José Elías García-Sánchez

Microorganisms direct identification from blood culture by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows a fast and reliable bacterial identification from culture plates. Direct analysis of clinical samples may increase its usefulness in samples in which a fast identification of microorganisms can guide empirical treatment, such as blood cultures (BC). Three hundred and thirty BC, reported as positive by the automated BC incubation device, were processed by conventional methods for BC processing, and by a fast method based on direct MALDI-TOF MS. Three hundred and eighteen of them yield growth on culture plates, and 12 were false positive. The MALDI-TOF MS-based method reported that no peaks were found, or the absence of a reliable identification profile, in all these false positive BC. No mixed cultures were found. Among these 318 BC, we isolated 61 Gram-negatives (GN), 239 Gram-positives (GP) and 18 fungi. Microorganism identifications in GN were coincident with conventional identification, at the species level, in 83.3% of BC and, at the genus level, in 96.6%. In GP, identifications were coincident with conventional identification in 31.8% of BC at the species level, and in 64.8% at the genus level. Fungaemia was not reliably detected by MALDI-TOF. In 18 BC positive for Candida species (eight C. albicans, nine C. parapsilosis and one C. tropicalis), no microorganisms were identified at the species level, and only one (5.6%) was detected at the genus level. The results of the present study show that this fast, MALDI-TOF MS-based method allows bacterial identification directly from presumptively positive BC in a short time (<30 min), with a high accuracy, especially when GN bacteria are involved.

El cine en la docencia de las enfermedades infecciosas y la microbiología clínica

El cine,desde sus comienzos,ha reflejado de forma constante la infeccion por su omnipresencia en la vida y por su trascendencia individual y social. Practicamente ninguna enfermedad infecciosa escapa a su ojo,hasta elpunto de que convenientemente enfocado y encauzado constituye un autentico tratado de infectologia,un recurso docente muy valioso capaz de complementar los metodos clasicos y de fomentar el espiritu critico de los alumnos.No puede desaprovecharse el enorme caudal de informacion,de imagenes,de sonidos,de consecuencias,de situaciones,de puntos de vista que proporciona y que pueden ser de gran utilidad tanto en la divulgacion como en la formacion en enfermedades infecciosas y en microbiologia clinica.

Update on pathogenesis and diagnosis of intravascular catheter-related infections.

Infections associated with the use of intravascular catheters cause a substantial morbidity and mortality. New knowledge in the pathogenesis of catheter related bloodstream infections has led to advances in the prevention and management of these infections. The purpose of the present chapter is to review the most relevant data published recently on pathogenesis and diagnosis of intravascular catheter-related infections. It is focus in four different aspects: a) pathogenesis of catheter-related infections and particularly factors affecting biofilm formation and modulation; b) pathogenesis of intravascular catheter-related infections caused by Staphylococcus lugdunensis; c) news on microbiological diagnosis of catheter-related bacteremia; and d) evaluation of current use of blood cultures in the era of continuous monitoring blood cultures systems.

P- and E-selectin blockade can control bacterial translocation and modulate systemic inflammatory response.

Bacterial translocation is an important phenomenon in clinical medicine and leads to an increase in patient morbidity and mortality by multiple organ failure. The selectin family plays an important role in the pathogenesis of inflammation, causing an increase in leukocyte-endothelium interactions and inducing a greater leukocytes migration. This study considered the effect of a sulfo derivative of Sialyl-Lewis(X), GM 1998-016, that will block the P- and E-selectins interaction with a ligand, the Sialyl-Lewis(X), valuing the modulation of the systemic inflammatory response and the induced translocation. Seventy-five Wistar male rats were injected intraperitoneally with Zymosan A and treated with different doses of GM 1998-016 according to study groups. Measurements of values of qualitative and quantitative microbiology, neutrophil infiltration (myeloperoxidase), oxygen free radicals (superoxide anion, superoxide dismutase, catalase, and gluthatione peroxidase), and cytokines (tumor necrosis factor-alpha and interleukin-1beta) were taken at different times after Zymosan administration. A significant decrease of bacterial translocation, both local (MLN) and systemic (p < .05), was observed, with a decrease in the neutrophil infiltration (p < .001), the oxygen free radicals production (p < .01) and the studied cytokines (p < .01). In conclusion, GM 1998-016 showed a protective effect in an in vivo experimental model of bacterial translocation, downregulating the inflammatory response and the leukocyte-endothelium interactions.

Activity of BAY y 3118, a Novel 4-Quinolone, against Brucella melitensis

We have tested the in-vitro activities of BAY y 3118, a new chlorofluoroquinolone, ciprofloxacin, sparfloxacin, streptomycin, tetracycline and rifampin against 59 strains of Brucella melitensis. BAY y 3118 (MIC90 0.12 mg/L) was twice as active as sparfloxacin and tetracycline (MIC90 0.25 mg/L). The activity of ciprofloxacin, rifampin and streptomycin (MIC90 0.5, 2, and 8 mg/L, respectively) was, respectively, four-, sixteen-, and more than sixty-fold lower than that of BAY y 3118.

Estudios de sensibilidad en bacterias anaerobias

The anaerobic bacteria resistance to antibiotics is increasing, and even has appeared against the most active of those, like metronidazol and carbapenems. This fact forces to make and periodical sensibility tests -at least in the most aggressive and virulent species, in cases that they are isolated from life locations and in the absence of therapeutic response- to check the local sensibility and to establish suitable empiric therapies, all based on multicentric studies carried out in order to this or well to check the activity of new antibiotics. For the laboratory routine, the easiest sensibility method is the E-test/MIC evaluator. Another alternative is microdilution, thats only normalized for Bacteroides. There are preliminary facts that allow the use of disc diffusion method in some species of Bacteroides and Clostridium. For the temporal and multicentric studies, the procedure is dilution in agar plate, the reference method.

Diagnóstico microbiológico de las infecciones intraabdominales

Intra-abdominal infections represent a large and wide group of diseases which include intra- and retro-peritoneal infections. Some of them could be defined as uncomplicated, where the infectious process is limited to the organ or tissue of origin (appendicitis, diverticulitis, cholecystitis…). Complications occur when the infection spreads to the peritoneum, triggering localised peritonitis and abdominal abscesses. Most intra-abdominal infections are due to perforation or inflammation of the intestinal wall. The microorganisms that cause these infections come from the gastrointestinal flora, and therefore produce polymicrobial infections mixed with a predominance of anaerobic bacteria. Microbiological diagnosis is essential to determine the aetiology and the susceptibility of antimicrobial agents of the microorganism involved, especially in nosocomial infections or in community infections in predisposed patients due to increasing bacterial resistance to antimicrobial agents, multidrug resistance and fungal involvement. Despite the advances in microbiological diagnosis, in the case of intra-abdominal infections it still remains direct, being based on stains and cultures, the most notable progress is the introduction of mass spectrometry (MALDI-TOF) for the rapid identification of the pathogens involved. This review will provide recommendations on the collection, transport and microbiological processing of clinical specimens. Comments on the pathogenesis, clinical and microbiological diagnosis of peritonitis primary, secondary, tertiary and peritonitis (and other infections) associated with peritoneal dialysis, intra-abdominal abscesses (intraperitoneal, retroperitoneal and visceral), biliary tract infections, appendicitis and diverticulitis are also presented.

Las bacterias anaerobias 150 años después de su descubrimiento por Pasteur

In 2011 we celebrated the 150th anniversary of the discovery of anaerobic bacteria by Louis Pasteur. The interest of the biomedical community on such bacteria is still maintained, and is particularly focused on Clostridium difficile. In the past few years important advances in taxonomy have been made due to the genetic, technological and computing developments. Thus, a significant number of new species related to human infections have been characterised, and some already known have been reclassified. At pathogenic level some specimens of anaerobic microflora, that had not been isolated from human infections, have been now isolated in some clinical conditions. There was emergence (or re-emergence) of some species and clinical conditions. Certain anaerobic bacteria have been associated with established infectious syndromes. The virulence of certain strains has increased, and some hypotheses on their participation in certain diseases have been given. In terms of diagnosis, the routine use of MALDI-TOF has led to a shortening of time and a cost reduction in the identification, with an improvement directly related to the improvement of data bases. The application of real-time PCR has been another major progress, and the sequencing of 16srRNA gene and others is currently a reality for several laboratories. Anaerobes have increased their resistance to antimicrobial agents, and the emergence of resistance to carbapenems and metronidazole, and multi-resistance is a current reality. In this situation, linezolid could be an effective alternative for Bacteroides. Fidaxomicin is the only anti-anaerobic agent introduced in the recent years, specifically for the diarrhoea caused by C.difficile. Moreover, some mathematical models have also been proposed in relation with this species.

The Pitt Bacteremia Score, Charlson Comorbidity Index and Chronic Disease Score are useful tools for the prediction of mortality in patients with Candida bloodstream infection

Candida bloodstream infection (CBI) is associated with high mortality. The aim of this study was to compare the utility of the combined use of the Pitt Bacteremia Score (PBS) and Charlson Comorbidity Index (CCI) or Chronic Disease Score (CDS) to predict mortality among patients with CBI. Thereby, all consecutive patients with CBI at our institution between 2010 and 2014 were included. The PBS was used to evaluate CBI severity and the CCI and CDS were used to assess comorbidities of patients with CBI. Logistic regression analysis was used to estimate odds ratios for 30‐day mortality in models including the PBS and CCI or CDS. A total of 189 CBI episodes were identified. Logistic regression models including the PBS and either CCI or CDS showed that the combined use of a comorbidity score and a severity score significantly predicted 30‐day mortality. The performance of the different models was similar. Aggregated scores of comorbidity (CCI and CDS) and disease severity (PBS) are useful for the prediction of 30‐day mortality risk in patients with CBI. Their use may facilitate the analysis of risk factors for poorer outcome and the development of an index for CBI mortality.

Comparative study of bacterial translocation control with nitric oxide donors and COX2 inhibitor

OBJECTIVE AND DESIGN To evaluate the beneficial effects of exogenous NO and an inhibitor of the COX2, and their action levels in a model of SIRS/bacterial translocation (BT) induced by Zymosan A(®). MATERIAL AND METHODS Ninety Wistar rats were submitted to different treatments, and after 12h and 24h they were anaesthetized in order to collect blood, mesenteric lymph nodes, and kidney for subsequent biochemical analyses and microbiological examinations. TREATMENTS A nitric oxide donor, Molsidomine(®), was compared with a COX2 inhibitor, Celecoxib(®). METHODS Zymosan A(®) was administered to Wistar rats. The animals were divided into 6 groups: one group for survival study, Group (1) No manipulation (BASAL); Group (2) vehicle of Zymosan A(®) given intraperitoneally (SHAM); Group I (control), with Zymosan A(®) (0.6g/kg) intraperitoneally; Group II (Molsidomine), with Molsidomine(®) (4mg/kg) through the penis dorsal vein, 30min prior to administration of the Zy(®) (0.6g/kg); Group III (Celecoxib), with Celecoxib(®) (400mg/kg) orally through a stomach tube, 6h prior to administration of the Zy (0.6g/kg). DETERMINATIONS The parameters survival, bacterial translocation, renal function, neutrophil accumulation, oxygen free radicals (OFR), detoxifying enzymes, and cytokines were measured at different times after Zymosan administration. RESULTS The model established induced a mortality rate of 100% and generated BT and systemic inflammatory response syndrome (SIRS) in all samples. It also significantly increased all variables, with p<.001 for MPO and all pro-inflammatory cytokines, and p<.01 for all OFR. Treatment with Molsidomine reduced mortality to 0%, decreased BT, MPO, pro-inflammatory cytokines and OFR (p<.001) significantly and increased IL-10 and IL-6 production. Moreover, the Celecoxib(®) showed a lower capacity for SIRS regulation. CONCLUSIONS The exogenous administration of NO prevented BT and controlled SIRS. Therefore these results suggest that Molsidomine could be used as a therapeutic strategy to protect against BT.