Benito Regueiro

Potential clinical utility of polymerase chain reaction in microbiological testing for sepsis

Objectives: To evaluate the potential improvement of antimicrobial treatment by utilizing a new multiplex polymerase chain reaction (PCR) assay that identifies sepsis-relevant microorganisms in blood. Design: Prospective, observational international multicentered trial. Setting: University hospitals in Germany (n = 2), Spain (n = 1), and the United States (n = 1), and one Italian tertiary general hospital. Patients: 436 sepsis patients with 467 episodes of antimicrobial treatment. Methods: Whole blood for PCR and blood culture (BC) analysis was sampled independently for each episode. The potential impact of reporting microorganisms by PCR on adequacy and timeliness of antimicrobial therapy was analyzed. The number of gainable days on early adequate antimicrobial treatment attributable to PCR findings was assessed. Measurements and Main Results: Sepsis criteria, days on antimicrobial therapy, antimicrobial substances administered, and microorganisms identified by PCR and BC susceptibility tests. Results: BC diagnosed 117 clinically relevant microorganisms; PCR identified 154. Ninety-nine episodes were BC positive (BC+); 131 episodes were PCR positive (PCR+). Overall, 127.8 days of clinically inadequate empirical antibiotic treatment in the 99 BC+ episodes were observed. Utilization of PCR-aided diagnostics calculates to a potential reduction of 106.5 clinically inadequate treatment days. The ratio of gainable early adequate treatment days to number of PCR tests done is 22.8 days/100 tests overall (confidence interval 15–31) and 36.4 days/100 tests in the intensive care and surgical ward populations (confidence interval 22–51). Conclusions: Rapid PCR identification of microorganisms may contribute to a reduction of early inadequate antibiotic treatment in sepsis.

Production of toxins by Escherichia coli strains isolated from calves with diarrhoea in Galicia (North-western Spain)

A total of 289 Escherichia coli colonies isolated from 78 diarrhoeic calves were studied for production of heat-labile (LT) and heat-stable (STa) enterotoxins, verotoxin (VT), cytotoxic necrotizing factor (CNF) and K99 antigen, and they were serotyped. Production of STa was detected in a single strain possessing both K99 and F41 antigens; the serotype was 09:K (A) 35. LT-producing strains were not detected. From 16 (20.5%) calves, 51 VT-producing colonies of E. coli were isolated. Production of the necrotic factor was detected in 33 E. coli colonies isolated from 14 (17.9%) calves. Serotype was a useful marker for production of VT and CNF. Among the 51 VT-producing colonies, 24 were untypable and the remainder belonged to serotypes O2:K?, O103:K--, O104:K?, O128:K?, O153:K-- and O157:K--:H7. Four of the 33 CNF-producing colonies were untypable and the majority of the remaining colonies belonged to serotypes O15:K14, O78:(K80), O123:K-- and O139:K--. Both VT and CNF were lethal for mice, but only CNF showed necrotizing reaction in rabbit skin. Our results indicate that VT-producing and CNF-producing E. coli strains are frequently isolated from diarrhoeic calves and that according to the serotypes exhibited, some of them might be considered potential pathogens for humans. The role of VT-producing and CNF-producing strains in calf diarhoea remains to be established.

Susceptibility trends of Bacteroides fragilis group and characterisation of carbapenemase-producing strains by automated REP-PCR and MALDI TOF

Susceptibility testing of clinical isolates of anaerobic bacteria is not considered, often, mandatory in routine clinical practice and the treatments are empirically established. Thus, periodic monitoring of the susceptibility patterns of anaerobic bacteria is advisable. The aim of this study was to update on resistance of Bacteroides fragilis group in our Institution with special attention to carbapenems reporting metallo-beta-lactamase producing strains for the first time in Spain, and to compare fingerprinting analysis results obtained by using automated rep-PCR (DiversiLab System) and MALDI-TOF MS. A total of 830 non-duplicated clinical isolates of the B. fragilis group recovered from the years 2006 to 2010 were studied. B. fragilis was the most prevalent species (59.5%). The total susceptibility of B. fragilis group isolates were: penicillin, 13.3%; amoxicillin/clavulanic, 89.6%; piperacillin-tazobactam, 91.8%; cefoxitin, 65.8%; ertapenem, 95.9%; imipenem, 98.2%; clindamycin, 53.4% and metronidazole, 96.4%. The percentage of sensitive isolates did not change significantly over time for amoxicillin/clavulanic, cefoxitin, clindamycin and metronidazole. A slight increase in the rate of resistance to ertapenem and imipenem was observed. Imipenem resistance and carbapenemase production were detected for the first time in our laboratory in the year 2007. No other report of carbapenemase-producing B. fragilis in our country has been previously published. Six imipenem-resistant isolates were MBL-producing and PCR positive for cfiA gene. Four of them were PCR positive for IS-like immediately upstream cfiA gene and two of them were negative. Both, automated rep-PCR (DiversiLab) and MALDI-TOF MS, revealed a great genetic diversity among carbapenem-producing strains suggesting the acquisition of novel resistance genes more than clonal dissemination of them. Both methods seem to be useful tools for fast and accurate identification and strain typing of B. fragilis group in the daily laboratory routine. Because of the relevant increase observed in Bacteroides species isolated from blood cultures and the appearance of carbapenemase-producing strains in our Institution, we recommend to test the antimicrobial susceptibility of the isolates, at least in the most severe patients.

Evaluación de una PCR multiplex en tiempo real para la detección de patógenos en el tejido valvular de pacientes con endocarditis

Un nuevo test multiplex basado en una reaccion en cadena de polimerasa en tiempo real LightCycler Septi- Fast® permite la identificacion de 25 especies bacterianas y fungicas directamente desde la sangre. El test SeptiFast® ha sido utilizado para el diagnostico etiologico rapido de la endocarditis infecciosa, pero no ha sido ensayado directamente sobre las vegetaciones cardiacas de pacientes intervenidos por endocarditis infecciosa. Se realizo un estudio prospectivo para analizar 15 muestras de tejido valvular con endocarditis infecciosa activa utilizando SeptiFast® y comparando su sensibilidad con el hemocultivo, el cultivo del tejido valvular y el SeptiFast® en sangre. La sensibilidad del SeptiFast® del tejido valvular fue del 100%, confirmo el diagnostico obtenido mediante hemocultivo en 13 casos y proporciono el diagnostico etiologico en 2 casos con hemocultivo negativo. La sensibilidad de SeptiFast en el tejido valvular fue superior al cultivo convencional de las vegetaciones y al SeptiFast en sangre.

Evaluation of a Multiplex Real-Time PCR Assay for Detecting Pathogens in Cardiac Valve Tissue in Patients With Endocarditis

With a novel real-time multiplex polymerase chain reaction test, the LightCycler SeptiFast® test, 25 bacterial and fungal species can be identified directly in blood. The SeptiFast® test has been used for rapid etiologic diagnosis in infectious endocarditis using blood samples but has not been evaluated directly on cardiac vegetations in patients being treated for infectious endocarditis. We prospectively analyzed 15 samples of heart valve tissue with active infectious endocarditis using the SeptiFast® test and compared the tests sensitivity with that of blood culture, valve tissue culture, and the SeptiFast® test in blood. The sensitivity of the SeptiFast test in heart valve tissue was 100%. The test results confirmed the diagnosis obtained using blood culture in 13 cases and identified the pathogen in 2 cases where blood culture tested negative. The sensitivity of the SeptiFast® test in heart valve tissue was greater than that obtained with conventional culture of vegetations or with the SeptiFast test in blood.

The role of the gut microbiota in schizophrenia: Current and future perspectives

Abstract Objectives: Schizophrenia is a poorly understood chronic disease. Its pathophysiology is complex, dynamic, and linked to epigenetic mechanisms and microbiota involvement. Nowadays, correlating schizophrenia with the environment makes sense owing to its multidimensional implications: temporal and spatial variability. Microbiota involvement and epigenetic mechanisms are factors that are currently being considered to better understand another dimension of schizophrenia. Methods: This review summarises and discusses currently available information, focussing on the microbiota, epigenetic mechanisms, technological approaches aimed at performing exhaustive analyses of the microbiota, and psychotherapies, to establish future perspectives. Results: The connection between the microbiota, epigenetic mechanisms and technological developments allows for formulating new approaches objectively oriented towards the development of alternative psychotherapies that may help treat schizophrenia. Conclusions: In this review, the gut microbiota and epigenetic mechanisms were considered as key regulators, revealing a potential new aetiology of schizophrenia. Likewise, continuous technological advances (e.g. culturomics), aimed at the microbiota-gut–brain axis generate new evidence on this concept.

Perspectiva histórica de la espectrometría de masas en microbiología

La espectrometria de masas (EM) es una tecnica de analisis que permite caracterizar muestras midiendo las masas (estrictamente las razones masa-carga) de las moleculas componentes. Cuenta con mas de un siglo de historia y evolucion tecnologica y a lo largo de los anos ha ampliado su alcance desde los isotopos a moleculas pequenas, moleculas organicas mas complejas y, en las ultimas decadas, macromoleculas (acidos nucleicos y proteinas). La EM MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) es una variante que permite el analisis de mezclas complejas de proteinas y que se ha aplicado recientemente a la identificacion de microorganismos en cultivo, convirtiendose en una herramienta rapida y eficaz para el diagnostico microbiologico que ha conseguido entrar en poco tiempo en la rutina de muchos servicios de microbiologia clinica. El gran impacto que ha tenido esta impulsando el desarrollo de nuevas aplicaciones en el campo de la microbiologia clinica.