The use of biomimetic surfaces to reduce single- and dual-species biofilms of Escherichia coli and Pseudomonas putida.

The ability of bacteria to adhere to and form biofilms on food contact surfaces poses serious challenges, as these may lead to the cross-contamination of food products. Biomimetic topographic surface modifications have been explored to enhance the antifouling performance of materials. In this study, the topography of two plant leaves, Brassica oleracea var. botrytis (cauliflower, CF) and Brassica oleracea capitate (white cabbage, WC), was replicated through wax moulding, and their antibiofilm potential was tested against single- and dual-species biofilms of Escherichia coli and Pseudomonas putida. Biomimetic surfaces exhibited higher roughness values (SaWC = 4.0 ± 1.0 µm and SaCF = 3.3 ± 1.0 µm) than the flat control (SaF = 0.6 ± 0.2 µm), whilst the CF surface demonstrated a lower interfacial free energy (ΔGiwi) than the WC surface (-100.08 mJ m-2 and -71.98 mJ m-2, respectively). The CF and WC surfaces had similar antibiofilm effects against single-species biofilms, achieving cell reductions of approximately 50% and 60% for E. coli and P. putida, respectively, compared to the control. Additionally, the biomimetic surfaces led to reductions of up to 60% in biovolume, 45% in thickness, and 60% in the surface coverage of single-species biofilms. For dual-species biofilms, only the E. coli strain growing on the WC surface exhibited a significant decrease in the cell count. However, confocal microscopy analysis revealed a 60% reduction in the total biovolume and surface coverage of mixed biofilms developed on both biomimetic surfaces. Furthermore, dual-species biofilms were mainly composed of P. putida, which reduced E. coli growth. Altogether, these results demonstrate that the surface properties of CF and WC biomimetic surfaces have the potential for reducing biofilm formation.

Hydroxytyrosol recovery from olive pomace: a simple process using olive mill industrial equipment and membrane technology.

In this work, pilot-scale nanofiltration was used to obtain aqueous solutions rich in hydroxytyrosol and tyrosol from olive oil by-products. A large-scale simple process involving olive mill standard machinery (blender and decanter) was used for the olive pomace pre-treatment with water. The aqueous extract was then directly fed to a nanofiltration unit and concentrated by reverse osmosis. Final concentration factors ranged between 7 and 9 for hydroxytyrosol and between 4 and 7 for tyrosol. The final aqueous solution, obtained as retentate stream of reverse osmosis, was highly concentrated in hydroxytyrosol and tyrosol and their concentrations remained stable over at least 14 months.

Effect of Hydrogen Peroxide on Cyanobacterial Biofilms.

Although a range of disinfecting formulations is commercially available, hydrogen peroxide is one of the safest chemical agents used for disinfection in aquatic environments. However, its effect on cyanobacterial biofilms is poorly investigated. In this work, biofilm formation by two filamentous cyanobacterial strains was evaluated over seven weeks on two surfaces commonly used in marine environments: glass and silicone-based paint (Sil-Ref) under controlled hydrodynamic conditions. After seven weeks, the biofilms were treated with a solution of hydrogen peroxide (H2O2) to assess if disinfection could affect long-term biofilm development. The cyanobacterial biofilms appeared to be tolerant to H2O2 treatment, and two weeks after treatment, the biofilms that developed on glass by one of the strains presented higher biomass amounts than the untreated biofilms. This result emphasizes the need to correctly evaluate the efficiency of disinfection in cyanobacterial biofilms, including assessing the possible consequences of inefficient disinfection on the regrowth of these biofilms.

Development of Antifouling Strategies for Marine Applications.

Marine biofouling is an undeniable challenge for aquatic systems since it is responsible for several environmental and ecological problems and economic losses. Several strategies have been developed to mitigate fouling-related issues in marine environments, including developing marine coatings using nanotechnology and biomimetic models, and incorporating natural compounds, peptides, bacteriophages, or specific enzymes on surfaces. The advantages and limitations of these strategies are discussed in this review, and the development of novel surfaces and coatings is highlighted. The performance of these novel antibiofilm coatings is currently tested by in vitro experiments, which should try to mimic real conditions in the best way, and/or by in situ tests through the immersion of surfaces in marine environments. Both forms present their advantages and limitations, and these factors should be considered when the performance of a novel marine coating requires evaluation and validation. Despite all the advances and improvements against marine biofouling, progress toward an ideal operational strategy has been slow given the increasingly demanding regulatory requirements. Recent developments in self-polishing copolymers and fouling-release coatings have yielded promising results which set the basis for the development of more efficient and eco-friendly antifouling strategies.

Characterization and biofouling potential analysis of two cyanobacterial strains isolated from Cape Verde and Morocco.

Cyanobacteria are new sources of value-added compounds but also ubiquitous and harmful microfoulers on marine biofouling. In this work, the isolation and identification of two cyanobacterial strains isolated from Cape Verde and Morocco, as well as their biofilm-forming ability on glass and Perspex under controlled hydrodynamic conditions, were performed. Phylogenetic analysis revealed that cyanobacterial strains isolated belong to Leptothoe and Jaaginema genera (Leptothoe sp. LEGE 181153 and Jaaginema sp. LEGE 191154). From quantitative and qualitative data of wet weight, chlorophyll a content and biofilm thickness obtained by optical coherence tomography, no significant differences were found in biofilms developed by the same cyanobacterial strain on different surfaces (glass and Perspex). However, the biofilm-forming potential of Leptothoe sp. LEGE 181153 proved to be higher compared with Jaaginema sp. LEGE 191154, particularly at the maturation stage of biofilm development. Three-dimensional biofilm images obtained from confocal laser scanning microscopy showed different patterns between both cyanobacterial strains and also among the two surfaces. Because standard methodologies to evaluate cyanobacterial biofilm formation, as well as two different optical imaging techniques, were used, this work also highlights the possibility of integrating different techniques to evaluate a complex phenomenon like cyanobacterial biofilm development.

Assessment of the Antibiofilm Performance of Chitosan-Based Surfaces in Marine Environments.

Marine biofouling is a natural process often associated with biofilm formation on submerged surfaces, creating a massive economic and ecological burden. Although several antifouling paints have been used to prevent biofouling, growing ecological concerns emphasize the need to develop new and environmentally friendly antifouling approaches such as bio-based coatings. Chitosan (CS) is a natural polymer that has been widely used due to its outstanding biological properties, including non-toxicity and antimicrobial activity. This work aims to produce and characterize poly (lactic acid) (PLA)-CS surfaces with CS of different molecular weight (Mw) at different concentrations for application in marine paints. Loligo opalescens pens, a waste from the fishery industry, were used as a CS source. The antimicrobial activity of the CS and CS-functionalized surfaces was assessed against Cobetia marina, a model proteobacterium for marine biofouling. Results demonstrate that CS targets the bacterial cell membrane, and PLA-CS surfaces were able to reduce the number of culturable cells up to 68% compared to control, with this activity dependent on CS Mw. The antifouling performance was corroborated by Optical Coherence Tomography since PLA-CS surfaces reduced the biofilm thickness by up to 36%, as well as the percentage and size of biofilm empty spaces. Overall, CS coatings showed to be a promising approach to reducing biofouling in marine environments mimicked in this work, contributing to the valorization of fishing waste and encouraging further research on this topic.

Hydrodynamic conditions affect the proteomic profile of marine biofilms formed by filamentous cyanobacterium.

Proteomic studies on cyanobacterial biofilms can be an effective approach to unravel metabolic pathways involved in biofilm formation and, consequently, obtain more efficient biofouling control strategies. Biofilm development by the filamentous cyanobacterium Toxifilum sp. LEGE 06021 was evaluated on different surfaces, glass and perspex, and at two significant shear rates for marine environments (4 s-1 and 40 s-1). Higher biofilm development was observed at 4 s-1. Overall, about 1877 proteins were identified, and differences in proteome were more noticeable between hydrodynamic conditions than those found between surfaces. Twenty Differentially Expressed Proteins (DEPs) were found between 4 s-1 vs. 40 s-1. On glass, some of these DEPs include phage tail proteins, a carotenoid protein, cyanophynase glutathione-dependent formaldehyde dehydrogenase, and the MoaD/ThiS family protein, while on perspex, DEPs include transketolase, dihydroxy-acid dehydratase, iron ABC transporter substrate-binding protein and protein NusG. This study contributes to developing a standardized protocol for proteomic analysis of filamentous cyanobacterial biofilms. This kind of proteomic analysis can also be useful for different research fields, given the broad spectrum of promising secondary metabolites and added-value compounds produced by cyanobacteria, as well as for the development of new antibiofilm strategies.

The Use of 3D Optical Coherence Tomography to Analyze the Architecture of Cyanobacterial Biofilms Formed on a Carbon Nanotube Composite.

The development of environmentally friendly antifouling strategies for marine applications is of paramount importance, and the fabrication of innovative nanocomposite coatings is a promising approach. Moreover, since Optical Coherence Tomography (OCT) is a powerful imaging technique in biofilm science, the improvement of its analytical power is required to better evaluate the biofilm structure under different scenarios. In this study, the effect of carbon nanotube (CNT)-modified surfaces in cyanobacterial biofilm development was assessed over a long-term assay under controlled hydrodynamic conditions. Their impact on the cyanobacterial biofilm architecture was evaluated by novel parameters obtained from three-dimensional (3D) OCT analysis, such as the contour coefficient, total biofilm volume, biovolume, volume of non-connected pores, and the average size of non-connected pores. The results showed that CNTs incorporated into a commercially used epoxy resin (CNT composite) had a higher antifouling effect at the biofilm maturation stage compared to pristine epoxy resin. Along with a delay in biofilm development, a decrease in biofilm wet weight, thickness, and biovolume was also achieved with the CNT composite compared to epoxy resin and glass (control surfaces). Additionally, biofilms developed on the CNT composite were smoother and presented a lower porosity and a strictly packed structure when compared with those formed on the control surfaces. The novel biofilm parameters obtained from 3D OCT imaging are extremely important when evaluating the biofilm architecture and behavior under different scenarios beyond marine applications.

Manufacturing PLA/PCL Blends by Ultrasonic Molding Technology.

Ultrasonic molding (USM) is a good candidate for studying the plasticization of polymer mixtures or other composite materials due to either the little amount of material needed for processing, low waste or the needed low pressure and residence time of the mold. Thus, the novelty of this research is the capability of USM technology to process PLA/PCL blends and their corresponding neat materials, encompassing all the production stages, from raw material to the final specimen. The major findings of the work revealed that the thermal properties of the blends were not affected by the USM process, although the crystallinity degree experienced variations, decreasing for PLA and increasing for PCL, which was attributed to the crystallization rate of each polymer, the high process speed, the short cooling time and the small particle size. The employed ultrasonic energy increased the molecular weight with low variations through the specimen. However, the degradation results aligned with the expected trend of these material blends. Moreover, this study also showed the effect pellet shape and dimensions have over the process parameters, as well as the effect of the blend composition. It can be concluded that USM is a technology suitable to successfully process PLA/PCL blends with the correct determination of process parameter windows.

Unveiling the Antifouling Performance of Different Marine Surfaces and Their Effect on the Development and Structure of Cyanobacterial Biofilms.

Since biofilm formation by microfoulers significantly contributes to the fouling process, it is important to evaluate the performance of marine surfaces to prevent biofilm formation, as well as understand their interactions with microfoulers and how these affect biofilm development and structure. In this study, the long-term performance of five surface materials-glass, perspex, polystyrene, epoxy-coated glass, and a silicone hydrogel coating-in inhibiting biofilm formation by cyanobacteria was evaluated. For this purpose, cyanobacterial biofilms were developed under controlled hydrodynamic conditions typically found in marine environments, and the biofilm cell number, wet weight, chlorophyll a content, and biofilm thickness and structure were assessed after 49 days. In order to obtain more insight into the effect of surface properties on biofilm formation, they were characterized concerning their hydrophobicity and roughness. Results demonstrated that silicone hydrogel surfaces were effective in inhibiting cyanobacterial biofilm formation. In fact, biofilms formed on these surfaces showed a lower number of biofilm cells, chlorophyll a content, biofilm thickness, and percentage and size of biofilm empty spaces compared to remaining surfaces. Additionally, our results demonstrated that the surface properties, together with the features of the fouling microorganisms, have a considerable impact on marine biofouling potential.

Characterization of planktonic and biofilm cells from two filamentous cyanobacteria using a shotgun proteomic approach.

Cyanobacteria promote marine biofouling with significant impacts. A qualitative proteomic analysis, by LC-MS/MS, of planktonic and biofilm cells from two cyanobacteria was performed. Biofilms were formed on glass and perspex at two relevant hydrodynamic conditions for marine environments (average shear rates of 4 s-1 and 40 s-1). For both strains and surfaces, biofilm development was higher at 4 s-1. Biofilm development of Nodosilinea sp. LEGE 06145 was substantially higher than Nodosilinea sp. LEGE 06119, but no significant differences were found between surfaces. Overall, 377 and 301 different proteins were identified for Nodosilinea sp. LEGE 06145 and Nodosilinea sp. LEGE 06119. Differences in protein composition were more noticeable in biofilms formed under different hydrodynamic conditions than in those formed on different surfaces. Ribosomal and photosynthetic proteins were identified in most conditions. The characterization performed gives new insights into how shear rate and surface affect the planktonic to biofilm transition, from a structural and proteomics perspective.

The Relative Importance of Shear Forces and Surface Hydrophobicity on Biofilm Formation by Coccoid Cyanobacteria.

Understanding the conditions affecting cyanobacterial biofilm development is crucial to develop new antibiofouling strategies and decrease the economic and environmental impact of biofilms in marine settings. In this study, we investigated the relative importance of shear forces and surface hydrophobicity on biofilm development by two coccoid cyanobacteria with different biofilm formation capacities. The strong biofilm-forming Synechocystis salina was used along with the weaker biofilm-forming Cyanobium sp. Biofilms were developed in defined hydrodynamic conditions using glass (a model hydrophilic surface) and a polymeric epoxy coating (a hydrophobic surface) as substrates. Biofilms developed in both surfaces at lower shear conditions contained a higher number of cells and presented higher values for wet weight, thickness, and chlorophyll a content. The impact of hydrodynamics on biofilm development was generally stronger than the impact of surface hydrophobicity, but a combined effect of these two parameters strongly affected biofilm formation for the weaker biofilm-producing organism. The antibiofilm performance of the polymeric coating was confirmed at the hydrodynamic conditions prevailing in ports. Shear forces were shown to have a profound impact on biofilm development in marine settings regardless of the fouling capacity of the existing flora and the hydrophobicity of the surface.

Effect of sub-lethal chemical disinfection on the biofilm forming ability, resistance to antibiotics and expression of virulence genes of Salmonella Enteritidis biofilm-surviving cells.

Although disinfection procedures are widely implemented in food environments, bacteria can survive and present increased virulence/resistance. Since little is known about these phenomena regarding biofilms, this study aimed to investigate the effect of chemical disinfection on biofilm-derived cells of Salmonella Enteritidis. Using a reference strain (NCTC 13349) and a food isolate (350), biofilm susceptibility to benzalkonium chloride (BAC), sodium hypochlorite (SH) and hydrogen peroxide (HP) was evaluated and biofilms were exposed to sub-lethal concentrations of each disinfectant. Biofilm-derived cells were characterized for their biofilm forming ability, antibiotic resistance and expression of virulence-associated genes. Except for a few instances, disinfectant exposure did not alter antibiotic susceptibility. However, SH and HP exposure enhanced the biofilm forming ability of Salmonella Enteritidis NCTC 13349. After BAC and HP exposure, biofilm-derived cells presented a down-regulation of rpoS. Exposure to BAC also revealed an up-regulation of invA, avrA and csgD on Salmonella Enteritidis NCTC 13349. The results obtained suggest that biofilm-derived cells that survive disinfection may represent an increased health risk.

Biofilm formation behaviour of marine filamentous cyanobacterial strains in controlled hydrodynamic conditions.

Marine biofouling has severe economic impacts and cyanobacteria play a significant role as early surface colonizers. Despite this fact, cyanobacterial biofilm formation studies in controlled hydrodynamic conditions are scarce. In this work, computational fluid dynamics was used to determine the shear rate field on coupons that were placed inside the wells of agitated 12-well microtiter plates. Biofilm formation by three different cyanobacterial strains was assessed at two different shear rates (4 and 40 s-1 ) which can be found in natural ecosystems and using different surfaces (glass and perspex). Biofilm formation was higher under low shear conditions, and differences obtained between surfaces were not always statistically significant. The hydrodynamic effect was more noticeable during the biofilm maturation phase rather than during initial cell adhesion and optical coherence tomography showed that different shear rates can affect biofilm architecture. This study is particularly relevant given the cosmopolitan distribution of these cyanobacterial strains and the biofouling potential of these organisms.

Prevalence of sarcopenia and associated factors in institutionalised older adult patients.

BACKGROUND & AIMS: Sarcopenia is a syndrome characterised by a progressive and generalised loss of skeletal muscle mass and strength with a risk of adverse outcomes such as physical disability, poor quality of life and death. The main aim of the present study was to establish the prevalence of sarcopenia using EWGSOP-defined criteria in institutionalised older adult patients in long-term care institutions. A secondary purpose was to identify the risk factors that develop Sarcopenia in this population. METHODS: A Multicentre cross-sectional study was conducted in 334 institutionalised older adult patients, where the prevalence of sarcopenia and its relation with certain risk factors were measured. Physical performance was measured by gait speed, muscle strength measured by a handheld dynamometer and skeletal muscle mass measured using bioimpedance analysis. Different variables were collected: body mass index (BMI), diseases documented in the clinical record, the numbers of falls, the level of activity and functional ability. RESULTS: Two hundred eighty five individuals were included. According the EWGSOP algorithm and the cut-off points proposed by Masanes et al. for the Spanish population, 118 (41.4%) participants presented sarcopenia, of which 32 patients (27%) suffered from moderate sarcopenia, 78 patients (66%) were identified as severe sarcopenia patients and only 8 (7%) were classified as sarcopenic obesity. More female residents (96 females (81.4%) vs. 22 males (18.6%), p < 0.0001) tended to be sarcopenic. Patients diagnosed with sarcopenia tended to be more functionally impaired and had a more unfavourable BMI than those who were not sarcopenic (Barthel score 40.93 vs, 49.22, p = 0.0034 and BMI 23.57 vs, 27.61, p < 0.0001). Results from regression analysis indicated that those older than 85 years old (OR 2.495, 95% CI 1.401-4.441), the female gender, (OR 3.215, 95%CI 1.635-6.324) and whose BMI was lower than 22 (OR 5.973, 95% CI 2.932-12.165) appeared to be associated with sarcopenia, whereas the Barthel Index and other factors were not. CONCLUSION: The present study suggests that sarcopenia is highly prevalent in patients living in long-term care institutions, especially in female patients. Our findings support that the muscle mass was negatively associated with poor nutritional status and poor capacity to develop basic activities of daily living that indicates high dependency of these patients and high necessity of care.

Single-Point Incremental Forming of Two Biocompatible Polymers: An Insight into Their Thermal and Structural Properties.

Sheets of polycaprolactone (PCL) and ultra-high molecular weight polyethylene (UHMWPE) were fabricated and shaped by the Single-Point Incremental Forming process (SPIF). The performance of these biocompatible polymers in SPIF was assessed through the variation of four main parameters: the diameter of the forming tool, the spindle speed, the feed rate, and the step size based on a Box⁻Behnken design of experiments of four variables and three levels. The design of experiments allowed us to identify the parameters that most affect the forming of PCL and UHMWPE. The study was completed by means of a deep characterization of the thermal and structural properties of both polymers. These properties were correlated to the performance of the polymers observed in SPIF, and it was found that the polymer chains are oriented as a consequence of the SPIF processing. Moreover, by X-ray diffraction it was proved that polymer chains behave differently on each surface of the fabricated parts, since the chains on the surface in contact with the forming tool are oriented horizontally, while on the opposite surface they are oriented in the vertical direction. The unit cell of UHMWPE is distorted, passing from an orthorhombic cell to a monoclinic due to the slippage between crystallites. This slippage between crystallites was observed in both PCL and UHMWPE, and was identified as an alpha star thermal transition located in the rubbery region between the glass transition and the melting point of each polymer.

Treatment Outcome in Male Gambling Disorder Patients Associated with Alcohol Use.

AIMS: The primary objective of this study was to analyze the association between alcohol consumption and short-term response to treatment (post intervention) in male patients with gambling disorder enrolled in a group cognitive behavioral therapy (CBT) program. METHODS: The sample consisted of 111 male individuals with a diagnosis of Gambling Disorder, with a mean age of 45 years (SD = 12.2). All participants were evaluated by a comprehensive assessment battery and assigned to CBT groups of 10-14 patients attending 16 weekly outpatient sessions lasting 90 min each. RESULTS: The highest mean pre- and post-therapy differences were recorded for the alcohol risk/dependence group on the obsessive/compulsive and anxiety dimensions of the SCL-90-R. As regards the presence of relapses and dropouts over the course of the CBT sessions, the results show a significant association with moderate effect size: patients with risk consumption or alcohol dependence were more likely to present poor treatment outcomes. CONCLUSIONS: Alcohol abuse was frequent in GD, especially in patients with low family income and high accumulated debts. High levels of somatization and high overall psychopathology (measured by the SCL-90-R) were associated with increased risk of alcohol abuse. Alcohol abuse was also associated with poor response to treatment.

Breast Cancer Stem Cell Culture and Enrichment Using Poly(ε-Caprolactone) Scaffolds.

The cancer stem cell (CSC) population displays self-renewal capabilities, resistance to conventional therapies, and a tendency to post-treatment recurrence. Increasing knowledge about CSCs' phenotype and functions is needed to investigate new therapeutic strategies against the CSC population. Here, poly(ε-caprolactone) (PCL), a biocompatible polymer free of toxic dye, has been used to fabricate scaffolds, solid structures suitable for 3D cancer cell culture. It has been reported that scaffold cell culture enhances the CSCs population. A RepRap BCN3D+ printer and 3 mm PCL wire were used to fabricate circular scaffolds. PCL design and fabrication parameters were first determined and then optimized considering several measurable variables of the resulting scaffolds. MCF7 breast carcinoma cell line was used to assess scaffolds adequacy for 3D cell culture. To evaluate CSC enrichment, the Mammosphere Forming Index (MFI) was performed in 2D and 3D MCF7 cultures. Results showed that the 60° scaffolds were more suitable for 3D culture than the 45° and 90° ones. Moreover, 3D culture experiments, in adherent and non-adherent conditions, showed a significant increase in MFI compared to 2D cultures (control). Thus, 3D cell culture with PCL scaffolds could be useful to improve cancer cell culture and enrich the CSCs population.

On the Rule of Mixtures for Predicting Stress-Softening and Residual Strain Effects in Biological Tissues and Biocompatible Materials.

In this work, we use the rule of mixtures to develop an equivalent material model in which the total strain energy density is split into the isotropic part related to the matrix component and the anisotropic energy contribution related to the fiber effects. For the isotropic energy part, we select the amended non-Gaussian strain energy density model, while the energy fiber effects are added by considering the equivalent anisotropic volumetric fraction contribution, as well as the isotropized representation form of the eight-chain energy model that accounts for the material anisotropic effects. Furthermore, our proposed material model uses a phenomenological non-monotonous softening function that predicts stress softening effects and has an energy term, derived from the pseudo-elasticity theory, that accounts for residual strain deformations. The model's theoretical predictions are compared with experimental data collected from human vaginal tissues, mice skin, poly(glycolide-co-caprolactone) (PGC25 3-0) and polypropylene suture materials and tracheal and brain human tissues. In all cases examined here, our equivalent material model closely follows stress-softening and residual strain effects exhibited by experimental data.

[Influence of immigration on tuberculosis transmission patterns in Castellón, Spain (2004-2007)]. / Influencia de la inmigración en los patrones de transmisión de la tuberculosis en Castellón (2004-2007).

BACKGROUND: This study aimed to identify tuberculosis transmission patterns in Castellón in a period of major demographic changes. METHODS: A prospective study of patients with positive culture in the province of Castellon over a 4-year period (2004-2007) was carried out. Cases were described by year and nationality and were compared with those reported to the Department of Public Health. We studied the population with available molecular patterns, identified through restriction fragment length polymorphism (RFLP) and analyzed the variables from patient clusters, based on data collected in surveys of the Department of Health and the Laboratory Management Program. RESULTS: According to data from the Department of Public Health, the overall rate of tuberculosis per 100,000 inhabitants in the province of Castellón was 15.7 in 2004, 19.9 in 2005, 18.2 in 2006 and 17.5 in 2007. In our laboratory, strains were identified from 301 patients, representing 77% (301/390) of reported cases and 94% (301/321) of reported cases with a positive culture. The percentage of tuberculosis among foreigners increased with age, exceeding 50% in 2007. Molecular studies were available in 95% of patients (286); 58% were Spanish and 42% were foreigners, of whom 54% were Romanians. The cluster percentage was 40%, with 30% of mixed clusters. According to conventional contact studies, 85% of patients in clusters had been considered isolated cases. CONCLUSIONS: The increased rate of tuberculosis in Castellón was mainly due to the increasing number of cases among foreigners, mostly Romanians. The availability of molecular studies in all patients with a positive culture allowed us to analyze how and where tuberculosis is transmitted in our province. Forty percent of the patients were grouped into clusters; of these, mixed clusters accounted for one third, indicating the high integration of immigrants in our area.