Presence of extended-spectrum beta-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.

One of the main public health problems nowadays is the increase of antimicrobial resistance, both in the hospital environment and outside it (animal environment, food and aquatic ecosystems, among others). It is necessary to investigate the virulence-associated factors and the ability of horizontal gene transfer among bacteria for a better understanding of the pathogenicity and the mechanisms of dissemination of resistant bacteria. Therefore, the objective of this work was to detect several virulence factors genes (fimA, papC, papG III, cnf1, hlyA and aer) and to determine the conjugative capacity in a wide collection of extended-spectrum beta-lactamases-producing E. coli isolated from different sources (human, food, farms, rivers, and wastewater treatment plants). Regarding virulence genes, fimA, papC, and aer were distributed throughout all the studied environments, papG III was mostly related to clinical strains and wastewater is a route of dissemination for cnf1 and hlyA. Strains isolated from aquatic environments showed an average conjugation frequencies of 1.15 x 10(-1) +/- 5 x 10(-1), being significantly higher than those observed in strains isolated from farms and food (p < 0.05), with frequencies of 1.53 x 10(-4) +/- 2.85 x 10(-4) and 9.61 x 10(-4) +/- 1.96 x 10(-3), respectively. The reported data suggest the importance that the aquatic environment (especially WWTPs) acquires for the exchange of genes and the dispersion of resistance. Therefore, specific surveillance programs of AMR indicators in wastewaters from animal or human origin are needed, in order to apply sanitation measures to reduce the burden of resistant bacteria arriving to risky environments as WWTPs.

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 degrees C and 55 degrees C) and thermal pretreatment (80 degrees 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 degrees C (the ratio of produced biogas volume per VS consumed was 119% higher than that at 35 degrees C), but similar VS removal (0.9% less elimination at 55 degrees C). On the other hand, sloe digestion was more effective at 35 degrees C (biogas/VS-consumed ratio 252% higher than that at 55 degrees 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 degrees C and 160% at 55 degrees 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.

The aim of this study was to determine the occurrence and patterns of resistance of extended-spectrum beta-plactamase (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 ChromlD ESBL agar and further phenotypic (antimicrobial study on MicroScan(C) 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 13-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. MIST E. coil 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.

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 -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 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.

Plasma immersion ion implantation technology has been utilized to enhance the photocatalytic activity of the anatase phase of TiO2 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. TiO2 thin films, deposited on polished stainless steel AISI 304 and silicon wafers, were implanted with nitrogen ions (N+/N-2(+)) at 20 kV energy and different temperatures between 250 and 350 degrees 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. (C) 2016 Elsevier B.V. All rights reserved.

This study presents a comprehensive approach of a clonal diversity analysis of 448 Extended-spectrum beta-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 beta-lactamase genes (bla(CTX-M), bla(SHV), bla(TEM), bla(OXA)). 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 beta-lactamase genes were bla(CTXM-14) (26%) and bla(CTXM-1) (21.4%), followed by bla(SHV-12), bla(CTX-M-15) and bla(TEM-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.

The aim of this study was to assess the routine food safety procedures carried out in catering services so as to provide a useful monitoring program for managers based on the relevant deviations detected in the observational survey and activities carried out in 15 catering services located in Navarra (region of northern Spain). The main hygiene standards, food handler practices and compliance with the HACCP system were evaluated by means of visual inspections (checklist), monitoring of critical control points (food storage and food processing temperatures, total polar compounds) and microbiological analysis of food-contact and hand-contact surfaces. While general high scores were obtained in the observational survey (73.3% above 36 points), none of the evaluated services had obtained the maximum rate of 48 points. The mayor deficiencies were related to the presence of dirty uniforms, improper hand washing or use of gloves, incorrect disinfection of vegetables and the missing records of stated critical points (temperatures and polar compounds in oil). The performed monitoring activities confirmed the safety of frying oil in all the kitchens, despite the fact that only 33% of them applied scientific methods to measure polar compounds. By contrast, holding temperatures were unsatisfactory in 77% of cold dishes and in 19% of hot meals. Despite the fact that 77% of food contact surfaces were considered acceptable (<4 CFU/cm2), high levels of total mesophilic microorganisms and Enterobacteriaceae were detected in hand-contact surfaces such as aprons and handles. The periodic visual supervision focusing on hygienic practices and the microbiological surveillance of surfaces of high risk for cross-contamination should provide valuable information for improving the knowledge, attitudes and practices of food handlers regarding food safety.

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.

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.

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 (<55 °C). However, the percentage of noncomplying dishes was reduced by a half after the training session (p < 0.001). Since the significant differences observed in recorded temperatures were related to the type of meal (with or without sauces) and the type of cooking procedure, a lower safe criterion for the retention of hot dishes was suggested if the temperature is continuously maintained over 55 °C until serving. With regard to cleaning and disinfection, 18.3% of the 600 analyzed surfaces did not meet the established cleaning criterion (¿100 CFU/25 cm2) in the first period, while in the second period this percentage was reduced to 13.6% in both catering businesses (p = 0.021). The dirtiest surfaces were equipment such as cutting boards and meat slicing machines (>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 ß-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 ß-lactamases (bla) genes were blaCTx-M (67.4%) followed by blaTEM (47%), blaSHV (17.4%) and blaOxA (8.3%); furthermore, two or more ß-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.

Controlling Listeria monocytogenes in smoked salmon is still a challenge for food business operators. The reported lowlevels and uneven distribution of the pathogenmake 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 (N95%). 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 foodmatrices, such as determining the maximumnumber of units per batch that can be pooled with effective detection of L. monocytogenes.

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.

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 (<10 cfu/g) and uneven pathogen distribution. A single company was responsible for 80% of the positive lots. All purchased products showed values higher than 4 C at retail and an average increase of 2.5 C or up to 6.2 C was recorded when isothermal or plastic shopping bags were used for transport, respectively. To avoid noncompliance of the Food Safety Objective for L. monocytogenes in seafood RTE products more efforts from all stakeholders are needed, with special attention so as to improve control and maintenance of refrigerators at retail and to enhance consumer education regarding food safety practices.

Patterns of resistance in ß-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 ß-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 ß-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 ß-lactam antibiotics. Results indicate a widespread distribution of ESBL-producing Enterobacteriaceae in meat products, with a high rate of ß-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/m(3) 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 high-school 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.

The catering business has increased in a extended way in recent years. Many of foodborne outbreaks have been originated in this sector, and therefore government agencies have reviewed current legislation establishing new systems to ensure food safety. The European Regulation 852/2004 and Real Decreto 3484/2000 in Spain, provide requirements for food enterprises to apply self-control systems, following the principles of the Hazard Analysis and Critical Control Points (HACCP). In contrast with other sectors of food industry, catering companies have found several difficulties for the implementation and application of HACCP. Among the difficulties, the figure of the food handler is one of the most important, because it has been reported deficiencies in knowledge, attitudes and behaviour, observing incorrect hygienic practices and lack of interest in food safety. Therefore, the subject of this review is to analyze the current status of HACCP in the catering business and to propose an action plan to improve food safety management.

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.

In order to evaluate the increase of L. monocytogenes concentration during home storage, a low level of the pathogen (<10 CFU/g) was inoculated in sliced ham. Two storage temperatures (5 °C and 9 °C) were selected from a previous study in 33 home refrigerators. Three days of storage or less were necessary to reach the regulated limited concentration (100 CFU/g), at both temperatures. When storage time was extended to 5 days, the pathogen achieved risky values (>103 CFU/g).