Factor H-related protein 1 promotes complement-mediated opsonization of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important human opportunistic pathogen responsible for a wide range of infections. The complement system is the main early host defense mechanism to control these infections. P. aeruginosa counteracts complement attack by binding Factor H (FH), a complement regulator that inactivates C3b, preventing the formation of the C3-convertase and complement amplification on the bacterial surface. Factor H-related proteins (FHRs) are a group of plasma proteins evolutionarily related to FH that have been postulated to interfere in this bacterial mechanism of resisting complement. Here, we show that FHR-1 binds to P. aeruginosa via the outer membrane protein OprG in a lipopolysaccharide (LPS) O antigen-dependent manner. Binding assays with purified components or with FHR-1-deficient serum supplemented with FHR-1 show that FHR-1 competes with FH for binding to P. aeruginosa. Blockage of FH binding to C3b deposited on the bacteria reduces FH-mediated cofactor activity of C3b degradation, increasing the opsonization of the bacteria and the formation of the potent chemoattractant C5a. Overall, our findings indicate that FHR-1 is a host factor that promotes complement activation, facilitating clearance of P. aeruginosa by opsonophagocytosis.

Environmental surveillance of Pseudomonas aeruginosa in recreational waters in tourist facilities of the Balearic Islands, Spain (2016-2019).

BACKGROUND: Pseudomonas aeruginosa is a major opportunistic human pathogen commonly connected with recreational water activities. Spain is a tourist destination where most of the establishments have swimming pool. Nevertheless, the prevalence of P. aeruginosa in public swimming pools in our country is unknown. This works aimed to survey the P. aeruginosa presence in tourist Spanish recreational waters. METHOD: Tourist recreational water in hotels in the Balearic Islands were visited for four years (2016-2019). The levels of selected parameters were determined, and their correlation with P. aeruginosa contamination investigated. RESULTS: We evaluated 11,014 samples from 254 facilities. Unacceptable levels of at least one legislated parameter were detected in 30.7% of cases, implicating closure in 15.9%, being P. aeruginosa the leading cause of closure. The prevalence of the pathogen was 14.2%, with lower presence in outer swimming pools. Disinfectant levels influence P. aeruginosa contamination, and bromine-maintained pools were more often contaminated than those treated with chlorine. Prevalence remained constant over the years, although it increased in 2019. CONCLUSIONS: P. aeruginosa prevalence in our recreational waters is similar to other countries, and the contamination rates depend on the installations and type and disinfectant levels. Corrective measures are still needed to improve pathogen control.

Pseudomonas aeruginosa and the Complement System: A Review of the Evasion Strategies.

The increasing emergence of multidrug resistant isolates of P. aeruginosa causes major problems in hospitals worldwide. This concern is particularly significant in bloodstream infections that progress rapidly, with a high number of deaths within the first hours and without time to select the most appropriate treatment. In fact, despite improvements in antimicrobial therapy and hospital care, P. aeruginosa bacteremia remains fatal in about 30% of cases. The complement system is a main defensive mechanism in blood against this pathogen. This system can mark bacteria for phagocytosis or directly lyse it via the insertion of a membrane attack complex in the bacterial membrane. P. aeruginosa exploits different strategies to resist complement attack. In this review for the special issue on "bacterial pathogens associated with bacteriemia", we present an overview of the interactions between P. aeruginosa and the complement components and strategies used by this pathogen to prevent recognition and killing by the complement system. A thorough understanding of these interactions will be critical in order to develop drugs to counteract bacterial evasion mechanisms.

Improvement of the Antibacterial Activity of Phage Lysin-Derived Peptide P87 through Maximization of Physicochemical Properties and Assessment of Its Therapeutic Potential.

Phage lysins are a promising alternative to common antibiotic chemotherapy. However, they have been regarded as less effective against Gram-negative pathogens unless engineered, e.g., by fusing them to antimicrobial peptides (AMPs). AMPs themselves pose an alternative to antibiotics. In this work, AMP P87, previously derived from a phage lysin (Pae87) with a presumed nonenzymatic mode-of-action, was investigated to improve its antibacterial activity. Five modifications were designed to maximize the hydrophobic moment and net charge, producing the modified peptide P88, which was evaluated in terms of bactericidal activity, cytotoxicity, MICs or synergy with antibiotics. P88 had a better bactericidal performance than P87 (an average of 6.0 vs. 1.5 log-killing activity on Pseudomonas aeruginosa strains treated with 10 µM). This did not correlate with a dramatic increase in cytotoxicity as assayed on A549 cell cultures. P88 was active against a range of P. aeruginosa isolates, with no intrinsic resistance factors identified. Synergy with some antibiotics was observed in vitro, in complex media, and in a respiratory infection mouse model. Therefore, P88 can be a new addition to the therapeutic toolbox of alternative antimicrobials against Gram-negative pathogens as a sole therapeutic, a complement to antibiotics, or a part to engineer proteinaceous antimicrobials.

Environmental surveillance of Legionella in tourist facilities of the Balearic Islands, Spain, 2006 to 2010 and 2015 to 2018.

BackgroundLegionnaires' disease is a respiratory illness often associated with hotels and travel. Spain is a major tourist destination and one of the European countries with most cases of Legionnaires' disease , both community- and travel-associated. However, the prevalence of Legionella in tourist facilities is unknown.AimThe present investigation aimed to survey the tourist facilities in the Balearic Islands, Spain, for Legionella prevalence.MethodsWe visited tourist facilities in the Balearic Islands in two different periods (2006-2010 and 2015-2018) and took water samples following national and international guidelines. Legionella was investigated by culture methods following international standards (ISO 11731:1998).ResultsWe evaluated 13,472 samples from 465 facilities. Bacteria of the Legionella genus were detected in 65.4% of the surveyed facilities. Contamination of the facilities was significantly higher during the second decade (54.5 vs 78.6%). The most frequent colonisers were L. pneumophila serogroup 2-14. We detected the pathogen in 15.9% and 6.9% of hot and cold water distribution systems samples, respectively. The Legionella contamination rate in cold water systems samples was higher when free chlorine levels were < 0.2 mg/L and at > 25 °C temperatures, while in the hot water systems samples, the contamination rate was higher at < 50 °C. Of the samples from hot tubs, 10.9% were contaminated.ConclusionLegionella prevalence in hotels in the Balearic Islands was high but the contamination rates depended on the installations. Corrective measures are still needed to improve Legionella control.

Determination of Legionella spp. prevalence in Spanish hotels in five years. Are tourists really at risk?

Legionella spp. is the etiological agent of the serious respiratory pneumonia known as Legionnaires' disease. This respiratory illness is frequently associated with travel and tourist resorts. Spain is an important tourist destination, and one of the top European countries concerning Legionnaires' disease cases, both community and travel associated. Still, the colonization of Legionella in our hotels remains scarce. Here, we surveyed 204 hotels in the Canary Islands, Spain, for five years (2015-2019), to determine the Legionella prevalence. Samples were obtained and analysed following national and international guidelines. We detected the pathogen in 140 of 2,318 samples (6.0%). The water distribution systems (WDS) were more colonized (7.4%) than the whirlpools (4.7%). Contamination levels were minimal (<3 log CFU L-1) in most of the cases, and only 3.6% of samples were highly contaminated minimal (>4 log CFU L-1). We isolated Legionella in 4.3% and 8.5% of cold and hot water distribution systems, respectively. The Legionella prevalence in cold water systems samples was higher when free chlorine levels were below 0.2 mg L-1, whereas in the hot water systems samples, the prevalence was higher at <50 °C. Legionella pneumophila was the most frequently isolated species, being the members of the serogroups 2-14 the most prevalent. The annual distribution showed a colonization pick in June, followed by the winter months. Regarding the geographical distribution, the presence of Legionella was more prevalent in the western islands. Our study concludes that Legionella contamination rates in samples from facilities of the Canary Islands is lower than most of the observed in other European studies. However, corrective measures are still needed to improve Legionella control.

Pseudomonas aeruginosa adaptation in cystic fibrosis patients increases C5a levels and promotes neutrophil recruitment.

Cystic fibrosis (CF) disease is characterized by an intense airway inflammatory response mediated by neutrophils and chronic respiratory infections caused by P. aeruginosa. High levels of the complement component C5a, the strongest neutrophil chemoattractant molecule, are commonly found in the CF lung and have been associated with a worsening of the disease. In this study, we investigated how the isolates from CF patients modulate the levels of C5a and identified the bacterial factors involved. We demonstrated that most isolates from airway chronic infections induce the production and accumulation of C5a, an effect attributable to the loss of C5a cleavage by the exoproteases alkaline protease (AprA) and elastase B (LasB). Furthermore, we found that lack of the bacterial protease-dependent C5a degradation is due to mutations in the master regulator LasR. Thus, complementation of a non-C5a-cleaving CF isolate with a functional wild-type LasR restored its ability to express both proteases, cleave C5a and reduce neutrophil recruitment in vitro. These findings suggest that the non-cleaving C5a phenotype acquired by the LasR variants frequently isolated in CF patients may account for the strong neutrophilia and general neutrophil dysfunction predisposing toward increased inflammation and reduced bacterial clearance described in CF patients.

Use of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry Analysis of Serum Peptidome to Classify and Predict Coronavirus Disease 2019 Severity.

BACKGROUND: Classification and early detection of severe coronavirus disease 2019 (COVID-19) patients is required to establish an effective treatment. We tested the utility of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to classify and predict the severity of COVID-19. METHODS: We used MALDI-TOF MS to analyze the serum peptidome from 72 patients with COVID-19 (training cohort), clinically classified as mild (28), severe (23), and critical (21), and 20 healthy controls. The resulting matrix of peak intensities was used for Machine Learning (ML) approaches to classify and predict COVID-19 severity of 22 independent patients (validation cohort). Finally, we analyzed all sera by liquid chromatography mass spectrometry (LC-MS/MS) to identify the most relevant proteins associated with disease severity. RESULTS: We found a clear variability of the serum peptidome profile depending on COVID-19 severity. Forty-two peaks exhibited a log fold change ≥1 and 17 were significantly different and at least 4-fold more intense in the set of critical patients than in the mild ones. The ML approach classified clinical stable patients according to their severity with 100% accuracy and correctly predicted the evolution of the nonstable patients in all cases. The LC-MS/MS identified 5 proteins that were significantly upregulated in the critical patients. They included the serum amyloid protein A2, which probably yielded the most intense peak detected by MALDI-TOF MS. CONCLUSIONS: We demonstrate the potential of the MALDI-TOF MS as a bench to bedside technology to aid clinicians in their decision making regarding patients with COVID-19.

Brote de gastroenteritis causado por norovirus en un hotel de Menorca, España / Norovirus outbreak causing gastroenteritis in a hotel in Menorca, Spain

OBJETIVO: Determinar el agente responsable de un brote de gastroenteritis ocurrido en un hotel de Menorca en septiembre de 2016. MÉTODOS: Se estudió la epidemiología de los casos y se investigaron muestras ambientales y clínicas para la presencia de microorganismos indicadores y patógenos. RESULTADOS: Se detectaron 151 casos: 123 afectaron a clientes y 28 a personal. Los análisis microbiológicos detectaron la presencia de norovirus genotipo II en heces de pacientes, así como en habitaciones y zonas comunes. El plan de control implementado permitió la erradicación del brote. CONCLUSIONES: Este estudio del brote causado por norovirus del genotipo II demuestra que una rápida actuación es crítica para controlar este tipo de brotes

OBJECTIVES: To establish the agent responsible for a gastroenteritis outbreak in a hotel in Menorca (Spain) in September 2016. METHODS: The study included epidemiological and laboratory analysis. Environmental and stool samples were examined for bacterial and viral pathogens. RESULTS: One hundred and fifty-one cases were detected, 123 among the tourists staying in the hotel and 28 affecting the staff. The presence of genotypeII norovirus was discovered in the microbiological studies of patient's faeces, as well as in the surface samples of rooms and common areas. The control plan implemented allowed for control of the outbreak. CONCLUSIONS: This study on a genotypeII norovirus outbreak reveals the importance of a rapid response for controlling these types of outbreaks

Norovirus outbreak causing gastroenteritis in a hotel in Menorca, Spain. / Brote de gastroenteritis causado por norovirus en un hotel de Menorca, España.

OBJECTIVES: To establish the agent responsible for a gastroenteritis outbreak in a hotel in Menorca (Spain) in September 2016. METHODS: The study included epidemiological and laboratory analysis. Environmental and stool samples were examined for bacterial and viral pathogens. RESULTS: One hundred and fifty-one cases were detected, 123 among the tourists staying in the hotel and 28 affecting the staff. The presence of genotypeII norovirus was discovered in the microbiological studies of patient's faeces, as well as in the surface samples of rooms and common areas. The control plan implemented allowed for control of the outbreak. CONCLUSIONS: This study on a genotypeII norovirus outbreak reveals the importance of a rapid response for controlling these types of outbreaks.

Molecular Analysis of the Contribution of Alkaline Protease A and Elastase B to the Virulence of Pseudomonas aeruginosa Bloodstream Infections.

Pseudomonas aeruginosa is a major cause of nosocomial bloodstream infections. This microorganism secretes two major proteases, alkaline protease A (AprA) and elastase B (LasB). Despite several in vitro studies having demonstrated that both purified proteases cleave a number of components of the immune system, their contribution to P. aeruginosa bloodstream infections in vivo remains poorly investigated. In this study, we used a set of isogenic mutants deficient in AprA, LasB or both to demonstrate that these exoproteases are sufficient to cleave the complement component C3, either soluble or deposited on the bacteria. Nonetheless, exoprotease-deficient mutants were as virulent as the wild-type strain in a murine model of systemic infection, in Caenorhabditis elegans and in Galleria mellonella. Consistently, the effect of the exoproteases on the opsonization of P. aeruginosa by C3 became evident four hours after the initial interaction of the complement with the microorganism and was not crucial to survival in blood. These results indicate that exoproteases AprA and LasB, although conferring the capacity to cleave C3, are not essential for the virulence of P. aeruginosa bloodstream infections.

Efficient management of a norovirus outbreak causing gastroenteritis in two hotels in Spain, 2014 / Control de un brote de gastroenteritis causado por norovirus en 2 hoteles en España, 2014

OBJECTIVES: To determine the aetiological agent causing a gastroenteritis outbreak in two hotels in Majorca (Spain) on August and September 2014. METHODS: An epidemiological study was carried out. Environmental and stool samples were analyzed for different pathogens, including norovirus. RESULTS: Epidemiological analysis detected 79 cases among the tourists hosted in the affected hotels over the period 18th August to 3rd September. They included 52 (attack rate: 6.4%) and 27 (attack rate: 3.0%) cases in hotel A and B, respectively. Seven of the staff members were also affected. Microbiological analyses detected genotype 2 norovirus in patient's stool samples, in rooms and in common areas' surfaces. The specific control plan rapidly implemented at the beginning of the outbreak, and further adapted for norovirus elimination, allowed to control the outbreak. CONCLUSIONS: Our study demonstrates that the outbreak was caused by genotype 2 norovirus, and reflects the importance of a rapid analysis and response for its control

OBJETIVOS: Determinar el agente etiológico responsable del brote de gastroenteritis producido en 2 hoteles de Mallorca (España) en agosto y septiembre de 2014. MÉTODOS: Se realizó un estudio epidemiológico y se analizaron muestras ambientales y de heces para la presencia de patógenos, incluyendo norovirus. RESULTADOS: El análisis epidemiológico detectó 79 casos entre los clientes hospedados en los hoteles desde el 18 de agosto al 3 de septiembre: 52 en el hotel A (tasa de ataque: 6,4%) y 27 en el B (tasa de ataque: 3,0%), así como en 7 miembros del personal. Los análisis microbiológicos detectaron norovirus genotipo 2 en las heces de los pacientes y en las superficies de las habitaciones y zonas comunes. El plan específico implementado rápidamente y adaptado para la eliminación de norovirus permitió el control del brote. CONCLUSIONES: En este brote causado por norovirus del genotipo 2 se refleja la importancia de un análisis y una respuesta rápida para su control

Efficient management of a norovirus outbreak causing gastroenteritis in two hotels in Spain, 2014.

OBJECTIVES: To determine the aetiological agent causing a gastroenteritis outbreak in two hotels in Majorca (Spain) on August and September 2014. METHODS: An epidemiological study was carried out. Environmental and stool samples were analyzed for different pathogens, including norovirus. RESULTS: Epidemiological analysis detected 79 cases among the tourists hosted in the affected hotels over the period 18th August to 3rd September. They included 52 (attack rate: 6.4%) and 27 (attack rate: 3.0%) cases in hotel A and B, respectively. Seven of the staff members were also affected. Microbiological analyses detected genotype 2 norovirus in patient's stool samples, in rooms and in common areas' surfaces. The specific control plan rapidly implemented at the beginning of the outbreak, and further adapted for norovirus elimination, allowed to control the outbreak. CONCLUSIONS: Our study demonstrates that the outbreak was caused by genotype 2 norovirus, and reflects the importance of a rapid analysis and response for its control.

Ultrafast and Ultrasensitive Naked-Eye Detection of Urease-Positive Bacteria with Plasmonic Nanosensors.

Identifying the pathogen responsible for an infection is a requirement in order to personalize antimicrobial treatments. Detecting bacterial enzymes, such as proteases, lipases, and oxidoreductases, is a winning approach for detecting pathogens at the point of care. In this Article, a new method for detecting urease-producing bacteria rapidly and at ultralow concentrations is reported. In this method, longsome bacteriological culture steps are substituted for a 10 min capture procedure with positively charged magnetic beads. The presence of urease-positive bacteria on the particles is then queried with a plasmonic signal generation step that generates blue- or red-colored nanoparticle suspensions upon addition of the enzyme substrate. These colorimetric signals, which can be easily identified by eye, are generated by the NH3-dependent assembly of gold nanoparticles in the presence of bovine serum albumin (BSA). The proposed method can detect Proteus mirabilis with a limit of detection of 101 cells mL-1, with a total assay time of 40 min, even in the presence of a large excess of urease-negative bacteria ( Pseudomonas aeruginosa). Furthermore, it does not require bulky equipment, and it can detect P. mirabilis at clinically relevant concentrations within minutes, making it suitable for detecting urease-positive pathogens at the point of care.

Interplay among Resistance Profiles, High-Risk Clones, and Virulence in the Caenorhabditis elegans Pseudomonas aeruginosa Infection Model.

The increasing prevalence of nosocomial infections produced by multidrug-resistant (MDR) or extensively drug-resistant (XDR) Pseudomonas aeruginosa is frequently linked to widespread international strains designated high-risk clones. In this work, we attempted to decipher the interplay between resistance profiles, high-risk clones, and virulence, testing a large (n = 140) collection of well-characterized P. aeruginosa isolates from different sources (bloodstream infections, nosocomial outbreaks, cystic fibrosis, and the environment) in a Caenorhabditis elegans infection model. Consistent with previous data, we documented a clear inverse correlation between antimicrobial resistance and virulence in the C. elegans model. Indeed, the lowest virulence was linked to XDR profiles, which were typically linked to defined high-risk clones. However, virulence varied broadly depending on the involved high-risk clone; it was high for sequence type 111 (ST111) and ST235 but very low for ST175. The highest virulence of ST235 could be attributed to its exoU+ type III secretion system (TTSS) genotype, which was found to be linked with higher virulence in our C. elegans model. Other markers, such as motility or pigment production, were not essential for virulence in the C. elegans model but seemed to be related with the higher values of the statistical normalized data. In contrast to ST235, the ST175 high-risk clone, which is widespread in Spain and France, seems to be associated with a particularly low virulence in the C. elegans model. Moreover, the previously described G154R AmpR mutation, prevalent in ST175, was found to contribute to the reduced virulence, although it was not the only factor involved. Altogether, our results provide a major step forward for understanding the interplay between P. aeruginosa resistance profiles, high-risk clones, and virulence.

Sensing Mg2+ contributes to the resistance of Pseudomonas aeruginosa to complement-mediated opsonophagocytosis.

Pseudomonas aeruginosa adaptation to survive in the host hinges on its ability to probe the environment and respond appropriately. Rapid adaptation is often mediated by two-component regulatory systems, such as the PhoP/PhoQ system that responds to Mg2+ ion concentration. However, there is limited information about the role of PhoQ in P. aeruginosa bloodstream infections. We used a murine model of systemic infection to test the virulence of a PhoQ-deficient mutant. Mutation of PhoQ impaired the virulence and the ability to cause bacteremia of P. aeruginosa. In the presence of blood concentrations of Mg2+ , a PhoQ mutant bound more C3 and was more susceptible to complement-mediated opsonophagocytosis than the parent strain, suggesting a direct effect of the Mg2+ on the modulation of expression of a bacterial component controlled by the PhoP/PhoQ system. Ligand blot analysis, C3 binding experiments and opsonophagocytosis assays identified this component as the outer membrane protein OprH, expression of which impaired the virulence of P. aeruginosa in a murine model of systemic infection. We demonstrate that expression of PhoQ is essential to detect Mg2+ and reduce the expression of OprH, a previously unrecognized C3 binding molecule that promotes the opsonophagocytosis of P. aeruginosa.

Augmented Reality for Real-Time Detection and Interpretation of Colorimetric Signals Generated by Paper-Based Biosensors.

Colorimetric tests are becoming increasingly popular in point-of-need analyses due to the possibility of detecting the signal with the naked eye, which eliminates the utilization of bulky and costly instruments only available in laboratories. However, colorimetric tests may be interpreted incorrectly by nonspecialists due to disparities in color perception or a lack of training. Here we solve this issue with a method that not only detects colorimetric signals but also interprets them so that the test outcome is understandable for anyone. It consists of an augmented reality (AR) app that uses a camera to detect the colored signals generated by a nanoparticle-based immunoassay, and that yields a warning symbol or message when the concentration of analyte is higher than a certain threshold. The proposed method detected the model analyte mouse IgG with a limit of detection of 0.3 µg mL-1, which was comparable to the limit of detection afforded by classical densitometry performed with a nonportable device. When adapted to the detection of E. coli, the app always yielded a "hazard" warning symbol when the concentration of E. coli in the sample was above the infective dose (106 cfu mL-1 or higher). The proposed method could help nonspecialists make a decision about drinking from a potentially contaminated water source by yielding an unambiguous message that is easily understood by anyone. The widespread availability of smartphones along with the inexpensive paper test that requires no enzymes to generate the signal makes the proposed assay promising for analyses in remote locations and developing countries.

OmpK26, a novel porin associated with carbapenem resistance in Klebsiella pneumoniae.

Clinical isolates of Klebsiella pneumoniae resistant to carbapenems are being isolated with increasing frequency. Loss of the expression of the major nonspecific porins OmpK35/36 is a frequent feature in these isolates. In this study, we looked for porins that could compensate for the loss of the major porins in carbapenem-resistant organisms. Comparison of the outer membrane proteins from two K. pneumoniae clinical isogenic isolates that are susceptible (KpCS-1) and resistant (KpCR-1) to carbapenems revealed the absence of OmpK35/36 and the presence of a new 26-kDa protein in the resistant isolate. An identical result was obtained when another pair of isogenic isolates that are homoresistant (Kpn-3) and heteroresistant (Kpn-17) to carbapenems were compared. Mass spectrometry and DNA sequencing analysis demonstrated that this new protein, designated OmpK26, is a small monomeric oligogalacturonate-specific porin that belongs to the KdgM family of porins. Insertion-duplication mutagenesis of the OmpK26 coding gene, yjhA, in the carbapenem-resistant, porin-deficient isolate KpCR-1 caused the expression of OmpK36 and the reversion to the carbapenem-susceptible phenotype, suggesting that OmpK26 is indispensable for KpCR-1 to lose OmpK36 and become resistant to these antibiotics. Moreover, loss of the major porin and expression of OmpK26 reduced in vitro fitness and attenuated virulence in a murine model of acute systemic infection. Altogether, these results indicate that expression of the oligogalacturonate-specific porin OmpK26 compensates for the absence of OmpK35/36 and allows carbapenem resistance in K. pneumoniae but cannot restore the fitness of the microorganism.

Microbiological levels of randomly selected food contact surfaces in hotels located in Spain during 2007-2009.

The aim of this study was to survey the microbial levels of food contact surfaces in hotels. Microbiological levels of 4611 surfaces (chopping machines, kitchenware, knives, worktops, and cutting boards) from 280 different facilities in Spain were determined in a 3-year period. The contact-plate technique was used throughout the survey. Overall, the mean of the log of total aerobic count cm(-2) was 0.62, better than those reported for child-care and assisted living facilities. Significant differences were detected among different types of surfaces, time of sampling, season, and year. The majority (74%) of food contact surfaces sampled in Spanish hotels was within the recommended standard of <1.3 log CFU cm(-2), and differences depend on several factors. Our results set a representative picture of the actual situation in our resorts and establish the basis for the development of educational programs to improve food handlers' knowledge of foodborne diseases and their transmission via food contact surfaces.

Emetic disease caused by Bacillus cereus after consumption of tuna fish in a beach club.

Several vomiting episodes were reported in December 2007 by the management of a beach club in Calvià. The subsequent case investigation confirmed tuna fish consumption a few hours before onset of emesis in all cases. Microbiological analyses detected high bacterial levels in ready-to-eat fish samples, indicating inappropriate cooking procedures. More important, elevated levels of Bacillus cereus were present both in raw and cooked fish. No other pathogens were detected, indicating B. cereus as the etiological agent. To our knowledge, this is the first case of emetic disease by B. cereus likely to be associated with fish consumption.