Reporter bacteriophage T7NLC utilizes a novel NanoLuc::CBM fusion for the ultrasensitive detection of Escherichia coli in water.

Rapid detection of bacteria responsible for foodborne diseases is a growing necessity for public health. Reporter bacteriophages (phages) are robust biorecognition elements uniquely suited for the rapid and sensitive detection of bacterial species. The advantages of phages include their host specificity, ability to distinguish viable and non-viable cells, low cost, and ease of genetic engineering. Upon infection with reporter phages, target bacteria express reporter enzymes encoded within the phage genome. In this study, the T7 coliphage was genetically engineered to express the newly developed luceriferase, NanoLuc (NLuc), as an indicator of bacterial contamination. While several genetic approaches were employed to optimize reporter enzyme expression, the novel achievement of this work was the successful fusion of the NanoLuc reporter to a carbohydrate binding module (CBM) with specificity to crystalline cellulose. This novel chimeric reporter (nluc::cbm) bestows the specific and irreversible immobilization of NanoLuc onto a low-cost, widely available crystalline cellulosic substrate. We have shown the possibility of detecting the immobilized fusion protein in a filter plate which resulted from a single CFU of E. coli. We then demonstrated that microcrystalline cellulose can be used to concentrate the fusion reporter from 100 mL water samples allowing a limit of detection of <10 CFU mL-1E. coli in 3 hours. Therefore, we conclude that our phage-based detection assay displays significant aptitude as a proof-of-concept drinking water diagnostic assay for the low-cost, rapid and sensitive detection of E. coli. Additional improvements in the capture efficiency of the phage-based fusion reporter should allow a limit of detection of <10 CFU per 100 mL.

Wegener's granulomatosis presenting as acute systemic vasculitis following 20 years of limited tracheobronchial disease.

OBJECTIVE: We report a patient with a 20-year history of apparently idiopathic airways stenoses, who presented with an antineutrophil cytoplasmic antibody (ANCA) associated, acute, systemic vasculitis with necrotising glomerulonephritis, subsequently diagnosed as Wegener's granulomatosis. METHODS: We present a case report and a review of the world literature on airway stenosis in Wegener's granulomatosis. RESULTS: To our knowledge, this is the first report of Wegener's granulomatosis manifesting as local airway disease for such a prolonged period, before transforming into a systemic vasculitis. CONCLUSIONS: This case highlights the need for physicians to be alert to the possibility of Wegener's granulomatosis as a cause of apparently idiopathic airway stenosis, and to be aware that systemic disease may occur in very long-standing, limited Wegener's granulomatosis.

Tn7: smarter than we thought.

A notable feature of transposable elements--segments of DNA that can move from one position to another in genomes--is that they are highly prevalent, despite the fact that their translocation can result in mutation. The bacterial transposon Tn7 uses an elaborate system of target-site selection pathways that favours the dispersal of Tn7 in diverse hosts as well as minimizing its negative effects.

Tn7 recognizes transposition target structures associated with DNA replication using the DNA-binding protein TnsE.

We report that the bacterial transposon Tn7 selects targets by recognizing features associated with DNA replication using the transposon-encoded DNA-binding protein TnsE. We show that Tn7 transposition directed by TnsE occurs in one orientation with respect to chromosomal DNA replication, indicating that a structure or complex involved in DNA replication is likely to be a critical determinant of TnsE insertion. We find that mutant TnsE proteins that allow higher levels of transposition also bind DNA better than the wild-type protein. The increased binding affinity displayed by the TnsE high-activity mutants indicates that DNA binding is relevant to transposition activity and suggests that TnsE interacts directly with target DNAs. In vitro, TnsE interacts preferentially with certain DNA structures, indicating a mechanism for the TnsE-mediated orientation and insertion preference. The pattern of TnsE-mediated insertion events around the Escherichia coli chromosome provides insight into how DNA replication forks proceed in vivo.

Tn7 transposes proximal to DNA double-strand breaks and into regions where chromosomal DNA replication terminates.

We report that the bacterial transposon Tn7 can preferentially transpose into regions where chromosomal DNA replication terminates. DNA double-strand breaks are associated with the termination of chromosomal replication; therefore, we directly tested the effect of DNA breaks on Tn7 transposition. When DNA double-strand breaks are induced at specific sites in the chromosome, Tn7 transposition is stimulated and insertions are directed proximal to the induced break. The targeting preference for the terminus of replication and DNA double-strand breaks is dependent on the Tn7-encoded protein TnsE. The results presented in this study could also explain the previous observation that Tn7 is attracted to events associated with conjugal DNA replication during plasmid DNA transfer.

Streptococcus pyogenes transmission among Air Force recruits: efficacy of surveillance and prophylaxis protocols.

The prevalence of Streptococcus pyogenes in Air Force recruits (n = 10,634) completing basic training at Lackland Air Force Base between November 1, 1993, and March 1, 1994, was determined. Throat cultures were obtained on the second day and the last day of training, approximately 6 weeks later. Although the prevalence on the second day was nearly identical between males (1.99%) and females (1.98%), males with positive cultures increased to 4.30%, whereas females with positive cultures increased to 2.92% at the end of training. Within flights, an increase was noted in male flights when members sought care and subsequently were positive for S. pyogenes. Within these flights, S. pyogenes prevalence significantly increased from 2.79 to 7.20% (p < 0.001) despite antibiotic therapy and prophylaxis treatment measures. Within female flights, the increase was from 2.68 to 3.35%, which was not statistically significant (p < 0.19). A total of 161 trainees were treated for S. pyogenes as a result of positive cultures; remarkably, 27.3% of these were still culture-positive on the last day of training.

Annular phase-matched dual-pump coherent anti-stokes Raman spectroscopy system for the simultaneous detection of nitrogen and methane.

The concentration and pressure dependence of dual-pump coherent anti-Stokes Raman spectroscopy (CARS) signals from nitrogen and methane was investigated. CARS spectra were acquired from a gas cell at pressures of 0.007 to 2.24 MPa and methane concentrations of 0.5 to 50%. The square root of the methane signal intensity divided by the nitrogen signal intensity was found to have a near-linear dependence on methane concentration at all pressures investigated. The pressure dependence of this integrated intensity ratio decreased with increasing pressure and became negligible at the highest pressures tested. The shot-to-shot variation at concentrations determined from single-laser-shot measurements was less than 7%. Single-laser-shot CARS spectra of nitrogen and methane were obtained from the cylinder of a firing direct-injection natural gas engine.

Pressure dependence of laser-induced fluorescence from acetone.

The use of laser-induced fluorescence (LIF) from acetone is becoming increasingly widespread as a diagnostic of mixing processes in both reacting and nonreacting flows. One of the major reasons for its increasing use is that the acetone LIF signal is believed to be nearly independent of pressure because of fast intersystem crossing from the first excited singlet state, from which the fluorescence signal originates, to the first excited triplet state, which does not fluoresce. To evaluate the use of acetone LIF at pressures higher than atmospheric, we have performed a study of acetone LIF in a flowing gas cell at pressures up to 8 atm. We used four different buffer gases: air, nitrogen, methane, and helium. Surprisingly, we find that the acetone fluorescence quantum efficiency increases slightly (~30%-50%) as the buffer-gas pressure increases from 0.6 to 5 atm for all four buffer gases. When the buffer gas is air, we observe a decrease in the acetone fluorescence quantum efficiency as the buffer-gas pressure is increased from 5 to 8 atm; for the other three buffer gases the quantum efficiency is constant to within experimental error in this pressure regime. The observed pressure dependence of the acetone fluorescence signal is explained by use of a four-level model. The increase in the fluorescence quantum efficiency with pressure is probably the result of incomplete vibrational relaxation coupled with an increase in the intersystem crossing rate with increasing vibrational excitation in the first excited singlet manifold.

Nerve blocks for regional anesthesia of the face.

Many trauma repairs and dermatologic procedures performed on the face require anesthesia of the involved area. The specific requirements for anesthesia and vasoconstriction vary, depending on the location, size and depth of the injury or lesion. Direct infiltration of anesthetic is very uncomfortable, and adequate anesthesia may require multiple injections. In addition, direct infiltration of a volume of anesthetic sufficient to produce adequate anesthesia can cause distortion of the tissue, hindering the identification of margins. Intraoral nerve blocks routinely used by dentists can achieve safe and effective regional anesthesia in the anterior portion of the face.

Simplified quantitation of urinary benzoylecgonine in cocaine addiction research and for related pharmacotherapeutic trials.

In clinical trials of potential pharmacotherapies for cocaine addiction, objective determination of subject relapse relies on qualitative urine analysis for benzoylecgonine, the major metabolite of cocaine. Unlike qualitative analysis, quantitative measurement allows differentiation between continued cocaine use and a single use, as well as identification of changes in the quantity of cocaine used at different times. The only quantitative technique that has been used is expensive and not generally feasible. This study was performed to modify an existing qualitative technique for use as a new simple and readily available quantitative method for identifying cocaine use among research subjects. Benzoylecgonine levels in 24-hour urine specimens collected from 11 cocaine-addicted subjects hospitalized in a research setting were measured semi-quantitatively by fluorescence polarization immunoassay. Accurate results required thorough mixing of urine specimens prior to analysis. At admission, eight subjects had urinary benzoylecgonine levels > or = 0.30 microgram/ml, the standard positive/negative cut-off used in qualitative analysis. The mean half-life of benzoylecgonine during initial elimination was 0.46 +/- 0.08 (SEM, n = 8) days. Benzoylecgonine (BE)/creatinine (C) levels remained > or = 0.30 microgramBE/mgC for 4.8 +/- 0.5 (n = 8) days and > or = 0.03 microgramBE/mgC for 10.5 +/- 1.5 (n = 8) days. Relapses in three subjects could be identified by quantitative analysis. This study indicates that quantitation of benzoylecgonine in daily urine specimens provides a sensitive, objective index to cocaine use.

Measuring the effect of the Rorschach color cards (VIII-X) on perceptual accuracy and special scores in differentiating borderline from schizophrenic protocols.

In this study, measures were developed that compared conventional Form accuracy and the ratio of Special Scores on Cards VIII-X (Color Cards) with overall Rorschach scores. Two new measures, a Color Card Perception Difference (CCPD) score and a Special Score Difference (SSD) score were used for this purpose. CCPD and SSD scores were calculated by subtracting component X + % scores and weighted Special Score ratios found on the overall Rorschach from those unique to Cards VIII-X. CCPD scores were found to be highly effective in differentiating a previously diagnosed borderline from a schizophrenic group. CCPD and SSD scores were correlated with MMPI scales for profile characteristics.

Redundant homosexual F transfer facilitates selection-induced reversion of plasmid mutations.

F plasmids use surface exclusion to prevent the redundant entry of additional F plasmids during active growth of the host cells. This mechanism is relaxed during stationary phase and nonlethal selections, allowing homosexual redundant plasmid transfer. Homosexual redundant transfer occurs in stationary-phase liquid cultures, within nongrowing populations on solid media, and on media lacking a carbon source. We examined the relationship between homosexual redundant transfer, which occurs between F+ hosts, and reversion of a plasmid-encoded lac mutant allele, lacI33omegalacZ. Sodium dodecyl sulfate (SDS) and mutations that prevent normal transfer to F- cells reduced redundant transfer and selection-induced reversion of the lacI33omegalacZ allele. A recA null mutation reduced redundant transfer and selection-induced reversion of the lacI33omegalacZ mutation. Conversely, a recD null mutation increased redundant transfer and selection-induced reversion of the lacI33omegalacZ allele. These results suggest an explanation for why SDS and these mutations affect reversion of the plasmid lacI33omegalacZ allele. However, a direct causal relationship between transfer and reversion remains to be established. These results suggest that Rec proteins play an active role in redundant transfer and/or that redundant transfer is regulated with the activation of recombination. Redundant homosexual plasmid transfer during a period of stress may represent a genetic response that facilitates evolution of plasmid-encoded functions through mutation, recombination, reassortment, and dissemination of genetic elements present in the populations.

Characterization of a new rho mutation that relieves polarity of Mu insertions.

We report the identification and characterization of a new rho mutation, rho614, that relieves polarity of Mu insertions in Escherichia coli. The mutation was identified by its ability to suppress the polarity of the Mu-mediated phi(lamB'-'lacZ)hyb61-4 fusion that is located at codon four of the lamB signal sequence. The rho614 mutation alters residue 80 in the proposed RNA-binding domain of Rho and is recessive to wild-type rho. We suggest that in the presence of the rho614 allele transcripts initiated at the Mu promoter PcM fail to terminate at presumptive Rho-dependent termination sites, namely rut1 and rut2, and continue into the 3' 'lamB gene allowing a LamB+ phenotype. This contention is supported by deletion analysis of the region and the observation that insertional inactivation of genes that reduce transcription from PcM, clpP (ATP protease), himA (IHF-alpha), and himD (IHF-beta), block the LamB+ phenotype. rho614, rho4 and rho201 alleles suppress the polarity of a malK::Mu insertion on the downstream lamB gene. However, the polarity of the phi(lamB'-'lacZ)hyb61-4 insertion is only suppressed by the rho614 mutation. We propose that the rho614 mutation allows suppression of transcriptional polarity without interfering with translation initiation signals of the truncated 'lamB gene. In addition to identifying a new rho mutation and Rho-dependent terminator sequence, this system provides a means of studying Rho protein/terminator relationships through the identification of new classes of rho mutations.

Redundant transfer of F' plasmids occurs between Escherichia coli cells during nonlethal selections.

Surface exclusion is the mechanism by which F plasmids prevent the redundant entry of additional F plasmids into the host cell during exponential growth. This mechanism is relaxed in cells that are in stationary phase. Using genetically marked F' plasmids and host strains, we extend this finding to Escherichia coli populations during extended nonlethal selection in bacterial lawns. We show that a high level of redundant transfer occurs between these nongrowing cells during the selection. This result has implications for the mechanism of adaptive mutagenesis.

Availability of reliable serum methadone determination for management of symptomatic patients.

Methadone, when used in the appropriate dose, prevents opioid withdrawal during the 24-hour period following medication. However, the appropriate dose for a given patient may be difficult to determine due to variations in methadone metabolism which is affected by many factors. Early opioid withdrawal, requiring a higher dose of methadone, is often difficult to diagnose because many of the symptoms are also symptoms of other syndromes common in the methadone maintenance population. In this study, ten patients in stable methadone maintenance treatment reporting > or = 4 Himmelsbach signs of abstinence were compared with ten patients reporting fewer symptoms. Until recently, accurate, precise, and affordable determination of serum methadone level has not been readily available from commercial laboratories. This study has found that such measures are now available. Serum specimens from each subject were sent to three commercial laboratories for determination of serum methadone level. Results from the three laboratories were highly correlated. No statistical correlation was found between serum methadone level and number of Himmelsbach signs. Of the subjects reporting four or more symptoms, 40% had low serum methadone levels ( < 150 ng/ml); 60% did not. Of the subjects reporting fewer than four symptoms, 90% had serum methadone levels > or = 150 ng/ml. Subjects with > or = 4 Himmelsbach signs had lower dose-adjusted serum methadone levels, the amount of methadone circulating per mg dose, (t = 1.54, p < .0702). Thus, for patients who report symptoms which could be attributable to opioid withdrawal, measurement of serum methadone level may help to differentiate complaints due to early abstinence from those due to other medical conditions.

Developmental patterns of laryngeal and respiratory function for speech production.

Estimates of subglottal air pressure, laryngeal airflow, and laryngeal airway resistance from syllable repetitions of children and adults were used in describing developmental changes in these variables and in hypothesizing corresponding changes in respiratory function. A trend was found for pressure and resistance to decrease with increases in flow from preschool age through adulthood. These patterns could be explained by the smaller size of laryngeal airway structures and increased expiratory muscle forces during speech in the younger subjects. When the subglottal air pressures were combined with published data on air volume expired during speech and recoil pressures of the respiratory system, hypothetical functions were derived for respiratory muscle forces and the work of speech breathing. These functions predict (a) a developmental shift from the use of net expiratory muscle force in the speech of preschool children to the adult patterns of combined inspiratory and expiratory muscle forces, and (b) the expiratory work of speech breathing is greater in preschool children than in older children and adults.

Real-time molecular epidemiologic analysis of an outbreak of Streptococcus pyogenes invasive disease in US Air Force trainees.

OBJECTIVE: To determine if molecular epidemiologic techniques, including comparative automated DNA sequencing of polymorphic virulence genes, could be used in the course of a bacterial disease outbreak to unambiguously determine clonal relationships among implicated strains. DESIGN: Strains recovered from all patients with invasive infections and a sample of carriers were analyzed by multilocus enzyme electrophoresis and automated DNA sequencing of a gene encoding an extracellular protease and a highly polymorphic part of the streptokinase gene. SETTING: A US Air Force training facility in San Antonio, Tex. PATIENTS: A squadron with about 800 Air Force trainees, including three recruits with invasive Streptococcus pyogenes infections. RESULTS: Multilocus enzyme electrophoresis and automated DNA sequencing of polymorphic virulence genes unambiguously defined person-to-person spread of an otherwise rare S pyogenes clone in the course of the disease outbreak and clarified strain relationships in real time. CONCLUSIONS: Molecular strain characterization techniques can be employed rapidly in a disease outbreak to definitively resolve complex relationships among pathogenic bacteria, infer patterns of clone spread, and help formulate rational public health control measures. The approach has broad applicability to other infectious agents.

Further studies on Pseudomonas aeruginosa LasA: analysis of specificity.

Full elastolytic activity in Pseudomonas aeruginosa is a result of the combined activities of elastase, alkaline proteinase, and the lasA gene product, LasA. The results of this study demonstrate that an active fragment of the LasA protein which is isolated from the culture supernatant fraction is capable of degrading elastin in the absence of elastase, thus showing that LasA is a second elastase produced by this organism. In addition, it is shown that LasA-mediated enhancement of elastolysis results from the separate activities of LasA and elastase upon elastin. The LasA protein does not affect the secretion or activation of a proelastase as previously proposed in other studies. Furthermore, LasA has specific proteolytic capability, as demonstrated by its ability to cleave beta-casein. Preliminary analysis of beta-casein cleavage in the presence of various protease inhibitors suggests that LasA may be classified as a modified serine protease.

Correlates of vestibular evaluation results during the first year of life.

Prenatal and perinatal correlates of abnormal auditory brainstem responses in neonates have been studied extensively. In contrast, vestibular function during the first year of life has received sparse attention. Using a specially modified vestibular test battery, 65 infants (17 low-risk, 48 high-risk) were initially evaluated during their first 6 months of life. Results revealed normal vestibular function in 46 infants (13 low-risk, 33 high-risk) and abnormal findings at either 3 or 6 months in 19 infants (4 low-risk, 15 high-risk). Correlations between vestibular results and variables such as auditory brainstem response results, birth history, and postnatal course in the neonatal intensive care unit were analyzed statistically. While some differences were mildly significant, none were highly significant. The lack of significant correlation between abnormal auditory brainstem response and vestibular results is of particular interest.