Cytotoxicity and Antimicrobial Resistance of Aeromonas Strains Isolated from Fresh Produce and Irrigation Water.

The genus Aeromonas has received constant attention in different areas, from aquaculture and veterinary medicine to food safety, where more and more frequent isolates are occurring with increased resistance to antibiotics. The present paper studied the interaction of Aeromonas strains isolated from fresh produce and water with different eukaryotic cell types with the aim of better understanding the cytotoxic capacity of these strains. To study host-cell pathogen interactions in Aeromonas, we used HT-29, Vero, J774A.1, and primary mouse embryonic fibroblasts. These interactions were analyzed by confocal microscopy to determine the cytotoxicity of the strains. We also used Galleria mellonella larvae to test their pathogenicity in this experimental model. Our results demonstrated that two strains showed high cytotoxicity in epithelial cells, fibroblasts, and macrophages. Furthermore, these strains showed high virulence using the G. mellonella model. All strains used in this paper generally showed low levels of resistance to the different families of the antibiotics being tested. These results indicated that some strains of Aeromonas present in vegetables and water pose a potential health hazard, displaying very high in vitro and in vivo virulence. This pathogenic potential, and some recent concerning findings on antimicrobial resistance in Aeromonas, encourage further efforts in examining the precise significance of Aeromonas strains isolated from foods for human consumption.

A multicentre study investigating parameters which influence direct bacterial identification from urine.

Rapid diagnosis is one of the best ways to improve patient management and prognosis as well as to combat the development of bacterial resistance. The aim of this study was to study parameters that impact the achievement of reliable identification using a combination of flow cytometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF-MS).The study was carried out in nine hospitals in Spain and included 1,050 urine samples with bacterial counts of 5x106 bacteria/ml. MALDI-ToF-MS-based identification was performed according to a previously described protocol. Valid identification by direct MALDI-ToF-MS was obtained in 72.8% of samples, in 80.3% of samples found to be positive by culture, 32.2% of contaminated samples, and 19.7% of negative samples. Among the positives samples with a valid identification the concordance at the species level was 97.2%. The parameters related to success of direct identification were: high bacterial count, the presence of Escherichia coli as a pathogen and rod-bacteria morphology provided by flow cytometry. The parameters related to failure were a high epithelial cell (EC) count, a high white blood cell (WBC) count and urine samples obtained from in-patients. In summary, this multicentre study confirms previously published data on the usefulness and accuracy of direct MALDI-ToF-MS-based identification of bacteria from urine samples. It seems important to evaluate not only the bacterial count, but also other parameters, such as EC and WBC counts, bacterial species and morphology, and the health care setting, to decide whether the sample is suitable for direct identification.

Evaluation of the VITEK-MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system for the identification of clinical Corynebacterium species / Evaluación del sistema VITEK-MS de espectrometría de masas MALDI-TOF para la identificación de corinebacterias de importancia clínica

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Acinetobacter baumannii in critically ill patients: Molecular epidemiology, clinical features and predictors of mortality / Acinetobacter baumannii en pacientes críticos: epidemiología molecular, características clínicas y predictores de mortalidad

INTRODUCTION: The main aim of this study was to assess changes in the epidemiology and clinical presentation of Acinetobacter baumannii over a 10-year period, as well as risk factors of mortality in infected patients. METHOD: Prospective, multicentre, hospital-based cohort studies including critically ill patients with A. baumannii isolated from any clinical sample were included. These were divided into a first period ('2000 study') (one month), and a second period ('2010 study') (two months). Molecular typing was performed by REP-PCR, PFGE and MSLT. The primary endpoint was 30-day mortality. RESULTS: In 2000 and 2010, 103 and 108 patients were included, and the incidence of A. baumanniicolonization/infection in the ICU decreased in 2010 (1.23 vs. 4.35 cases/1000 patient-days; p < 0.0001). No differences were found in the colonization rates (44.3 vs. 38.6%) or infected patients (55.7 vs. 61.4%) in both periods. Overall, 30-day mortality was similar in both periods (29.1 vs. 27.8%). The rate of pneumonia increased from 46.2 in 2000 to 64.8% in 2010 (p < 0.001). Performing MSLT, 18 different sequence types (ST) were identified (18 in 2000, 8 in 2010), but ST2 and ST79 were the predominant clones. ST2 isolates in the ICU increased from 53.4% in the year 2000 to 73.8% in 2010 (p = 0.002). In patients with A. baumanniiinfection, the multivariate analysis identified appropriate antimicrobial therapy and ST79 clonal group as protective factors for mortality. CONCLUSIONS: At 10 years of the first analysis, some variations have been observed in the epidemiology of A. baumannii in the ICU, with no changes in mortality. Epidemic ST79 clone seems to be associated with a better prognosis and adequate treatment is crucial in terms of survival

INTRODUCCIÓN: El principal objetivo fue evaluar los cambios en la epidemiología a lo largo de un periodo de 10años, así como la presentación clínica y los factores predictores de mortalidad en los pacientes críticos infectados por Acinetobacter baumannii. MÉTODO: Estudio de cohortes prospectivo y multicéntrico en el que se incluyeron pacientes críticos con A. baumannii aislado de cualquier muestra clínica. Se consideró un primer período («estudio de 2000») (un mes) y un segundo («estudio de 2010») (2 meses). La tipificación molecular se realizó mediante REP-PCR, PFGE y MSLT. La variable resultado primaria fue la mortalidad a los 30días. RESULTADOS: En 2000 y 2010 se incluyeron 103 y 108 pacientes, respectivamente, y la incidencia de colonización/infección por A. baumannii en la UCI disminuyó en 2010 respecto al 2000 (1,23 vs. 4,35 casos/1.000 pacientes-días; p < 0,0001). No se encontraron diferencias en la tasa de colonización (44,3 vs. 38,6%) o infección (55,7 vs. 61,4%) en ambos periodos. En general, la mortalidad a los30 días fue similar en ambos periodos (29,1 vs. 27,8%). La tasa de neumonía aumentó desde el 46,2% en 2000 al 64,8% en 2010 (p < 0,001). Mediante MSLT, se identificaron 18 tipos de secuencias diferentes (ST) (18 en 2000, 8 en 2010), pero ST2 y ST79 fueron los clones predominantes. La identificación de ST2 aumentó en la UCI desde el 53,4% en 2000 al 73,8% en 2010 (p = 0,002). En los pacientes infectados, el tratamiento antimicrobiano apropiado y el grupo clonal ST79 fueron factores protectores de mortalidad en el análisis multivariante. CONCLUSIONES: A los 10 años del primer análisis se han observado algunos cambios en la epidemiología de A. baumannii en la UCI, sin cambios en la mortalidad. El clon ST79 epidémico parece estar asociado con un mejor pronóstico, y el tratamiento adecuado es crucial en términos de supervivencia

Acinetobacter baumannii in critically ill patients: Molecular epidemiology, clinical features and predictors of mortality.

INTRODUCTION: The main aim of this study was to assess changes in the epidemiology and clinical presentation of Acinetobacter baumannii over a 10-year period, as well as risk factors of mortality in infected patients. METHOD: Prospective, multicentre, hospital-based cohort studies including critically ill patients with A. baumannii isolated from any clinical sample were included. These were divided into a first period ("2000 study") (one month), and a second period ("2010 study") (two months). Molecular typing was performed by REP-PCR, PFGE and MSLT. The primary endpoint was 30-day mortality. RESULTS: In 2000 and 2010, 103 and 108 patients were included, and the incidence of A. baumannii colonization/infection in the ICU decreased in 2010 (1.23 vs. 4.35 cases/1000 patient-days; p<0.0001). No differences were found in the colonization rates (44.3 vs. 38.6%) or infected patients (55.7 vs. 61.4%) in both periods. Overall, 30-day mortality was similar in both periods (29.1 vs. 27.8%). The rate of pneumonia increased from 46.2 in 2000 to 64.8% in 2010 (p<0.001). Performing MSLT, 18 different sequence types (ST) were identified (18 in 2000, 8 in 2010), but ST2 and ST79 were the predominant clones. ST2 isolates in the ICU increased from 53.4% in the year 2000 to 73.8% in 2010 (p=0.002). In patients with A. baumannii infection, the multivariate analysis identified appropriate antimicrobial therapy and ST79 clonal group as protective factors for mortality. CONCLUSIONS: At 10 years of the first analysis, some variations have been observed in the epidemiology of A. baumannii in the ICU, with no changes in mortality. Epidemic ST79 clone seems to be associated with a better prognosis and adequate treatment is crucial in terms of survival.

Sternal wound infection caused by Gordonia bronchialis: identification by MALDI-TOF MS.

INTRODUCTION: Gordonia spp. infections are uncommon. However, a few clinical cases have been reported in the literature, particularly those involving immunocompromised hosts. Advanced microbiology diagnosis techniques, such as matrix-assisted laser desorption ionization-time of flight MS (MALDI-TOF MS), have been recently introduced in clinical microbiology laboratories in order to improve microbial identification, resulting in better patient management. CASE PRESENTATION: Here, we present a new clinical case of persistent wound infection caused by Gordonia bronchialis in a 64-year-old woman after a mitral valve replacement, using two MALDI-TOF-based systems for identifying this micro-organism. CONCLUSION: Both MALDI-TOF systems were able to identify Gordonia spp.; thus, providing a useful tool that overcomes the current limitations of phenotypic identification associated with this micro-organism. Although the technique validation deserves additional verification, our study provides guidance about MALDI-TOF as a fast and easy method for Gordonia spp. identification.

Epidemiologic and clinical impact of Acinetobacter baumannii colonization and infection: a reappraisal.

Acinetobacter baumannii is one of the most important antibiotic-resistant nosocomial bacteria. We investigated changes in the clinical and molecular epidemiology of A. baumannii over a 10-year period. We compared the data from 2 prospective multicenter cohort studies in Spain, one performed in 2000 (183 patients) and one in 2010 (246 patients), which included consecutive patients infected or colonized by A. baumannii. Molecular typing was performed by repetitive extragenic palindromic polymerase chain reaction (REP-PCR), pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). The incidence density of A. baumannii colonization or infection increased significantly from 0.14 in 2000 to 0.52 in 2010 in medical services (p < 0.001). The number of non-nosocomial health care-associated cases increased from 1.2% to 14.2%, respectively (p < 0.001). Previous exposure to carbapenems increased in 2010 (16.9% in 2000 vs 27.3% in 2010, p = 0.03). The drugs most frequently used for definitive treatment of patients with infections were carbapenems in 2000 (45%) and colistin in 2010 (50.3%). There was molecular-typing evidence of an increase in the frequency of A. baumannii acquisition in non-intensive care unit wards in 2010 (7.6% in 2000 vs 19.2% in 2010, p = 0.01). By MSLT, the ST2 clonal group predominated and increased in 2010. This epidemic clonal group was more frequently resistant to imipenem and was associated with an increased risk of sepsis, although not with severe sepsis or mortality. Some significant changes were noted in the epidemiology of A. baumannii, which is increasingly affecting patients admitted to conventional wards and is also the cause of non-nosocomial health care-associated infections. Epidemic clones seem to combine antimicrobial resistance and the ability to spread, while maintaining their clinical virulence.

Phenotypic and molecular characterization of Arcanobacterium haemolyticum isolated from clinical samples.

The aim of this study was to assess the phenotypic and genotypic diversity of 56 Arcanobacterium haemolyticum isolates isolated from 51 patients attending primary health care centres and emergency units in the health area of Santander (Cantabria, northern Spain). Phenotypic characterization was based on morphological, biochemical, and antigenic tests. Species identification was confirmed by amplification and sequencing of the 16S rDNA gene. Antimicrobial susceptibility testing was determined by microdilution following the Clinical and Laboratory Standards Institute recommendations for coryneform bacteria. Genetic diversity was evaluated using BOX-PCR and pulsed-field gel electrophoresis. Eighty percent of the isolates had an identical BOX-PCR pattern, suggesting the spread of a single clone. The present report provides extensive information on the phenotypic and genotypic characterization of A. haemolyticum.

Comparison of different culture media and growth conditions for recognition of Arcanobacterium haemolyticum.

Detection of Arcanobacterium haemolyticum is based upon typical beta-hemolysis and colony morphology, but it may go undetected if only conventional sheep blood agar media for detection of beta-hemolytic streptococci are used. The influence of different culture media, atmospheres, and times of incubation for the recognition of colonies of 47 isolates of A. haemolyticum was studied. After 48 h of incubation, trypticase soy agar with 5% horse blood in 5% CO(2) was the best medium.

[Study of celiac disease in the pediatric population of Cantabria (Spain) and first-degree relatives]. / Estudio de la enfermedad celíaca en la población pediátrica de Cantabria y sus familiares de primer grado.

BACKGROUND: Celiac disease (CD) is an autoimmune disease that affects genetically predisposed individuals. The HLA-DQ2 heterodimer is present in nearly 90% of patients while HLA-DQ8 is found in the remaining 10%. AIM: To study the characteristics of CD in pediatric patients in Cantabria and their first-degree relatives, with special emphasis on factors related to haplotype, serology, and forms of clinical presentation. PATIENTS AND METHODS: Eighty-six patients with CD and 215 first-degree relatives were HLA genotyped. Clinical, laboratory, immunologic, and histological data were obtained from all patients. RESULTS: Clinical presentation was classical in 95% of the patients and mono-symptomatic in the remaining 5%. Anti-gliadin antibodies (AGA) and anti-transglutaminase antibodies (ATGA) were positive in 95% of the patients and negative in 5% (all with IgA deficiency). DQ2 was found in 71% of the patients (homozygotes or heterozygotes) and DQ8 was found in 9.5%. No heterodimers of risk were found in 22%. CD was found in six relatives (three were positive for AGA and four were positive for ATGA). Forty-nine percent of the relatives carried the DQ2 heterodimer and 15% the DQ8 heterodimer; no heterodimers of risk were found in 40%. CONCLUSIONS: The most prevalent HLA found in patients with CD in the autonomous region of Cantabria was DQ2 (71%). This prevalence is clearly lower than that reported in other Spanish regions. The prevalence of CD among first-degree relatives was similar to that found in other studies performed in Spain (2.8%). Our data support the need for systematic study of the first-degree relatives of patients with CD.

Estudio de la enfermedad celíaca en la población pediátrica de Cantabria y sus familiares de primer grado / Study of celiac disease in the pediatric population of Cantabria (Spain) and first-degree relatives

Introducción: La enfermedad celíaca (EC) es una entidad mediada por fenómenos autoinmunes, que se presenta en sujetos susceptibles genéticamente. El 90% de los pacientes presenta el heterodímero HLA-DQ2, y el 10% restante suele presentar el HLA-DQ8. Objetivo: Estudiar las características de la EC en la población pediátrica de Cantabria y en sus familiares de primer grado, fundamentalmente en los aspectos relacionados con el haplotipo, la serología y sus formas de presentación clí nica. Pacientes y métodos: Estudio de 86 pacientes celíacos y 215 familiares de primer grado. Se recogieron datos clínicos, analíticos, inmunológicos, histológicos y de tipificación genómica. Resultados: El 95% de los caso se iniciaron con clínica clásica y el 5% eran formas monosintomáticas. Un 95% presentaba positividad a anticuerpos antigliadina (AAG) y antitransglutaminasa (AATG), y eran negativos el 5% (todos con déficit de IgA). Genotípicamente, un 71% eran portadores del DQ2 (incluidos los homocigotos y los heterocigotos), y un 9,5% del DQ8. Un 22% no presentaba heterodímero de riesgo. En el estudio familiar se hallaron 6 familiares con EC (3 AAG positivos y 4 AATG positivos). Del total, el 49% de los familiares portaba el DQ2, un 15% el DQ8, y un 40% no presentaba el heterodímero de riesgo. Conclusiones: El HLA más prevalente en nuestra comunidad fue el DQ2 (71%), claramente menor que lo publicado en nuestro medio. La prevalencia de EC en familiares de primer grado fue similar al resto de España (2,8%). Nuestros datos apoyan la necesidad del estudio sistemático en familiares de primer grado de pacientes celíacos

Background: Celiac disease (CD) is an autoimmune disease that affects genetically predisposed individuals. The HLA-DQ2 heterodimer is present in nearly 90% of patients while HLA-DQ8 is found in the remaining 10%. Aim: To study the characteristics of CD in pediatric patients in Cantabria and their first-degree relatives, with special emphasis on factors related to haplotype, serology, and forms of clinical presentation. Patients and methods: Eighty-six patients with CD and 215 first-degree relatives were HLA genotyped. Clinical, laboratory, immunologic, and histological data were obtained from all patients. Results: Clinical presentation was classical in 95% of the patients and mono-symptomatic in the remaining 5%. Anti-gliadin antibodies (AGA) and anti-transglutaminase antibodies (ATGA) were positive in 95% of the patients and negative in 5% (all with IgA deficiency). DQ2 was found in 71% of the patients (homozygotes or heterozygotes) and DQ8 was found in 9.5%. No heterodimers of risk were found in 22%. CD was found in six relatives (three were positive for AGA and four were positive for ATGA). Forty-nine percent of the relatives carried the DQ2 heterodimer and 15% the DQ8 heterodimer; no heterodimers of risk were found in 40%. Conclusions: The most prevalent HLA found in patients with CD in the autonomous region of Cantabria was DQ2 (71%). This prevalence is clearly lower than that reported in other Spanish regions. The prevalence of CD among first-degree relatives was similar to that found in other studies performed in Spain (2.8%). Our data support the need for systematic study of the first-degree relatives of patients with CD

Association of migraine and ESR1 G325C polymorphism.

Migraine is a genetically complex disorder in which sexual hormones influence the phenotype. ESR1 G594A polymorphism has been associated with migraine in Australians. We performed a case-control study with G594A and G325C polymorphisms to determine whether ESR1 is associated with migraine in our population. An association between G594A and migraine could not be demonstrated here. By contrast, we observed that the C325 allele conferred a 1.6 (95% confidence interval=1.1-2.4) higher risk for suffering from migraine in women than the G allele. Women carrying the C352C genotype were over 3 times more likely to suffer from migraine than those carrying the G325G genotype. Therefore, we conclude that ESR1 G325C polymorphism is associated with migraine in our population.

Sensitivity to bee venom antigen phospholipase A2: association with specific HLA class I and class II alleles and haplotypes in beekeepers and allergic patients.

Bee venom hypersensitivity is a clinical entity of outstanding importance because bee stings are a leading cause of mortality worldwide. Individuals with immediate-type bee venom hypersensitivity, beekeepers, and healthy controls were examined for HLA-DRB1, DQB1, and DQA1 alleles by sequence-specific oligonucleotide probe typing. Defined hypersensitivity to bee venom antigen phospholipase A2 (vbPLA2) is significantly associated with the presence of susceptible HLA class II alleles: DRB1*0101 (RR = 2.7, p < 3 x 10(-3)), DRB1*0103 (RR = 21.2, p < 7.5 x 10(-11)), DQA1*0101 (RR = 1.2, p < 38.52 x 10(-10)), and DQB1*0501 (RR = 4, p < 2.18 x 10(-10)). Some HLA class I alleles were also associated with risk to bee venom allergy: A*01 (RR = 2.4, p < 7.5 x 10(-4)), B*57 (RR = 35.1, p < 3.5 x 10(-7)), and B*5901 (p < 3.5 x 10(-5)), but they are probably of secondary significance. Three- (DRB1*0103-DQA1*0101-DQB1*0501) (RR = 21.24, p < 7.5 x 10(-11)) and five-loci (A*01-B*59-DRB1*0103-DQA1*0101-DQB1*0501) (p < 2.3 x 10(-6)) extended haplotypes are also significantly carried by vbPLA2 allergic patients. When HLA allele frequencies from patients are compared with those from beekeepers, only HLA-DRB1*0103 (RR = 11.7, p < 8.5 x 10(-5)) and HLA-DQA1*0101 (p < 0.02) were significantly increased in the former. These observations emphasize the importance of the DRB1*0103-DQA1*0101-DQB1*0501 haplotype as a strong candidate for susceptibility to vbPLA2 hypersensitivity, at least in our region.