Comparison of biotyping, ribotyping, and pulsed-field gel electrophoresis for investigation of a common-source outbreak of Burkholderia pickettii bacteremia.

Over a 3-month period, six immunocompromised patients developed one or more episodes of Burkholderia pickettii bacteremia and/or catheter infection. Vials of a commercially available, "sterile" saline for injection which had been used for flushing the patients' indwelling intravenous devices were implicated as the common source of the organisms. No further cases were diagnosed once the use of this saline was discontinued. Twenty-six isolates, including 9 outbreak-related strains from case patients and contaminated saline as well as 17 control strains, were tested comparatively by biotyping, ribotyping with EcoRI and HindIII, and pulsed-field gel electrophoresis (PFGE) with SpeI. Macrorestriction analysis revealed nine PFGE groups and was more discriminating than ribotyping (seven ribotypes) and biotyping (two biovars). Among the outbreak-related isolates, one B. pickettii type was found by the three typing methods. Furthermore, PFGE was useful for subdividing ribotypes and for distinguishing isolates involved in the outbreak from all epidemiologically unrelated strains.     

Assessment of Resolution and Intercenter Reproducibility of Results of Genotyping Staphylococcus aureus by Pulsed-Field Gel Electrophoresis of SmaI Macrorestriction Fragments: a Multicenter Study

Twenty well-characterized isolates of methicillin-resistant Staphylococcus aureus were used to study the optimal resolution and interlaboratory reproducibility of pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. Five identical isolates (one PFGE type), 5 isolates that produced related PFGE subtypes, and 10 isolates with unique PFGE patterns were analyzed blindly in 12 different laboratories by in-house protocols. In several laboratories a standardized PFGE protocol with a commercial kit was applied successfully as well. Eight of the centers correctly identified the genetic homogeneity of the identical isolates by both the in-house and standard protocols. Four of 12 laboratories failed to produce interpretable data by the standardized protocol, due to technical problems (primarily plug preparation). With the five related isolates, five of eight participants identified the same subtype interrelationships with both in-house and standard protocols. However, two participants identified multiple strain types in this group or classified some of the isolates as unrelated isolates rather than as subtypes. The remaining laboratory failed to distinguish differences between some of the related isolates by utilizing both the in-house and standardized protocols. There were large differences in the relative genome lengths of the isolates as calculated on the basis of the gel pictures. By visual inspection, the numbers of restriction fragments and overall banding pattern similarity in the three groups of isolates showed interlaboratory concordance, but centralized computer analysis of data from four laboratories yielded percent similarity values of only 85% for the group of identical isolates. The differences between the data sets obtained with in-house and standardized protocols could be the experimental parameters which differed with respect to the brand of equipment used, imaging software, running time (20 to 48 h), and pulsing conditions. In conclusion, it appears that the standardization of PFGE depends on controlling a variety of experimental intricacies, as is the case with other bacterial typing procedures.The use of electric field pulsing techniques in conjunction with agarose gel electrophoresis for discrimination of large DNA molecules was introduced by Schwarz and Cantor in 1984 (9). During the past decade the methodology has been adapted and improved by various research groups to the point that pulsed-field gel electrophoresis (PFGE) for bacterial strain typing is now utilized with relative ease in a variety of laboratories (1). The combination of contour-clamped homogeneous field electrophoresis and PFGE for the molecular analysis of Staphylococcus aureus has been reported since the late 1980s (7, 19). At present, PFGE is considered to have both the reproducibility and resolving power of a standard technique for the epidemiological typing of bacterial isolates (10, 15).Molecular typing systems can identify different strains within a species, generating data useful for taxonomic or epidemiologic purposes (10, 14). A frequently observed shortcoming of typing systems in general is their lack of reproducibility: most typing systems do not provide a definitive strain identification, which is usually due to the variability of the technique and the lack of large databases containing fragment patterns from a wide variety of organisms to which unknowns can be compared. These problems were recently described in detail for two molecular typing systems. A multicenter study on random amplification of polymorphic DNA for discrimination of S. aureus strains revealed a lack of interlaboratory reproducibility among the banding patterns generated by the participating centers, although the epidemiological interpretation of the data was similar for all the centers involved (16). For PFGE, a similar lack of interlaboratory reproducibility of patterns was observed, although the interpretation of the experimental data also differed per participating center (2). The latter study analyzed 12 different methicillin-resistant S. aureus (MRSA) strains with different techniques optimized in each center and different sources and types of equipment. Since interlaboratory discrepancies with respect to classification of the strains were observed, the study concluded that there is a clear need for standardization of the technique, including the construction of a panel of reference strains to assist the individual researcher in the optimization of the PFGE protocol.The aim of the present study was to compare the fragment patterns of a well-defined collection of MRSA isolates in 12 laboratories using in-house and a standard set of PFGE parameters to determine whether standardization of experimental parameters (DNA preparation and switching protocols) would improve intercenter reproducibility of PFGE analysis.     

Neocortical and hippocampal EEG in normal and lateral hypothalamic-damaged rats

Cortical and hippocampal EEG were correlated with behavior in rats before and after bilateral hypothalamic (LH) damage. In Stage 1 of recovery (aphagia and adipsia), the neocortex showed continuous large amplitude slow activity. It did not desynchronize during spontaneous acts such as grooming as well as during tail pinch-induced struggling or orienting, even though a slow form of hippocampal theta accompanied these acts. However, during Stage 2 (anorexia), the neocortical EEG did desynchronize when such theta appeared. Therefore, as behavioral recovery progresses after LH damage, there appears to be a concomitant recovery of cortical participation in such behavior. Early in recovery, LH rats, unlike normals, showed slow (3–4.5 Hz) atropine-sensitive hippocampal theta during automatisms such as grooming as well as during immobility. Thus, LH damage, while temporarily abolishing fast. noncholinergic theta, appears to release slow cholinergic theta. Later in recovery, faster atropine-resistant (noncholinergic) theta becomes functional again.     

Working conditions and health behavior as causes of educational inequalities in self-rated health: an inverse odds weighting approach

Objective:Using a novel mediation method that presents unbiased results even in the presence of exposure–mediator interactions, this study estimated the extent to which working conditions and health behaviors contribute to educational inequalities in self-rated health in the workforce.Methods:Respondents of the longitudinal Survey of Health, Ageing, and Retirement in Europe (SHARE) in 16 countries were selected, aged 50–64 years, in paid employment at baseline and with information on education and self-rated health (N=15 028). Education, health behaviors [including body mass index (BMI)] and working conditions were measured at baseline and self-rated health at baseline and two-year follow-up. Causal mediation analysis with inverse odds weighting was used to estimate the total effect of education on self-rated health, decomposed into a natural direct effect (NDE) and natural indirect effect (NIE).Results:Lower educated workers were more likely to perceive their health as poor than higher educated workers [relative risk (RR) 1.48, 95% confidence interval (CI) 1.37–1.60]. They were also more likely to have unfavorable working conditions and unhealthy behaviors, except for alcohol consumption. When all working conditions were included, the remaining NDE was RR 1.30 (95% CI 1.15–1.44). When BMI and health behaviors were included, the remaining NDE was RR 1.40 (95% CI 1.27–1.54). Working conditions explained 38% and health behaviors and BMI explained 16% of educational inequalities in health. Including all mediators explained 64% of educational inequalities in self-rated health.Conclusions:Working conditions and health behaviors explain over half of the educational inequalities in self-rated health. To reduce health inequalities, improving working conditions seems to be more important than introducing health promotion programs in the workforce.     

Characterization of xenobiotic-metabolizing enzymes and nitrosamine metabolism in the human esophagus

Esophageal cancer has been associated with tobacco smoking, and nitrosamines are possible causative agents for this cancer. The present study investigated the metabolism of the tobacco carcinogens N'- nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK), and N-nitrosodimethylamine (NDMA), as well as the presence of xenobiotic-metabolizing enzymes in human esophageal tissues from individuals in the United States and Huixian, Henan Province, China (a high-risk area for esophageal cancer). All esophageal microsomal samples activated NNN and the metabolic rate was 2-fold higher in the esophageal samples from China than the USA. All microsomal samples activated NDMA. However, most of the microsomal samples did not activate NNK. Troleandomycin (an inhibitor of cytochrome P450 3A) decreased the formation of NNN-derived keto acid by 20-26% in the esophageal microsomes. The activities for NADPH: cytochrome c reductase, ethoxycoumarin O-deethylase, NAD(P)H: quinone oxidoreductase and glutathione S-transferase were present in the esophageal samples. Coumarin 7-hydroxylase (a representative activity for P450 2A6) activity was not detected in the esophageal microsomal samples. The activities for nitrosamine metabolism and xenobiotic- metabolizing enzymes were decreased (by 30-50%) in the squamous cell carcinomas compared with their corresponding non-cancerous mucosa. The presence of activation and detoxification enzymes in the esophagus may play an important role in determining the susceptibility of the esophagus to the carcinogenic effect of nitrosamines. Our results suggest that P450s 3A4 and 2E1 are involved in the activation of NNN and NDMA, respectively, in the human esophagus.      

Community-acquired methicillin-resistant Staphylococcus aureus clones circulating in Belgium from 2005 to 2009: changing epidemiology

The present study reports the evolution of the demographic characteristics and the molecular epidemiology of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) in Belgium from 2005 to 2009. Four hundred and ten CA-MRSA isolates were prospectively collected and screened for the presence of Panton–Valentin leucocidin (PVL) and toxic shock syndrome toxin 1 (TSST-1) encoding genes, while clinical information were recorded. PVL- and TSST-1-positive isolates were genotyped by pulsed-field gel electrophoresis (PFGE). Staphylococcal cassette chromosome mec (SCCmec) type, spa type and multilocus sequence type (MLST) were determined on representative isolates. One hundred and fifty-nine (39 %) isolates were PVL-positive. PVL-positive isolates were significantly more frequently isolated from skin or soft tissue than PVL-negative isolates, causing mainly subcutaneous abscesses and furuncles. Patients with PVL-positive CA-MRSA were significantly younger than patients with PVL-negative CA-MRSA. Eighty-seven percent of the PVL-positive isolates belonged to a limited number (n?=?7) of PFGE types belonging to sequence types (ST) ST80, ST8, ST30, ST5, ST152, ST338 and a new ST, a single-locus variant of ST1. A temporal evolution of the distribution of these PFGE types was observed, characterised by (1) the dissemination of the ST8-SCCmecIV arcA-positive (USA300) genotype and (2) a genetic diversification. Forty-seven (11 %) strains were TSST-1-positive, of which 65 % clustered into four PFGE types, all belonging to ST5. The epidemiology of CA-MRSA in Belgium is changing, as the rapid diffusion of the USA300 clone seems to occur, together with a clonal diversification.