efficient technique for the fabrication of thin-films with antibacterial activity. The LbL coatings are composed of a positive polyelectrolyte such as Poly(allylamine hydrochloride) (PAH) and an anionic polyelectrolyte such as Poly(sodium 4-styrene sulfonate) solution (PSS). In addition, these polyelectrolytes can be also used as an adequate encapsulating agent of specific metal oxide precursors such as titanium dioxide (TiO2) and iron oxide (Fe2O3) nanoparticles, making possible the fabrication of hybrid thin films composed of organic polymeric chains related to the polyelectrolytes and inorganic structure associated to the metal oxide nanoparticles. Four different LbL coatings have been fabricated and a comparative study about the resultant topographical, optical and wettability properties is presented by using light interferometry, atomic force microscopy (AFM), UV-Vis spectroscopy and water contact angle (WCA) measurements. In addition, X-ray fluorescence (XRF) has been also employed in order to corroborate the presence of metal oxide precursors inside the polymeric chains of the polyelectrolytes. Finally, the antibacterial tests have demonstrated that LbL coatings composed of metal oxide nanoparticles produce an enhancement in the efficacy and antibacterial activity.

According to the search for alternatives to replace antibiotics in animal production suggested in the antimicrobial resistance action plans around the world, the objective of this work was to evaluate the bactericidal effect of kaolin–silver nanomaterial for its possible inclusion as an additive in animal feed. The antibacterial activity of the C3 (kaolin–silver nanomaterial) product was tested against a wide spectrum of Gram-negative and Gram-positive bacteria (including multidrug resistant strains) by performing antibiograms, minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC), as well as growth inhibition curves against seven strains causing infections in animals. The C3 product generated inhibition halos in all the tested strains, and a higher activity against Gram-negative bacteria was found, with MBC values ranged from 7.8 µg/mL (P. aeruginosa) to 15.6 µg/mL (E. coli and Salmonella). In contrast, it was necessary to increase the concentration to 31.3 µg/mL or 250 µg/mL to eliminate 99.9% of the initial population of S. aureus ATCC 6538 and E. faecium ATCC 19434, respectively. Conversely, the inhibition growth curves showed a faster bactericidal activity against Gram-negative bacteria (between 2 and 4 h), while it took at least 24 h to observe a reduction in cell viability of S. aureus ATCC 6538. In short, this study shows that the kaolin–silver nanomaterials developed in the framework of the INTERREG POCTEFA EFA183/16/OUTBIOTICS project exhibit antibacterial activity against a wide spectrum of bacteria. However, additional studies on animal safety and environmental impact are necessary to evaluate the effectiveness of the proposed alternative in the context of One Health.

Previous studies on hospital food waste have focused on raising awareness among patients about this problem. The aim of the study was to quantify the food waste in a flexible and inflexible ordering system from a hospital located in the north of Spain in order to implement specific modifications to reduce the waste. The avoidable waste of 15 dishes was determined in the flexible (choice menu) and inflexible (basal diet) ordering system by weighing the avoidable waste from the same dish and diet by conglomerate. Milk, chicken and lunch fish generated more than 25% of plate waste and were classified as critical dishes, with the choice menu being the one that obtained the lowest percentages of waste. The implemented modifications in the case of milk (reducing the serving size) did not decrease the waste percentage. By contrast, the new chicken recipes and the increased fish variety in the inflexible ordering system decreased the plate waste in both dishes from 35.7% to 7.2% and from 29.5% to 12.8%, respectively. Identifying critical dishes, implementing actions to reduce the food waste and monitoring the progress are essential measures to decrease plate waste in hospitals.

Presence of extended-spectrum β-lactamase (ESBL-E), AmpC-producing and carbapenemase-producing (CPE) Enterobacteriaceae has been observed not only in the clinical environment, but also in the out-of-hospital environment. The objective of this study was to isolate and characterize strains of ESBL, AmpC, and CPE present in feces of healthy carriers in Navarra (n = 125). Despite the fact that no CPE strains were isolated, 16% and 11.2% of the studied population were ESBL-E and AmpC carriers, respectively. No significant differences were found by gender or age; young people (5-18 years old) showed the highest ESBL-E prevalence (31.8%). The isolates corresponded to E. coli (57.1%), Enterobacter spp. (28.6%), and Citrobacter freundii (14.3%), and all strains showed multidrug-resistant profiles. High resistance against cephalosporins, penicillins, and monobactams, and sensitivity to carbapenems, quinolones, and aminoglycosides were observed. With respect to ESBL producers, 52.4% were CTX-M-type (19.0% CTX-M-14, 9.5% CTX-M-1, and 28.6% CTX-M-15) and 47.6% were TEM-type (38.1% TEM-171). These results confirm the extensive dissemination of these resistances among a healthy population and pose the need to implement control measures and strategies according to the One Health approach in order to prevent the increase of severe and untreatable infections in a not far future.

The current article aimed to evaluate the in vitro mutagenicity of ten fried meat-based food extracts obtained from different catering companies from Navarra (Spain). A miniaturized 6-well version of the Ames test in Salmonella typhimurium TA98, and the in vitro micronucleus test (OECD TG 487) in TK6 cells were performed. None of the ten extracts of fried meat-based food induced gene mutations in S. typhimurium TA98 with or without metabolic activation, but five induced chromosomal aberrations after 24 h treatment of TK6 without metabolic activation. More studies are needed to check the biological relevance of these in vitro studies.

Deep frying is one of the most used worldwide methods in food preparation, but controlling the oil quality (temperature and formation of polar compounds) is crucial. The main objective of this work was to assess the practices of food handlers with regard to the frying processes in social food services located in Navarra (a region of northern Spain). The study was performed in two phases: in the first one, a self-administrable questionnaire regarding the usual practices on food deep-frying processes was sent to the food services through the main social catering companies of Navarra participating in the study. In the second one, in situ monitoring of the frying practices was performed as verification tools of frying practices reported by food services and to check the oil quality. Almost half of the fryers exceeded the maximum recommended temperature to avoid the formation of toxic compounds (175 ◦C). Despite only one the fryers exceeded the maximum limit of polar compounds established by current Spanish regulation, the obtained values indicated that the oil had begun to degrade in 20% of the fryers. Oil temperature is an important factor that affects the quality of the oil. In addition, significant differences were found between the different frequencies of change or types of oils. We have noticed a lack of knowledge in relation to the risks associated to the bad management of frying oil. Therefore, it would be desirable to improve food handlers training in relation to this matter. Defining a periodic frequency of oil change according to its use and periodic controls of temperature and polar compounds (as part of the Hazard Analysis and Critical Control Point system), could be adequate tools to improve management of frying oil in food services.

The aim of this study was to determine the occurrence and patterns of resistance of extended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae in food products purchased in Navarra, northern Spain. A total of 174 samples of fish and chicken were analyzed from September 2015 to September 2016, including raw and ready-to-eat products: trout (n = 25), salmon (n = 28), panga (n = 13), chicken nuggets and chicken scalopes (n = 32), sushi (n = 31) and sliced cooked poultry (n = 45). Cefpodoxime-resistant strains were isolated on ChromID ESBL agar and further phenotypic (antimicrobial study on MicroScan© NM37 panel) and genotypic characterization (multiplex PCR, sequencing and multi-locus sequence typing, MLST) was performed to confirm and characterize ESBL producers. Raw chicken and sushi have been determined as the most risky products regarding transmission of ESBL-producing Enterobacteriaceae (occurrence 53.1% and 19.4%, respectively), while sliced cooked poultry products appear to be a safe product in this aspect. With regard to raw fish, prevalence in salmon was lower (3.6%) than in trout and panga (16.0%). Ninety-eight per cent of ESBL isolates (n = 50) show multidrug-resistant profiles, highlighting the high resistances against quinolones and tetracyclines observed in chicken isolates, as well as against ertapenem and chloramphenicol in sushi strains. Predominant β-lactamase type was SHV-12 (50.1%), followed by TEM-type (24.5%) and CTX-M (20.8%). In addition, CTX-M type was only detected in chicken products. The phylogenetic study showed the prevalence of groups A (35%), F (25%) and B1 (15%), usually related to nonvirulent strains. MLST E. coli isolates (n = 20) were grouped into 5 clonal complexes (CC) and 15 sequence types (ST), showing high clonal diversity. ST117 was the prevalent sequence type, while the human pathogen ST131 was not detected in this study. The high prevalence of ESBL-producing multidrug-resistant Enterobacteriaceae detected in products of widespread consumption such as chicken and sushi, increases the concern regarding human exposure to superbugs and encourages the need to improve surveillance of this public health issue.

Barley malt and sloe are two raw materials used in the production of beer and pacharan (a Spanish spirit drink), respectively. These organic wastes have been used as substrate of anaerobic digestion to assess the effect of temperature (35 °C and 55 °C) and thermal pretreatment (80 °C for 1.5 h). Viable microbiological isolates have also been identified through metabolic tests and a kinetic model which includes biogas generation and volatile solids (VS) removal has been proposed. Temperature studies on barley malt yielded higher productivities at 55 °C (the ratio of produced biogas volume per VS consumed was 119% higher than that at 35 °C), but similar VS removal (0.9% less elimination at 55 °C). On the other hand, sloe digestion was more effective at 35 °C (biogas/VS-consumed ratio 252% higher than that at 55 °C), while VS elimination can be considered the same. Attending to the effect of thermal pretreatment on productivity, barley malt showed no improvement while sloe was enhanced (144% at 35 °C and 160% at 55 °C). Kinetic modelling fits to the experimental results, showing differences in maximum specific growth rate of biomass according with the temperature conditions. In all cases the adjustment was good (R 2 > 0.96). Microbiological characterization identified bacterial population, both strict and facultative anaerobes.

Prerequisite programs are considered the most efficient tool for a successful implementation of self-control systems to ensure food safety. The main objective of this study was to evaluate the implementation of these programs in 15 catering services located in Navarra and the Basque Country (regions in northern Spain), through on-site audits and microbiological analyses. The implementation of the prerequisite program was incomplete in 60% of the sample. The unobserved temperature control during both the storage and preparation of meals in 20% of the kitchens reveals misunderstanding in the importance of checking these critical control points. A high level of food safety and hygiene (absence of pathogens) was observed in the analyzed meals, while 27.8% of the tested surfaces exceeded the established limit for total mesophilic aerobic microorganisms (≤100 CFU/25 cm²). The group of hand-contact surfaces (oven door handles and aprons) showed the highest level of total mesophilic aerobic microorganisms and Enterobacteriaceae, and the differences observed with respect to the food-contact surfaces (work and distribution utensils) were statistically significant (P < 0.001). With regard to the food workers' hands, lower levels of microorganisms were observed in the handlers wearing gloves (that is, for Staphylococcus spp we identified 43 CFU/cm2 on average compared with 4 CFU/cm2 (P < 0.001) for those not wearing and wearing gloves, respectively). For a proper implementation of the prerequisites, it is necessary to focus on attaining a higher level of supervision of activities and better hygiene training for the food handlers, through specific activities such as informal meetings and theoretical-practical sessions adapted to the characteristics of each establishment.

Mobile genetic elements play an important role in the dissemination of antibiotic resistant bacteria among human and environmental sources. Therefore, the aim of this study was to determine the occurrence and patterns of integrons and insertion sequences of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolated from different sources in Navarra, northern Spain. A total of 150 isolates coming from food products, farms and feeds, aquatic environments, and humans (healthy people and hospital inpatients), were analyzed. PCRs were applied for the study of class 1, 2, and 3 integrons (intI1, intI2, and intI3), as well as for the determination of insertion sequences (IS26, ISEcp1, ISCR1, and IS903). Results show the wide presence and dissemination of intI1 (92%), while intI3 was not detected. It is remarkable, the prevalence of intI2 among food isolates, as well as the co-existence of class 1 and class 2 (8% of isolates). The majority of isolates have two or three IS elements, with the most common being IS26 (99.4%). The genetic pattern IS26–ISEcp1 (related with the pathogen clone ST131) was present in the 22% of isolates (including human isolates). In addition, the combination ISEcp1–IS26–IS903–ISCR1 was detected in 11 isolates being, to our knowledge, the first study that describes this genetic complex. Due to the wide variability observed, no relationship was determined among these mobile genetic elements and β-lactam resistance. More investigations regarding the genetic composition of these elements are needed to understand the role of multiple types of integrons and insertion sequences on the dissemination of antimicrobial resistance genes among different environments.

Mobile genetic elements play an important role in the dissemination of antibiotic resistant bacteria among human and environmental sources. Therefore, the aim of this study was to determine the occurrence and patterns of integrons and insertion sequences of extended-spectrum &beta, lactamase (ESBL)-producing Escherichia coli isolated from different sources in Navarra, northern Spain. A total of 150 isolates coming from food products, farms and feeds, aquatic environments, and humans (healthy people and hospital inpatients), were analyzed. PCRs were applied for the study of class 1, 2, and 3 integrons (intI1, intI2, and intI3), as well as for the determination of insertion sequences (IS26, ISEcp1, ISCR1, and IS903). Results show the wide presence and dissemination of intI1 (92%), while intI3 was not detected. It is remarkable, the prevalence of intI2 among food isolates, as well as the co-existence of class 1 and class 2 (8% of isolates). The majority of isolates have two or three IS elements, with the most common being IS26 (99.4%). The genetic pattern IS26&ndash, ISEcp1 (related with the pathogen clone ST131) was present in the 22% of isolates (including human isolates). In addition, the combination ISEcp1&ndash, IS26&ndash, IS903&ndash, ISCR1 was detected in 11 isolates being, to our knowledge, the first study that describes this genetic complex. Due to the wide variability observed, no relationship was determined among these mobile genetic elements and &beta, lactam resistance. More investigations regarding the genetic composition of these elements are needed to understand the role of multiple types of integrons and insertion sequences on the dissemination of antimicrobial resistance genes among different environments.

Coffee consumption decreases the risk of oxidative stress-related diseases. The by-product obtained after brewing process (spent coffee) also has antioxidant capacity. Spent coffee and coffee brews (filter and espresso) extracts were obtained from Arabica and Robusta coffees, respectively. Spent coffee showed slightly high amounts in chlorogenic acids, but caffeine content was similar to their respective coffee brew. All samples exhibited strong protection activity against indirect acting mutagen 2-AF (≤92%), whereas the protection against NPD (direct mutagen) was 12–35% (Ames Test). The growth inhibition of common food-borne pathogen and food spoilage microorganisms by coffee extracts was also studied. Spent coffee showed antimicrobial activity, mainly against Gram-positive bacteria ( Staphylococcus aureus, Listeria monocytogenes ) and yeast ( Candida albicans ). The role of phenolic acids, caffeine and melanoidins in the antimutagenic and antimicrobial activities is discussed. Thus, spent coffee extracts could be a potential source of bioactive compounds, thereby becoming a promising new functional food ingredient.

This study presents a comprehensive approach of a clonal diversity analysis of 448 Extended-spectrum β-lactamase (ESBL)-producing E. coli isolated from environmental, human and food samples in Spain. The phenotypic confirmation of ESBL production was performed by disc diffusion and microdilution methods, while Polymerase Chain Reaction (PCR) and sequencing were used for the molecular characterization of β-lactamase genes (blaCTX-M, blaSHV, blaTEM, blaOXA). Clonal relationship of isolates was determined by multi-locus sequence typing (MLST). Multidrug resistant strains were present in all the studied niches, with percentages above 50.0%. The most prevalent β-lactamase genes were blaCTXM-14 (26%) and blaCTXM-1 (21.4%), followed by blaSHV-12, blaCTX-M-15 and blaTEM-42. MLST isolates were grouped into 26 clonal complexes (CC) and 177 different sequence types (ST) were detected. Despite the high clonal diversity observed, CC10 was the prevalent and the only CC detected in all niches, while other complexes as CC131 were mainly associated to human isolates. The observed prevalence and diversity of these resistant bacteria across the different environments encourages a One Health approach to prevent and control ESBL dissemination between environment and consumers.

Plasma immersion ion implantation technology has been utilized to enhance the photocatalytic activity of the anatase phase of TiO thin films deposited by cathodic arc evaporation PVD. The main objective of this study is to shift the light absorbance of the titania in order to obtain antibacterial activity under visible light irradiation. TiO thin films, deposited on polished stainless steel AISI 304 and silicon wafers, were implanted with nitrogen ions (N/N ) at 20 kV energy and different temperatures between 250 and 350 °C. The antibacterial activity of nitrogen implanted titania coatings has been monitored for Escherichia coli under visible light irradiation. Additionally ultra violet/visible spectrophotometry tests have been carried out to measure the changes in the light absorbance of the doped films. Further characterization has been performed, including X-ray photoelectron spectroscopy, X-ray diffraction and glow discharge optical emission spectrometry. As a result of Nitrogen implantation, the light absorption peak shifted from ultra violet region (UV-A) to visible wavelength range, which led to an increase of the antibacterial efficacy under visible light irradiation., This work was developed in the framework of the industrial project ACTIONIC (IDI-20140-230-231) supported by the Ministerio de Economía y Competitividad (MINECO), through Centro Desarrollo Tecnológico Industrial (CDTI), and the Government of Navarra through the project DECOBAC.

Proper application of HACCP in catering services involves monitoring decisive critical points. The purpose of this study was to assess food temperatures and surface hygiene control in two catering services in Navarra (Spain) at two different time periods: the first one after implementation of the HACCP system and the second period, after the initial supervision through audits and a specific training session regarding temperatures of products and hygienic conditions of surfaces and equipment because the majority of detected nonconformities were related to these parameters. The recorded temperatures of 650 cooked food products within the first period showed that only 65.1% of the hot dishes had a temperature higher than 65 °C, in accordance with Spanish legislation, and 12.9% of them showed a risky holding temperature (26%) compared to utensils for distribution (12.0%). As the impact of dirty surfaces on the hygienic quality of a finished product will depend on which step was being taken during dish elaboration when equipment or utensil was used, it is suggested that more restrictive limits be established regarding utensils and equipment that are in direct contact with the finished product (≤1 CFU/cm2). Results of the study demonstrate that a specific training session on these items has improved the temperature control of prepared meals and the effectiveness of cleaning and disinfection, essentials for guaranteeing the hygienic quality of prepared foods.

Water environments play an important role in the dissemination of antibiotic-resistant bacteria among humans, animals and agricultural sources. In order to assess the spread of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, we analyzed 279 effluent samples from 21 wastewater treatment plants in Navarra (northern Spain). A total of 185 cefpodoxime-resistant bacteria were isolated on ChromID ESBL agar plates, with high predominance of Escherichia coli among isolated species (73%). ESBL production was determined by different methods, concluding its presence in 86.5% of the isolates by the combination disk test, 75.7% by double-disk synergy test and 73.5% by MicroScan(®) NM37 automated system. PCR and sequencing analysis showed that the predominant β-lactamases (bla) genes were blaCTx-M (67.4%) followed by blaTEM (47%), blaSHV (17.4%) and blaOxA (8.3%); furthermore, two or more β-lactamases genes were found in 34.9% of the isolates. The results demonstrate the high prevalence of ESBL-producing Enterobacteriaceae in effluent water from wastewater treatment plants and confirm the need to optimize current disinfection procedures and to improve management of wastewater in an effort to minimize reservoirs of resistant bacteria. Further studies are needed for examining the presence of these bacteria in other environments and for determining the potential dissemination routes of these resistances as well as their impact on human health.

The aim of this study was to obtain data from refrigerated ready-to-eat seafood products at retail in Spain (young eels, crabstick and smoked salmon), regarding prevalence and levels of Listeria monocytogenes, storage temperatures and the impact of transport conditions (type of bag) on the temperature of the product. The one-year surveillance period was carried out according to the EC Regulation No. 2073/2005, taking 5 units/batch and analyzing 250 samples following ISO 11290-1/A1 and ISO 11290-2/A methodologies. Low prevalence of L. monocytogenes was observed in surimi products, while 4.8% of smoked salmon samples were positive for Listeria with low levels (

Patterns of resistance in β-lactamase-producing Enterobacteriaceae family were investigated in isolates from 141 meat products (beef, poultry and pork) purchased in Spain. The strains that grow in ChromID ESBL agar plates were confirmed using the paired disk diffusion method. Resistance to amoxicillin/clavulanic acid, ceftazidime, ceftriaxone, aztreonam, cefpodoxime, gentamicin, doxycycline, cotrimoxazol, norfloxacin, piperacillin/tazobactam, fosfomycin and cefoxitin were tested following CLSI recommendations. Minimum inhibitory concentrations were determined by the MicroScan® NM37 panel and β-lactamase genes were detected using multiplex PCR and sequencing. Results show poultry as the meat product having the highest prevalence (84%), with Escherichia coli being the predominant bacteria (71.3%). Predominant β-lactamase types were CTX-M (37.8%), followed by CTX-M + TEM combination (20.7%), TEM (17%), SHV (12.2%), TEM + SHV combination (10.9%) and OXA (1.2%). 93.9% of the strains were resistant to one or more β-lactam antibiotics. Results indicate a widespread distribution of ESBL-producing Enterobacteriaceae in meat products, with a high rate of β-lactam resistance and a low rate of AmpC cephalosporinase-producing strains.

A draft-tube spouted bed bioreactor was developed to investigate the microbial degradation of aqueous phenol using a cyclodextrin-based support material. Bacteria from activated sludge were acclimated to phenol in a continuous stirred tank bioreactor, and then immobilized onto the hydrogel particles within the spouted bed bioreactor. Microorganisms obtained under different operating conditions in both bioreactors were isolated and characterized. Batch phenol degradation assays performed on isolated dominant strains showed that Acinetobacter baumannii was the most resistant to phenol. Microbial population distribution in bioreactors was not only affected by phenol concentration, but also by oxygen availability, the system configuration and the presence of intermediates formed during phenol metabolization. A maximum elimination capacity of 2.79 kg-phenol/m3 d was achieved in the spouted bed bioreactor, with Comamonas acidovorans being the dominant strain during high degradation periods.

The evaluation of implementing the HACCP system in contract catering companies and assessment of the knowledge, attitudes and practices of the food handlers were the main objectives of this study. It was conducted in 20 companies throughout Spain, with visual inspection of facilities and food handler activities, as well as the distribution of a self-administrable questionnaire among the 105 participant catering workers. In addition, samples of surfaces and finished dishes were taken from each kitchen for microbiological evaluation. The results obtained from the questionnaires reflect extensive knowledge and correct behaviors among respondents with a middle- or highschool educational level, stability in the same workplace and positions of greater responsibility. However, the observation of hygiene practices revealed systematic incorrect procedures in 60% of the kitchens. Most observed deviations were related to the lack of HACCP training and information, the temperature of finished dishes, the storage areas and proper cleaning and disinfection. In summary, this study confirms the difficulties inherent in catering companies to effectively implement the HACCP system, such as the lack of well-trained personnel, lack of motivation or adhesion to said system on the part of the workers, and the lack of financial and economic resources to address the deficiencies in the facilities. Specific educational programs and grants are need for an adequate implementation of HACCP in this sector.

In order to evaluate the increase of L. monocytogenes concentration during home storage, a low level of the pathogen ( 10 3 CFU/g).

A one dimensional model was developed to assess the risk of listeriosis in Navarra (Spain) due to the consumption of smoked fish (salmon and trout) and sliced cooked ham (vacuum or non-vacuum-packed). Data regarding serving size, proportion of consumers, time of storage, refrigerator temperature, prevalence and counts of Listeria monocytogenes were loaded into the model in order to describe the variability, using Monte Carlo simulation. The final output of the model was the estimation of the annual number of listeriosis cases, the consumption of cooked ham being the responsible for the higher prediction of cases. Different “what if” scenarios have been studied in order to assess the likely impact of strategies in the estimated cases. Temperature storage at 4 °C through the food chain (including storage at home) has been demonstrated to be the best tool to decrease the risk of this serious illness.

The prevalence of Listeria monocytogenes in ready-to-eat products of markets in Northern Spain was studied, being analyzed 783 samples of deli meat products, smoked fish and pâte. RTE smoked fish was the most frequently contaminated food category (25% positive), with high occurrence in some brands (60% of lots positive). Significant differences in prevalence were found in in-store-packaged deli meat products (8.5%) with respect to manufacturer vacuum-packaged presentation (2.7%). Serological and molecular characterization of L. monocytogenes isolates confirmed the persistence of several clusters within manufacturing environments, as the same pulsotype was repeatedly isolated from different lots of the same brand and from different sliced products of the same store. These results reflect the need to improve hygiene and disinfection programs by addressing more accurate cleaning practices and continuous education of food workers.

Prerequisite programs are considered the most efficient tool for a successful implementation of self-control systems to ensure food safety. The main objective of this study was to evaluate the implementation of these programs in 15 catering services located in Navarra and the Basque Country (regions in northern Spain), through on-site audits and microbiological analyses. The implementation of the prerequisite program was incomplete in 60% of the sample. The unobserved temperature control during both the storage and preparation of meals in 20% of the kitchens reveals misunderstanding in the importance of checking these critical control points. A high level of food safety and hygiene (absence of pathogens) was observed in the analyzed meals, while 27.8% of the tested surfaces exceeded the established limit for total mesophilic aerobic microorganisms (≤100 CFU/25 cm²). The group of hand-contact surfaces (oven door handles and aprons) showed the highest level of total mesophilic aerobic microorganisms and Enterobacteriaceae, and the differences observed with respect to the food-contact surfaces (work and distribution utensils) were statistically significant (P0.001). With regard to the food workers' hands, lower levels of microorganisms were observed in the handlers wearing gloves (that is, for Staphylococcus spp we identified 43 CFU/cm2 on average compared with 4 CFU/cm2 (P0.001) for those not wearing and wearing gloves, respectively). For a proper implementation of the prerequisites, it is necessary to focus on attaining a higher level of supervision of activities and better hygiene training for the food handlers, through specific activities such as informal meetings and theoretical-practical sessions adapted to the characteristics of each establishment.

Brucellosis is a highly contagious bacterial zoonosis that affects millions of people worldwide. Brucella is highly infectious, especially when aerosolized. The infection induces severe protracted diseases, which are both debilitating and incapacitating, hence, Brucella melitensis has been considered a potential biological warfare agent. In the battle against Brucella, it is crucial to know its chemical-structure and biochemistry-metabolic characteristics. It is well known that Brucella, as well as many other intracellular bacterial pathogens, has evolved to survive and even proliferate within monocytes and macrophages cells. Depending on the route of entry (complement, Fc, lectin or fibronectin receptors), the fate of the bacteria will vary; it may even segregate from the endocytic route towards the endoplasmic reticulum. This intracellular "non regular" behaviour of Brucella makes treatment difficult. Most antibiotics, although effective in vitro, do not actively pass through cellular membranes, or, once inside, may not reach the discrete intracellular niche where the bacteria is hidden. Therefore, complete eradication of the microorganisms is difficult to achieve, and the incidence of relapses is rather high. Taking these data into consideration, this review will evaluate the past, current and new trends in the control of brucellosis, paying special attention to the drug delivery systems as potential vectors for targeting these intracellular sites where the organisms are located.

The incidence of Listeria monocytogenes in a vegetable processing plant was investigated over a 23-month period. Frozen ready-to-eat vegetable samples, well as the plant environment, were sampled. The molecular subtyping techniques, Random Amplified Polymorphic DNA (RAPD) and Restriction Endonuclease Analyses (REA), were performed to help investigate the origin and routes of Listeria dissemination. The low and sporadic incidence of L. monocytogenes made it impossible to establish an epidemiological sequence in the processing plant, though a case of cross-contamination between tomato and ratatouille was detected. Listeria innocua subtyping, however, allowed us to determine the prevalence of several strains in vegetables, and their presence on machinery samples suggested the possibility of cross-contamination during processing. The low incidence of L. monocytogenes indicated that the risk of listeriosis transmission by vegetable consumption is low. On the other hand, the isolation of the same strain of L. innocua in several surveys pointed out the risk of colonisation on surfaces and machinery. The persistence of Listeria spp. is a cause for concern as can lead to future contamination of vegetables processed in the plant and to a possible increased risk for health. Therefore, periodic controls for the presence of Listeria spp. and a further review of the cleaning and disinfection procedures used in frozen vegetable plants are recommended.

The activities of a short therapeutic regimen with azithromycin and the classic treatment doxycycline with streptomycin were compared and evaluated in mice infected with Brucella melitensis. In a chronic model, starting therapy 31 days after challenge, azithromycin (10 days, 50 mg/kg/day) significantly reduced the infection (2.9 logs, day 48 post-infection). The effectiveness of doxycycline (21 days, 50 mg/kg/12 hourly) was greater than azithromycin (4.1 logs of reduction, day 48 post-infection), and when doxycycline was administered for a period of 45 days, all the animals were bacteriologically cured from day 78. The combination with streptomycin (14 days, 10 mg/kg/day) did not improve the effect of any of the regimens. In an acute model infection, treatments with doxycycline or doxycycline-streptomycin, for a period of 3 days, starting 1 day after lethal challenge, were able to protect all the mice. In contrast, only 50% of the mice treated with azithromycin survived the challenge. In conclusion, although a short oral treatment with azithromycin was able to reduce the infection significantly, it was not able to cure the animals as effectively as the classic regimen with doxycycline administered for a longer period of time.

Fecal pathogen distributions in fresh-cut leafy vegetables are essential to develop suitable sampling plans so as to detect pathogen contaminations. In this study, a typical fresh-cut lettuce process was reproduced at pilot scale with different initial inoculum levels of Salmonella on lettuce (6–7, 4 and 1 log CFU/g). The pathogen was determined in all processed lettuce samples (n ≥ 50) and obtained count data were used to fit different probability distributions. The study showed that Salmonella is homogenously distributed on fresh-cut leafy vegetables as a result of processing (mainly washing) at all contamination levels. Negative binomial and Poisson-lognormal distributions were suitable to describe pathogen distribution at the high and medium levels. coefficient of variation modified ( CV *) indicated no overdispersion (i.e. clustering). Nevertheless, further research will be needed to assess the effect of using disinfectants in washing water on the final distribution pattern of pathogens in processed fresh-cut leafy vegetables.

Controlling Listeria monocytogenes in smoked salmon is still a challenge for food business operators. The reported low levels and uneven distribution of the pathogen make it necessary to apply good sampling plans and analytical procedures in order to detect contaminated batches. However, analyses are expensive and due to current economic situations, usually only one sample per lot is checked instead of the n=5 samples established in Regulation (EC) No 2073/2005. This work proposes the investigation of L. monocytogenes by pooling pre-enriched Half Fraser from several units from a batch (n=6). The results obtained from a preliminary in-house validation (n=51 pools) showed that the performance characteristics (sensitivity and specificity) are very high (>95%). This high reliability, together with reduced costs (nearly half), suggests that the wet pooling test could be a good cost-effective approach for investigating L. monocytogenes in smoked salmon. Additional research is needed before this approach can be used for other food matrices, such as determining the maximum number of units per batch that can be pooled with effective detection of L. monocytogenes.

Liposomes of stable multilamellar type, which previously demonstrated great efficiency in antibiotic transport, were used in this study as transport vehicles of antigenic extracts of Brucella melitensis (HS: complex of lipopolysaccharide/phospholipids/outer membrane proteins). The incorporation of HS into positively charged liposomes produced a protective effect against experimental murine brucellosis when they were administered 1 day before or 2 days after infection, as the number of colony-forming units in the spleen was reduced in relation to the untreated control group (P < 0.01). On the other hand, the use of HS-free or bound in liposomes with negative net charge did not produce a significant effect. Moreover, the incorporation of HS into cationic liposomes eliminated the toxicity of the lipopolysaccharide.

The performance of a draft-tube spouted bed bioreactor (DT-SBB) packed with hydrogel particles for biomass immobilization has been used to treat a phenolic wastewater in continuous mode. The biomass support particles were made of a cyclodextrin-based polymer and then seeded with an acclimated mixed culture. Due to the low density of such particles and also the bioreactor design, the pumping energy required to maintain a moving bed of the resulting bioparticles was very low and a recirculation flow was not necessary. The inlet phenol concentration and the phenol inlet loading rate were the operating variables studied. Phenol removal efficiency was used to monitor the process. The DTSBB showed a high removal capacity (up to 2.8 kg-phenol/md) with a high efficiency (>99%). The removal capacity of the DT-SBB was found to be limited by the availability of dissolved oxygen in periods with high phenol elimination rate. The removal efficiency decreased significantly for higher loading rates (>3.1 kg/md) because of phenol inhibition. The evolution of the distribution of microbial populations was also investigated. A predominance of gram-negative bacteria (especially the genera Pseudomonas and Acinetobacter) was observed during the periods of maximum degradation.

The susceptibility of 440 Listeria monocytogenes strains isolated from food (n=401) and clinical cases (n=39) between 1995 and 2005 was determined by standard agar dilution and E-test methods. Antimicrobial drugs currently used in veterinary and human therapy were tested, and they included penicillin G, ampicillin, cephalothin, gentamicin, chloramphenicol, tetracycline, doxycycline, trimethoprim, erythromycin, and clindamycin. The sensitivity of strains was established using Clinical and Laboratory Standards Institute (formerly NCCLS) breakpoints and MIC50 (the MIC for 50% of the strains) to MIC90 values. In general, isolates were susceptible to the majority of the antimicrobials tested, including beta-lactamics and aminoglycosides, which are normally used in the treatment of listeriosis. Resistance to tetracycline and doxycycline was found in five strains isolated from fresh trout belonging to the same fish farm. Molecular analysis by restriction endonuclease analysis showed a similar profile, suggesting the persistence of a strain well adapted to the presence of tetracycline in the environment of a fish farm, which is frequently used in aquaculture in order to prevent infections of fish.

The presence of Listeria spp. was investigated in a total of 3685 food samples obtained from different industries and markets of Northern Spain in the last 4 years. The samples analyzed include fresh raw products (meat, milk and poultry) and treated products (cooked and cured meats, frozen vegetables and smoked salmon). Occurrence of Listeria spp. varied from 8.1% in soft cheese to 76.3% in raw poultry samples. The highest incidence of L. monocytogenes also occurred in raw poultry (36.1% positive samples). Despite this high incidence of contamination, these kinds of products carry a low risk of listeriosis transmission because of the heat treatment prior to consumption. On the other hand, the ready-to-eat products (RTE) tested in this study showed incidences that could pose serious health problems, taking into account that the storage conditions may allow for rapid growth of the pathogen. It was also found that up to 75.5% of the L. monocytogenes strains isolated in this study belonged to serogroup 1, mainly serotype 1/2a, while the clinical cases observed in Navarra in the same period of time belonged mainly to serotype 4b/4bx.

The presence of Listeria spp. was investigated in 369 samples of cooked meat products and 52 of smoked salmon. Incidences of 17.6% for cooked meat and 38.5% for smoked salmon samples were found. All Listeria monocytogenes isolates (34 from meat products and 16 from smoked salmon) were typed serologically and by random amplified polymorphic DNA (RAPD) typing using primers HLWL74 (5'-ACGTATCTGC-3'), HLWL85 (5'-ACAACTGCTC-3'), and OMP-01 (5'-GTI'GGTGGCT-3'). Strains from cooked meat products were characterized and compared in relation to their origin. The detection of identical strains in products of different type and brand packed on the same date suggested cross-contamination, probably during the slicing process. All L monocytogenes isolates from smoked salmon were indistinguishable by serotyping and RAPD, suggesting that this strain was highly disseminated and adapted to the treatment used for the preservation of this food. RAPD subtypes were analyzed using GelCompar version 4.1 software and the unweighted pair method using arithmetic averages, and six groups with at least 78% similarity were established. Serotyping and RAPD results were in concordance, although RAPD showed a higher discriminatory power with L. monocytogenes isolates from meat products. RAPD is an easy method that could be useful to detect cross-contamination occurring during postprocessing manipulations.

The success of the use of liposomes as drug carriers depends on both their formulation and the method of preparation. We have carried out a series of in vitro studies using different formulations and preparation methods, with the aim of obtaining a type of liposome which is efficient in the treatment of brucellosis. On the basis of results obtained in studies of stability at 37 degrees C in the presence of serum lipoproteins and of the activation of phagocytic cells and antibiotic transport to the interior of monocytes infected with Brucella abortus, we conclude that the most suitable vesicles are positively charged, stable plurilamellar vesicles (phosphatidylcholine, 30% cholesterol, and 10% stearylamine). Gentamicin incorporated into these cationic liposomes completely eliminated all of the intracellular Brucella organisms (4.6 logs), while free gentamicin was capable of reducing the number of intracellular bacteria by only 0.3 log.

Brucella melitensis and B. ovis outer membrane blebs contained a protein displaying a temperature-dependent molecular mass upshift from 25 kDa to 30 kDa. A fraction of the protein tightly bound to LPS did not show the molecular mass upshift which was also blocked by exposure of the protein to Zwittergent 314. The B. melitensis heat-modifiable protein and Escherichia coli OmpA shared antigenic determinants. These data indicate that the Brucella group 3 outer membrane proteins belonged to the OmpA family of proteins.

We monitored the incidence of human listeriosis in Navarre, a region in north of Spain between 1995 and 2005, and carried out the characterisation of Listeria monocytogenes isolates obtained from clinical samples and ready-to-eat products (sliced cooked meat, smoked salmon and liver pate). The active surveillance requesting hospitals to notify all listeriosis cases (n=40) yielded higher incidence rates (average annual rate 0.65/100,000 inhabitants, range 0.18-1.18/100,000 inhabitants) than expected. Pregnant women were the largest group affected (n=13, 32.5% of the cases), with a peak in incidence during the last three years of the study period. From the 40 human cases we obtained 33 Listeria isolates. Serological and molecular characterisation by PFGE identified 20 different pulsotypes, which on three occasions enabled us to link sporadic cases into clusters. Although we could not identify the incriminated food product we found two clinical pulsotypes among smoked salmon and cooked meat isolates. Surveillance of listeriosis in Spain should be improved and coordinated with other European Union Member States in order to better estimate the burden of disease and to prevent foodborne outbreaks.

The detection of Brucella bacteremia by subculture does not always correlate with a positive signal in the BACTEC NR730 nonradiometric system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.). The effect of the inoculum size, pH, sodium polyanetholesulfonate, carbon sources (i-erythritol, sodium pyruvate, monosodium glutamate, D-glucose, and L-alanine), and urea in the release of CO2 was evaluated by using the reference strain Brucella melitensis 16M. In standard NR6 vials with or without blood, inocula 5 to 10 times larger (at least 265 CFU per vial) than those usually found in the blood of patients with brucellosis were necessary to produce a positive growth value (GV) in 4 days or less, and similar results were obtained with vials supplemented with the substrates listed above. GVs were consistently lower in vials with sodium polyanetholesulfonate than in vials without this agent. Vials with no blood inoculated with 265 CFU per vial showed turbidity 1 day before GVs became positive, proving that the major limiting detection factor was the low level of release of CO2 and not an inadequate growth medium. In NR6 vials buffered to pH 6.2, GVs became positive faster and were higher than those in standard vials. NR6 vials at pH 6.2 with 0.3% sodium pyruvate yielded a positive GV in the first day of bacterial turbidity.