Finished Whole-Genome Sequences of Clostridium butyricum Toxin Subtype E4 and Clostridium baratii Toxin Subtype F7 Strains.

Clostridium butyricum and Clostridium baratii species have been known to produce botulinum toxin types E and F, respectively, which can cause botulism, a rare but serious neuroparalytic disease. Here, we present finished genome sequences for two of these clinically relevant strains.

Finished Whole-Genome Sequences of Two Clostridium botulinum Type A(B) Isolates.

Clostridium botulinum secretes a potent neurotoxin that causes devastating effects when ingested, including paralysis and death if not treated. In the United States, some clinically significant strains produce toxin type A while also harboring a silent B gene. These are the first two closed genome sequences published for this subset.

Finished Whole-Genome Sequence of Clostridium argentinense Producing Botulinum Neurotoxin Type G.

Here, we present a closed genome sequence for Clostridium argentinense strain 89G, the first strain identified to produce botulinum neurotoxin type G (BoNT/G). Although discovered in 1970, to date, there have been no reference quality sequences publicly available for this species.

Characterizing the fecal microbiota of infants with botulism.

BACKGROUND: Infant botulism is the most prevalent form of botulism in the USA, representing 68.5 % of cases reported from 2001-2012. Infant botulism results when botulinum toxin-producing clostridia (BTPC) colonize the infant gut with concomitant in vivo production of the highly potent botulinum neurotoxin (BoNT). The gut microbiota of infants with botulism is largely uncharacterized; therefore, it remains unclear whether the microbiota profile of these patients are distinct in composition, abundance, or diversity. To address this uncertainty, we employed 16S rRNA gene profiling to characterize the fecal microbiota in 14 stool samples among laboratory-confirmed and non-confirmed infant botulism cases. RESULTS: Seven bacterial phyla were identified among all 14 infant stool samples examined. Compared to samples from non-confirmed cases, the fecal microbiota of infant botulism patients displayed significantly higher Proteobacteria abundance. Of the 20 bacterial families identified, Enterobacteriaceae was significantly more abundant in samples from infants with botulism. Firmicutes abundance and the abundance ratio of Firmicutes/Proteobacteria was significantly lower in samples from infants with botulism. Lactobacillus spp. abundance was notably reduced in 12 of the 14 samples. Clostridium botulinum and Clostridium baratii were identified in low relative abundances in confirmed and non-confirmed samples based on their 16S rRNA gene profiles, although their toxigenicity remained undetermined. No significant differences were observed in the number of operational taxonomic units (OTUs) observed or in fecal microbiota diversity between laboratory-confirmed and non-confirmed samples. Correlations between individual phylum abundances and infant age were variable, and no significant differences were shown in number of OTUs observed or in fecal microbiota diversity between samples delineated by overall mean age. CONCLUSIONS: Significant differences in Proteobacteria, Firmicutes, and Enterobacteriaceae abundances were identified in the fecal microbiota of infants with botulism when compared to samples from non-confirmed cases. Fecal microbiota diversity was not significantly altered in infants with botulism, and a limited presence of BTPC was shown. It could not be determined whether the fecal microbiota profiles shown here were comparable prior to patient illness, or whether they were the direct result of infant botulism. The results of this study do, however, provide a detailed and descriptive observation into the infant gut microbiota after intestinal colonization by BTPC.

Composition and stability of bacterial communities associated with granular activated carbon and anthracite filters in a pilot scale municipal drinking water treatment facility.

Granular activated carbon (GAC) is an alternative filter substrate for municipal water treatment as it provides a high surface area suitable for microbial colonization. The resulting microbial growth promotes biodegradation of organic materials and other contaminants from influent waters. Here, the community structure of the bacteria associated with three GAC and two anthracite filters was examined over 12 months to monitor changes in community composition. Nearly complete 16S rRNA genes were polymerase chain reaction amplified for terminal restriction fragment length polymorphism (T-RFLP) analyses. The identity of commonly occurring peaks was determined through the construction of five representative 16S rRNA clone libraries. Based on sequence analysis, the bacterial communities associated with both anthracite and GAC filters appear to be composed of environmentally derived bacteria, with no known human pathogens. Analysis of similarity tests revealed that significant differences in bacterial community structure occurred over time, with filter substrate playing an important role in determining community composition. GAC filters exhibited the greatest degree of bacterial community variability over the sampling period, while anthracite filters showed a lower degree of variability and less change in community composition. Thus, GAC may be a suitable biologically active filter substrate for the treatment of municipal drinking water.

The laboratory-clinical interface: point-of-care testing.

POC testing provides an opportunity for clinicians and laboratorians to work together to consider how best to serve the patients within an individual institution. Each health system has unique characteristics relative to patient population, as well as a unique laboratory structure. If physicians, nurses, laboratorians, and pathologists work collaboratively, the best interests of patients will be served. In some institutions that cater to specific patient groups, POC testing may offer clear and distinct advantages. In other institutions with sophisticated transport systems and established rapid response capabilities, the quality resulting from central laboratory testing may outweigh any advantages of bedside testing. Clearly, attention to regulatory issues, QC issues, the importance of proper documentation, proficiency testing, performance enhancement, and cost-effectiveness is requisite. As the technology for diagnostic testing advances through more microcomputerization, microchemistry, and enhanced test menus, the concept of POC testing will need perpetual revisiting. We hope that the information provided here will aid clinicians, laboratorians, and administrators in their quest to best serve their patients.

Cord lactate, pH, and blood gases from healthy neonates.

Lactate, pH, pO2, and pCO2 were determined in arterial, venous, and free-flowing mixed umbilical cord blood obtained from deliveries of apparently healthy neonates. The goals of this study were to establish reference ranges for lactate and pH against which results in cases of high-risk labor and delivery could be compared, to see how the gases correlated with these values, and to determine whether easily accessible mixed umbilical cord blood can serve as the sample in lieu of cord arterial or cord venous blood. Arterial and venous cord lactates were 2.98 mmol/l (+/- 1.40) and 2.80 mmol/l (+/- 1.35), respectively, from 85 cords obtained from vaginal and cesarean deliveries. Mixed cord blood lactate, obtained on 48 cords, was 2.72 mmol/l (+/- 1.28) versus 3.14 and 2.97 mmol/l for the arterial and venous samples from those cords, respectively, and correlated quite well with lactate from the venous specimens (r = 0.97). Differences of > 0.5 mmol/l occurred between mixed and arterial cord bloods in 21 patients, and between mixed and venous cord bloods in 6 of the 48 patients, respectively. We conclude that (1) less than 2.5% of deliveries of apparently healthy neonates have arterial, venous, or mixed cord lactates > or = 7.0 mmol/l and pH < or = 7.15, (2) neither cord venous pO2 nor pCO2 correlate well with cord venous lactate, and (3) readily available mixed cord blood is a satisfactory specimen for the measurement of venous cord latate.

Critical care profiling for informed treatment of severely ill patients.

An important advance in critical care medicine is the availability of key biochemical test results at the patient's bedside within the brief period when they can be used to guide resuscitation and cardiovascular stabilization effectively. Frequently the menu of key tests, called a "critical care profile (CCP)," must be available to the attending clinician within 5 minutes to guide therapy in real time. Recent developments that make it possible to deliver a CCP with these 5 minutes include the ability to measure all of the analytes of the critical care profile on a whole-blood sample, and point-of-care testing. Today the results of a critical care profile can be available to the attending clinician within 5 minutes of his or her request by sampling approximately 200 microL of whole blood with a critical care instrument at or near a patient's bedside. Tests frequently recommended for a critical care profile include glucose, pO2, pCO2, pH, lactate, ionized calcium, potassium, sodium, ionized magnesium, and either hemoglobin concentration or hematocrit. Hospital areas that need critical care profile support include the emergency department, the operating room, and the intensive care unit.

Characterization of a new ion selective electrode for ionized magnesium in whole blood, plasma, serum, and aqueous samples.

Results from a novel ion selective electrode (ISE) for ionized magnesium (Mg2+) correlate well with atomic absorption spectroscopy on aqueous solutions containing from 0.1-3.0 mmol MgCl2/L. Day to day precision (coefficient of variation) of the electrode on protein-based controls is < 4%; aqueous-based controls < 6%. The new ISE is selective for Mg2+ with a selectivity constant for Ca2+ (KMgCa) of 8 x 10(-2). Adding pathophysiologic concentrations of Cd2+, Ca2+, Cu2+, Fe3+, K+, Na+, or Zn2+ to serum and aqueous solutions gave negligible to minimal changes in measured Mg2+. Ligand binding studies in aqueous solution indicate that pathophysiologic concentrations of different anions (e.g. heparin, lactate, bicarbonate, phosphate, acetate and sulfate) bind to Mg2+, effectively reducing its concentration in solution. Likewise, silicon (as either found in Vacuutainer tubes or as chlorosilane) failed to exert any significant effect on measured Mg2+. Addition of Intralipid (up to 500 mg/dL) gave negligible to minimal changes in Mg2+. Mg2+ measurements on whole blood, plasma, and serum for a given human subject's samples are virtually identical, at least within the reference range for Mg2+. Typically, Mg2+ is 71% of TMg, but varies from subject to subject; i.e. Mg2+ cannot be predicted from TMg. Clinical studies revealed that the Mg2+/TMg ratio could be remarkably consistent in sequential samples (e.g., throughout the course of coronary bypass surgery) taken from one patient, but that this ratio could differ dramatically from the ratio in sequential samples taken from another. Mg2+ is held within a narrow range (0.53-0.67 mmol/L) in normal, healthy subjects when compared to TMg (0.70-0.96 mmol/L).

A new method for the rapid determination of ionized Mg2+ in whole blood, serum and plasma.

A novel ion selective electrode (ISE) for ionized magnesium (IMg2+) in whole blood (WB), plasma (PL) and serum (S) has now been designed and characterized in normal human subjects, diseased and pregnant subjects. Using this ISE on various levels of aqueous Mg2+ solutions (0.1-3.0mM), mean Mg2+ values are within 94.6 and 99.2% of their targets. The linearity of the ISE (0.1-3.0mM) in aqueous solution and human PL and S ranges between 92.0 and 99.3%. The ISE is highly selective for IMg2+, yielding measurements in less than 2 min, and exhibiting no or negligible effects from physiologic concentrations of Ca2+,Na+,K+ or H+. Ligand binding studies indicate that pathophysiologic concentrations of most smaller molecular weight anions fail to interfere with IMg2+ measurements in aqueous solution, PL or S. Likewise, pathophysiologic concentrations of heavy metals or lipids do not interfere with measurements for IMg2+. Comparison of healthy, normal WB, PL and S IMg2+, using the ISE, with measurements of ultrafilterable Mg by atomic absorption spectroscopy (AAS) are excellent. The mean value for IMg2+ in normal WB, PL and S is 0.58-0.60mM (range = 0.53-0.67mM). Compared to total Mg (TMg) (0.75-0.96mM) and ICa2+ (1.10-1.30mM), IMg2+ is held in a narrow, tight range, representing 71% of TMg. Preliminary studies on plasma and serum IMg2+ during open heart surgery and term pregnancy demonstrate significant abnormalities in IMg2+ and % IMg2+. The new ISE for IMg2+ should prove invaluable and revolutionary in studies of Mg metabolism in both healthy and diseased states.

New whole-blood testing for laboratory support of critical care at cardiac transplant centers and US hospitals.

Whole-blood analytical techniques include ion-specific, substrate-specific, amperometric, and impedance electrodes. These allow direct measurement of critical analytes simultaneously in whole blood without centrifugation, resulting in a response time of 2 to 5 minutes. Cardiac transplantation centers rely heavily on whole-blood instruments to provide rapid response tests essential for cardiovascular management. A survey of 81 blood gas laboratories in 25 cardiac transplantation centers and 73 blood gas laboratories in 36 general hospitals showed an increase in testing for potassium, free calcium, and glucose by blood gas laboratories since 1982 and extensive use of satellite laboratories near areas serving critically ill patients. The following three recent developments are improving the availability of laboratory results: (1) direct whole-blood measurement is gaining acceptance, (2) instrumentation designed specifically for rapid critical care profiling is being used extensively, and (3) testing is moving closer to patients. These trends suggest a significant change in laboratory support of critical care in the United States.

Conjugated bilirubin versus direct bilirubin in neonates.

Conjugated and direct bilirubin were measured in 288 samples from 107 neonates less than 15 days old. Retrospective analysis of the medical records showed that 53 neonates were hepatobiliary-normal, 42 patients had no obvious evidence of hepatobiliary disease but had received total parenteral nutrition, and 12 were clearly hepatobiliary-abnormal. Neither the mean values nor the distributions of either the conjugated bilirubin, as measured by a multilayered slide, or the direct bilirubin, measured by a solution diazo procedure, differed when comparing the hepatobiliary-normal population to that receiving total parenteral nutrition. However, as would be predicted, the hepatobiliary-abnormal population differed significantly from both of these groups using either direct bilirubin or conjugated bilirubin results. Samples obtained from hepatobiliary-abnormal neonates were reviewed in chronologic sequence if direct bilirubin results differed from conjugated bilirubin with respect to classification of normality/abnormality. In two of three neonates with developing cholestasis, conjugated bilirubin exceeded its upper limit of normal earlier than did direct bilirubin. Conjugated bilirubin returned to normal earlier than direct bilirubin for two neonates with an improving clinical status. Conjugated bilirubin measurement was judged to be more responsive to developing or resolving cholestasis then direct bilirubin. In addition, conjugated bilirubin measurement from the slide is known to agree well with that determined by high performance liquid chromatography fractionation of bilirubin and is less susceptible to interference than is direct bilirubin measurement, characteristics that recommend its use over direct bilirubin analysis in a clinical setting.

Unconjugated hyperbilirubinemia is overestimated in neonates with cholestasis. A more reliable method is proposed.

Unconjugated bilirubin was determined in 458 serum samples from 160 neonatal and pediatric patients by using two approaches. The first approach was to calculate indirect bilirubin by subtracting direct bilirubin, as measured by a diazo solution assay, from total bilirubin quantified by a Jendrassik-Grof procedure. The second approach was actually to measure the unconjugated bilirubin fraction using a multilayered slide. For samples containing little or no conjugated bilirubin, correlation between the calculated indirect bilirubin value and the unconjugated bilirubin measured by the slide was judged acceptable. Samples containing increased levels of conjugated bilirubin, however, yielded discrepancies between the approaches including differences of up to 5-10 mg/dL and more. Moreover, the magnitudes of the differences correlated with the amounts of conjugated bilirubin present in the samples. The unconjugated bilirubin values given by the slide were found to correlate more closely than the calculated indirect values with results obtained by an HPLC procedure that may be regarded as the most reliable method available. Therefore, we find that the slide provides a more accurate measure of unconjugated bilirubin than does the indirect bilirubin value in specimens from pediatric patients having evidence of cholestasis. The authors also believe that the magnitude of the difference in the values could make a difference in therapeutic strategies.

Conjugated bilirubin: a better indicator of impaired hepatobiliary excretion than direct bilirubin.

A prototype KODAK EKTACHEM Clinical Chemistry Slide (BuBc) provided a measurement of serum conjugated bilirubin which was at least as sensitive to developing conjugated hyperbilirubinemia as values provided by a direct (diazo) bilirubin assay. Under conditions where the impairment of hepatobiliary excretion was relieved in patients being treated for various hepatobiliary diseases, conjugated bilirubin was cleared from serum more rapidly than alkaline phosphatase, the delta bilirubin fraction, and bilirubin measured by the direct and total bilirubin assays. It is concluded that the conjugated bilirubin measurement provided by the BuBc Slide appears to be an earlier indicator of relief from hepatobiliary cholestasis, or conversely of residual impairment, than direct bilirubin, total bilirubin, or alkaline phosphatase.

Development of a layered-coating technology for clinical chemistry.

A genetic overview of how a layered-coating technology works is presented. Coating scientists can enhance specificity and sensitivity of clinical chemistry assays via the substantial flexibility they have in their choice of coating designs. Multiple layers in a coating provide multiple environments in which sequential chemical and physical reactions can be made to occur. Filtration, selective absorption, reactant and/or product immobilization, and the ability to control reaction kinetics via diffusion rates, layer proximities, and intralayer environments are some of the operative mechanisms that may be employed to improve upon assay accuracy and ease of performance. How these mechanistic alternatives provide the basis for a variety of clinical assays is illustrated. Emphasis is placed on unique features provided by this technology to the clinical laboratory. Performance data are compared to familiar liquid-phase assays.

A modified orcinol test for the specific determination of RNA.

The standard orcinol test for estimating RNA is modified and developed as a specific method for the determination of RNA in the presence of DNA and proteins. The main differences in the procedure of the modified test when compared to that of the standard test are preincubation of the samples with H2SO4 before addition of the orcinol reagent, decreased concentration of orcinol, no addition of FeCl3.6H2O, and quantitation of RNA at its maximum absorbance under these conditions at 500 nm where interferences from DNA and proteins were minimal.