Experimental Search for Neutron to Mirror Neutron Oscillations as an Explanation of the Neutron Lifetime Anomaly.

An unexplained >4σ discrepancy persists between "beam" and "bottle" measurements of the neutron lifetime. A new model proposed that conversions of neutrons n into mirror neutrons n^{'}, part of a dark mirror sector, can increase the apparent neutron lifetime by 1% via a small mass splitting Δm between n and n^{'} inside the 4.6 T magnetic field of the National Institute of Standards and Technology Beam Lifetime experiment. A search for neutron conversions in a 6.6 T magnetic field was performed at the Spallation Neutron Source which excludes this explanation for the neutron lifetime discrepancy.

Shuttle mutagenesis: a mini-transposon for producing PhoA fusions with exported proteins in Neisseria gonorrhoeae.

Shuttle mutagenesis is a method for producing stable mini-transposon (mTn) insertions into the genome of Neisseria gonorrhoeae (gonococcus, Gc) and other microbes. Using an mTn3 derivative, we have produced an mTn (mTnCmPhoA) which contains a phoA' gene lacking its N-terminal signal sequence useful for isolating genes which encode exported proteins. mTnCmPhoA was characterized in Gc and Escherichia coli using a cloned target containing the Gc genes, opaE1, pilA and pilB. PhoA+ Gc containing pilB::mTnCmPhoA insertions confirm that PilB is an exported protein in Gc. This system will be useful for isolating and characterizing extracytoplasmic virulence factors from Gc and other bacterial pathogens.

Shuttle mutagenesis: two mini-transposons for gene mapping and for lacZ transcriptional fusions in Neisseria gonorrhoeae.

Shuttle mutagenesis is a system we developed for producing stable transposon insertions in Saccharomyces cerevisiae [Seifert et al., Proc. Natl. Acad. Sci. USA 83 (1986) 735-739; Hoekstra et al., Methods Enzymol. 194 (1991) 329-342] and Neisseria gonorrhoeae (Gc) [Seifert et al., J. Bacteriol. 172 (1990) 40-46] by transposition in Escherichia coli and transformation into yeast or Gc. In developing the system for use in Gc, a series of mini-transposons (mTn) were derived from mTn3 which confer resistance to chloramphenicol in Gc (mTnCm) (Seifert et al., 1990). Herein, we describe the creation of two mTnCm derivatives for use in Gc. One of these transposons, mTnCmNS, contains the infrequently occurring NheI and SpeI restriction sites to localize genes on the gonococcal macro-restriction map which was recently developed using these restriction sites [Bihlmaier et al., Mol. Microbiol. 5 (1991) 2529-2539; Dempsey et al., J. Bacteriol. 173 (1991) 5476-5486]. The mTnCmLac was developed to generate lacZ transcriptional fusions using transposition. It contains at its end a promoterless lacZ gene which is expressed once the element has transposed downstream from a promoter in a cloned gene. In adapting the use of mTnCmLac to the shuttle mutagenesis system, we have identified some factors which affect the transformation of Gc using cloned chromosomal fragments containing the large heterologous insertion, mTnCmLac. Using mTnCmLac, we have created Gc variants containing a pilE::mTnCmLac fusion to determine that pilE transcription in Gc is not auto-regulated.

Transcriptional control of gonococcal pilE expression: involvement of an alternate sigma factor.

The pilE gene encoding Neisseria gonorrhoeae (Gc) pilin contains two putative promoter sequences 5' to the transcription start point (tsp), a Pribnow box and an RpoN-binding consensus sequence. Sequence analysis shows that the nucleotide (nt) sequence of the pilE promoter region is completely conserved among eight different Gc isolates. Using a pilE::lacZ transcriptional fusion, we demonstrate that the RpoN sigma factor can function in Escherichia coli to increase pilE transcription when the NifA activator from Klebsiella is present in trans. In addition, over-production of the native pilin protein using RpoN and NifA is lethal to E. coli. Finally, we show that the RpoN sigma factor decreases the basal expression of pilE when an activator is not present. These results suggest that, in Gc, pilE transcription may be regulated by RpoN in conjunction with an activator and that sigma 70 can also act to direct transcription of pilE.

Large-scale SNP scoring from unamplified genomic DNA.

Discoveries from the Human Genome Project (HGP) continue to spur changes in medical technology that will lead to new diagnostic procedures in the clinical lab. As more single nucleotide polymorphisms (SNPs) are discovered and correlated to human diseases, demands for genetic tests will increase. The enormity of the number of SNPs makes developing inexpensive and reliable high-throughput methods for SNP scoring imperative. High-throughput screening (HTS) means, at a minimum, a production rate of thousands of assays per day. Ideally, the technology will be easy, inexpensive and amenable to automation. The Invader assay offers a simple diagnostic platform to detect single nucleotide changes with high specificity and sensitivity from unamplified, genomic DNA. The Invader assay uses a structure-specific 5' nuclease (or flap endonuclease) to cleave sequence-specific structures in each of two cascading reactions. The cleavage structure forms when two synthetic oligonucleotide probes hybridise in tandem to a target. One of the probes cycles on and off the target and is cut by the nuclease only when the appropriate structure forms. These cleaved probes then participate in a second Invader reaction involving a dye-labelled fluorescence resonance energy transfer (FRET) probe. Cleavage of this FRET probe generates a signal, which can be readily analysed by fluorescence microtitre plate readers. The two cascading reactions amplify the signal significantly; each original target molecule can lead to more than 10(6) cleaved signal probes in one hour. This signal amplification permits identification of single base changes directly from genomic DNA without prior target amplification. The sequences of the oligonucleotide components of the secondary reaction are independent of the target of interest and permit the development of universal secondary reaction components useful to identify any target.

Community-acquired methicillin-resistant Staphylococcus aureus colonization in healthy children attending an outpatient pediatric clinic.

BACKGROUND: We previously showed that children attending an inner city pediatric emergency department were sometimes asymptomatically colonized with clindamycin-susceptible community-acquired methicillin-resistant Staphylococcus aureus (MRSA) and borderline methicillin-resistant S. aureus (BRSA) as well. We wished to ascertain whether healthy children attending an outpatient clinic were colonized with these organisms. Therefore to estimate the prevalence of community-acquired MRSA and BRSA nasal colonization in a well child population, we cultured children attending an inner city pediatric outpatient clinic. STUDY DESIGN: This was a prospective cross-sectional study conducted from January to August, 1999, at a primary care outpatient facility at the University of Chicago. The target population was 500 healthy children < or = 16 years of age who attended this facility to receive well child care. RESULTS: One hundred twenty-two (24.4%) children were colonized with S. aureus. Three of the 122 (2.5%) S. aureus isolates were MRSA; they came from children who lacked predisposing risk factors and were susceptible to clindamycin, gentamicin, trimethoprim-sulfamethoxazole, rifampin and ciprofloxacin. Two (1.6%) additional S. aureus isolates were BRSA; both children had predisposing risk factors for MRSA colonization. The mecA gene was present in the 3 MRSA isolates and absent in both BRSA isolates. CONCLUSIONS: These data document that a reservoir of asymptomatic MRSA colonization exists among healthy children who lack traditional risk factors for MRSA infections.

Current trends in community-acquired methicillin-resistant Staphylococcus aureus at a tertiary care pediatric facility.

BACKGROUND: The prevalence of community-acquired methicillin-resistant Staphylococcus aureus (MRSA) infections increased at the University of Chicago Children's Hospital (UCCH) from 10 per 100,000 admissions from 1988 to 1990 to 259 per 100,000 admissions from 1993 to 1995. Because this increase may have represented a one time occurrence or a limited disease outbreak, we updated our previous observations at UCCH in 1998 and 1999 to see whether this trend had continued. DESIGN: Prospective observational study. RESULTS: Twenty-three hospitalized children had an MRSA isolate during the 1-year study period. Ten were community-acquired, equally distributed between children with predisposing risk factors and those without. The overall prevalence of community-acquired MRSA was 208 per 100,000 admissions. Seven of the 10 community-acquired MRSA isolates were susceptible to clindamycin. Skin and soft tissue infections predominated among the children with a community-acquired MRSA isolate. Pulsed field gel electrophoresis of the 10 community-acquired MRSA isolates revealed 8 distinct patterns; these data suggest that multiple clones were circulating at UCCH. CONCLUSION: MRSA are no longer confined to children with established risk factors. The prevalence of community-acquired MRSA among children without identified risk factors is high in our institution.

Methicillin-resistant and borderline methicillin-resistant asymptomatic Staphylococcus aureus colonization in children without identifiable risk factors.

BACKGROUND: The recent evolution in the epidemiology of methicillin-resistant asymptomatic Staphylococcus aureus (MRSA) infections in children, whereby children without traditional risk factors for MRSA have been hospitalized in increasing numbers, prompted us to establish whether a parallel increase in "asymptomatic" MRSA colonization had occurred. METHODS: We cultured the nares and perineum of 500 children attending our Pediatric Emergency Department. RESULTS: One hundred thirty-two (26.4%) of these children were colonized with S. aureus. Eleven (8.3%) of the S. aureus isolates were MRSA; 4 (36.4%) of the 11 subjects colonized with MRSA had no risk factors. Seven (5.3%) of the 132 S. aureus isolates were borderline methicillin-resistant S. aureus (BRSA); 5 (71.4%) of the 7 subjects colonized with BRSA had no MRSA risk factors. CONCLUSIONS: These findings indicate that MRSA and BRSA isolates are circulating in the community and that MRSA isolates are no longer confined to children with frequent contact with a health care environment.

Antimicrobial resistance in staphylococci. Epidemiology, molecular mechanisms, and clinical relevance.

Staphylococcal infections continue to pose important clinical problems in children and adults. Antibiotic resistance among the staphylococci has rendered therapy of these infections a therapeutic challenge. Despite early, uniform susceptibility to penicillin, staphylococci acquired a gene elaborating beta-lactamase that rendered penicillin inactive and that is borne by nearly all clinical isolates. "Penicillinase-resistant beta-lactams," such as methicillin, were introduced in the early 1960s, but resistance to them has become an increasing concern. The mechanism of the so-called "methicillin resistance" is complex. Moreover, once confined to the ecology of hospitals and other institutions, a recent increase in community-acquired methicillin-resistant S. aureus infections has been observed. Glycopeptides, until now the only uniformly reliable therapeutic modality, have been increasingly used for therapy of staphylococcal infections. The recent recognition of clinical isolates with reduced susceptibility to glycopeptides is of concern.