The Pathogen-annotated Tracking Resource Network (PATRN) system: a web-based resource to aid food safety, regulatory science, and investigations of foodborne pathogens and disease.

Investigation of foodborne diseases requires the capture and analysis of time-sensitive information on microbial pathogens that is derived from multiple analytical methods and sources. The web-based Pathogen-annotated Tracking Resource Network (PATRN) system (www.patrn.net) was developed to address the data aggregation, analysis, and communication needs important to the global food safety community for the investigation of foodborne disease. PATRN incorporates a standard vocabulary for describing isolate metadata and provides a representational schema for a prototypic data exchange standard using a novel data loading wizard for aggregation of assay and attribution information. PATRN currently houses expert-curated, high-quality "foundational datasets" consisting of published experimental results from conventional assays and next generation analysis platforms for isolates of Escherichia coli, Listeria monocytogenes, and Salmonella, Shigella, Vibrio and Cronobacter species. A suite of computational tools for data mining, clustering, and graphical representation is available. Within PATRN, the public curated data repository is complemented by a secure private workspace for user-driven analyses, and for sharing data among collaborators. To demonstrate the data curation, loading wizard features, and analytical capabilities of PATRN, three use-case scenarios are presented. Use-case scenario one is a comparison of the distribution and prevalence of plasmid-encoded virulence factor genes among 249 Cronobacter strains with similar attributes to that of nine Cronobacter isolates from recent cases obtained between March and October, 2010-2011. To highlight PATRN's data management and trend finding tools, analysis of datasets, stored in PATRN as part of an ongoing surveillance project to identify the predominant molecular serogroups among Cronobacter sakazakii isolates observed in the USA is shown. Use-case scenario two demonstrates the secure workspace available for private users to upload and analyze sensitive data, and for collating cross-platform datasets to identify and validate congruent datapoints. SNP datasets from WGS assemblies and pan-genome microarrays are analyzed in a combinatorial fashion to determine relatedness of 33 Salmonella enterica strains to six strains collected as part of an outbreak investigation. Use-case scenario three utilizes published surveillance results that describe the incidence and sources of O157:H7 E. coli isolates associated with a produce pre-harvest surveillance study that occurred during 2002-2006. In summary, PATRN is a web-based integrated platform containing tools for the management, analysis and visualization of data about foodborne pathogens.

Three R's of bacterial evolution: how replication, repair, and recombination frame the origin of species.

The genetic diversity of bacteria results not only from errors in DNA replication and repair but from horizontal exchange and recombination of DNA sequences from similar and disparate species as well. New individuals carrying adaptive changes are thus being spawned constantly among the population at large. When new selection pressures appear, these are the individuals that survive, at the expense of the general population, to forge new populations. Depending on the severity and uniqueness of the selection pressure, this could lead to new speciation. It is becoming more and more evident that, as nucleotide sequences of numerous loci from many bacterial strains continue to amass, horizontal transfer has played a key role in configuring the Escherichia coli chromosome. Here, we examine views, both old and new, for the role of recombination in the evolution of bacterial chromosomes. We present novel phylogenetic evidence for horizontal transfer of three genes involved in DNA replication and repair (mutS, uvrD, and polA). These data reveal a prominent role for horizontal transfer in the evolution of genes known to play a key role in the fidelity of DNA replication and, thus, ultimate survival of the organism. Our data underscore that recombination plays both a diversifying and a homogenizing role in defining the structure of the E. coli genome.

Phylogenetic evidence for horizontal transfer of mutS alleles among naturally occurring Escherichia coli strains.

mutS mutators accelerate the bacterial mutation rate 100- to 1,000-fold and relax the barriers that normally restrict homeologous recombination. These mutators thus afford the opportunity for horizontal exchange of DNA between disparate strains. While much is known regarding the mutS phenotype, the evolutionary structure of the mutS(+) gene in Escherichia coli remains unclear. The physical proximity of mutS to an adjacent polymorphic region of the chromosome suggests that this gene itself may be subject to horizontal transfer and recombination events. To test this notion, a phylogenetic approach was employed that compared gene phylogeny to strain phylogeny, making it possible to identify E. coli strains in which mutS alleles have recombined. Comparison of mutS phylogeny against predicted E. coli "whole-chromosome" phylogenies (derived from multilocus enzyme electrophoresis and mdh sequences) revealed striking levels of phylogenetic discordance among mutS alleles and their respective strains. We interpret these incongruences as signatures of horizontal exchange among mutS alleles. Examination of additional sites surrounding mutS also revealed incongruous distributions compared to E. coli strain phylogeny. This suggests that other regional sequences are equally subject to horizontal transfer, supporting the hypothesis that the 61.5-min mutS-rpoS region is a recombinational hot spot within the E. coli chromosome. Furthermore, these data are consistent with a mechanism for stabilizing adaptive changes promoted by mutS mutators through rescue of defective mutS alleles with wild-type sequences.

Effect of regular physical exercise on resting nasal resistance.

OBJECTIVE: This study was conducted to determine (1) if long-term regular training changes resting nasal resistance in humans and (2) if the changes are related to the structural component or mucosal component of nasal resistance. METHODS: We used a case-control study to compare a group of 16 athletes to 15 sedentary people of similar age. Nasal resistance was measured by computerized head-out body plethysmograph posterior rhinometry. Physical activity was evaluated by the Baecke questionnaire. RESULTS: The p values (t-test) were very significant for the Baecke sports and total scores (p < .0001) but not for the other variables: age, untreated nasal resistances, decongested nasal resistances, and Baecke work and leisure scores. There were no significant correlations between nasal resistances and indexes of physical activity in all subjects (Pearson's correlation coefficient). The subjects with extremely low and high sports and total scores were paired and studied with the Signed test and the Wilcoxon signed rank test. No significant relationship was found between the nasal resistances and the Baecke scores. CONCLUSIONS: Resting nasal resistances in a group of endurance-trained athletes are identical to those found in a group of sedentary individuals, and this relationship stands for both the structural and mucosal components of nasal resistance. A new study of the same parameters is warranted to follow a cohort of sedentary subjects as they enroll in a physical training program.

Promiscuous origin of a chimeric sequence in the Escherichia coli O157:H7 genome.

A novel sequence of 2.9 kb in the intergenic region between the mutS and rpoS genes of Escherichia coli O157:H7 and closely related strains replaces a sequence of 6.1 kb in E. coli K-12 strains. At the same locus in Shigella dysenteriae type 1, a sequence identical to that in O157:H7 is bounded by the IS1 insertion sequence element. Extensive polymorphism in the mutS-rpoS chromosomal region is indicative of horizontal transfer events.

Argyremia in septal cauterization with silver nitrate.

OBJECTIVE: The purpose of this study was to demonstrate silver absorption in blood and hair specimens after septal cauterization with silver nitrate and to discuss the potential toxicity of silver. METHOD: A prospective study of 11 volunteers without any known occupational exposure to silver products or past history of septal cauterization with silver nitrate was undertaken. Subjects were recruited in an academic tertiary care centre from October 1996 to September 1997. The study population consisted of five patients with anterior epistaxis and six healthy volunteers without any bleeding problem. Cauterization was done with one or two silver nitrate applicators directly on the bleeding vessel or Kiesselbach's area. Blood was sampled before cauterization and at specified times after application, while hair strands were sampled only 3 months later. Measurements of silver concentration in whole blood and in hair segments were obtained by inductively coupled plasma mass spectrometry. RESULTS: Silver concentrations in whole blood increased significantly after cauterization (p = .02). The measured peak level seemed to correlate with the number of applicators used. No significant increase in silver concentration was observed in hair samples. CONCLUSIONS: Effective silver absorption occurs with only one or two silver nitrate applicators. Hair has not been as reliable as whole blood to document an acute and fragmentary exposure. The indiscriminate use of silver nitrate is a potential source of silver intoxication.

Trends in hospital stay for five congenital malformations in the province of Quebec (1983-1995).

OBJECTIVE: The aim of this study was to examine the evolution of hospital stays and utilization of outpatient facilities for the treatment of five congenital malformations (choanal atresia, isolated cleft lip, isolated cleft palate, branchial cyst-sinus and thyroglossal duct cyst-sinus). METHOD: A retrospective review of MED-ECHO data for the province of Quebec from 1983 to 1995 was conducted. RESULTS: For three of the five studied malformations (cleft palate, branchial cyst-sinus, and thyroglossal duct cyst-sinus), there has been a trend toward reduced hospital stay and increased utilization of out-patient facilities. CONCLUSION: There has been a general tendency to reduce hospital stays even in procedures with potential airway and bleeding complications.

Consensus on ambulatory care among Quebec otolaryngologists.

OBJECTIVES: The aims of this study were to obtain consensus on the suitability (or nonsuitability) of outpatient facilities for 15 ENT surgical procedures, to identify the factors involved in the decision making, and to use the Delphi method to evaluate it for this purpose. METHOD: A prospective study of 26 practising otolaryngologists in the Province of Quebec, using the Delphi method (3 rounds), was conducted. Fifteen adult and nine paediatric surgical procedures were included. RESULTS: Consensus (85% agreement) was obtained for eight procedures: septoplasty, rhinoplasty, endoscopic sinus surgery, otoplasty, mastotympanoplasty, lymph node biopsy, and branchial cyst excision (outpatient procedures) and tracheostomy (inpatient procedure). CONCLUSION: The Delphi method has been useful in initiating a process of rationalization about outpatient surgery.

Detection of mutator subpopulations in Salmonella typhimurium LT2 by reversion of his alleles.

Defects in the methyl-directed mismatch repair lead to both the hypermutability phenotype and removal of a barrier to genetic exchange between species. Mutator bacteria carrying such defects occur frequently among bacterial pathogens, suggesting that subpopulations of mutators are contained within pathogen clones and give rise to the genetic variants that are acted upon by selective forces to allow survival or successful infection. We report here on the detection of the mutator subpopulation in Salmonella typhimurium and determination of its frequency in laboratory cultures. The analysis involved screening for mutators among revertants of S. typhimurium histidine auxotrophs selected for the His+ phenotype, since the frequency of mutators is expected to be increased in the selected mutant population they helped to spawn. The increases in spontaneous reversion of histidine mutations were first measured in isogenic strains carrying mismatch repair-defective mutH, mutL, mutS, or uvrD alleles, relative to their mismatch repair-proficient counterparts. Screening for the mutator phenotype in nearly 12,000 revertants of repair-proficient strains carrying his mutations highly stimulated for reversion in mutator backgrounds, the base substitution in hisG428 and frameshift in hisC3076, yielded five mutator strains (0.04%). The his+ reversion mutations contained within the newly-arisen mutator strains were characteristic of the predominant nucleotide changes expected in such mutators, as assessed by comparison with the spectra for reversion events in wild-type and mismatch correction-defective backgrounds. The results show that subpopulations of mutators, residing in normal populations at a finite frequency, can be culled from the culture by strong selection for a required phenotype. We calculate that the frequency of mutators in the unselected population of S. typhimurium is 1-4x10-6, an incidence 10-fold lower than that expected based on studies of laboratory cultures of Escherichia coli.

High mutation frequencies among Escherichia coli and Salmonella pathogens.

Here it is reported that the incidence of mutators among isolates of pathogenic Escherichia coli and Salmonella enterica is high (over 1 percent). These findings counter the theory, founded on studies with laboratory-attenuated strains, that suggests mutators are rare among bacterial populations. Defects in methyl-directed mismatch repair underlie all mutator phenotypes described here. Of nine independently derived hypermutable strains, seven contained a defective mutS allele. Because these mutant alleles increase the mutation rate and enhance recombination among diverse species, these studies may help explain both the rapid emergence of antibiotic resistance and the penetrance of virulence genes within the prokaryotic community.

The thymine-thymine pyrimidine-pyrimidone(6-4) ultraviolet light photoproduct is highly mutagenic and specifically induces 3' thymine-to-cytosine transitions in Escherichia coli.

We have constructed single-stranded, M13-based vectors that contain a specifically located thymine-thymine pyrimidine-pyrimidone(6-4) UV photoproduct and have used these to estimate the frequency and accuracy of DNA replication past this adduct in uvrA6 cells of Escherichia coli. Both the normal and the Dewar valence photoisomer of the (6-4) adduct were studied. In the absence of SOS induction, vectors carrying the photoproducts were rarely replicated; relative to the lesion-free control, 1.9% of vectors carrying the normal (6-4) isomer produced plaques, and with the Dewar valence isomer the proportion was 0.4%. In SOS-induced cells, these frequencies rose to 22.1% and 12.3%, respectively. The error frequency of replication past the normal isomer in SOS-induced cells was high; in a random sample of 185 progeny phage analyzed, 169 (91%) contained mutations, all of which were targeted. Equally striking, a high proportion of the mutations (158/169; 93%) were of only one type, namely 3' T----C transitions. Both the error frequency and the specificity were much reduced with the Dewar valence isomer; overall, 74/140 (53%) of the phage analyzed were mutant, and of these only 34 (46%) entailed the 3' T----C transition. We speculate that the high error frequency and specificity arise from the formation of a stable T-G base pair, involving hydrogen bonds at O-2 and N-3 in the pyrimidone ring. Potential hydrogen bonds at these sites are coplanar in the normal but not in the Dewar isomer, perhaps explaining the reduced specificity of mutagenesis with the latter adduct.

T-T cyclobutane dimers are misinstructive, rather than non-instructive, mutagenic lesions.

The lesions produced by SOS-dependent mutagens in Escherichia coli are commonly referred to as nonpairing or non-instructive. Although these terms are likely to be appropriate for some lesions, particularly the abasic site, for others, such as the cyclobutane dimer, their suitability is open to question. To address this question, we have compared the error frequencies and spectra that result when a uniquely located T-T sequence, carried in a single-stranded vector, contains either a cis-syn or a trans-syn cyclobutane dimer, or when either the 5'T or 3'T is converted to an abasic site. The data suggest that the high accuracy with which the dimer-containing templates are replicated is unlikely to be the consequence of polymerase preference for the non-instructive insertion of dAMP. Similarly, mispairing, rather than non-pairing, is likely to cause mutations. Cyclobutane dimers seem therefore to be misinstructive rather than non-instructive lesions, and the common feature shared by SOS-inducing lesions is more their ability to block replication than inability to form correct base pairs.

SOS-dependent replication past a single trans-syn T-T cyclobutane dimer gives a different mutation spectrum and increased error rate compared with replication past this lesion in uninduced cells.

We have transfected SOS-induced and uninduced cells of a uvrA6 strain of Escherichia coli with single-stranded M13mp7-based vectors that carried a single trans-syn T-T cyclobutane dimer at a unique site. Unlike constructs carrying the cis-syn isomer of this lesion, these vectors could be replicated with modest efficiency (14%) in the absence of SOS induction and therefore provided an opportunity to measure directly the influence of such induction on error rate and mutation spectrum. We found that translesion synthesis in the absence of SOS induction was remarkably accurate; only 4% of the replicated bacteriophage contained mutations, which were exclusively targeted single T deletions. In SOS-induced cells, error frequency increased to 11% and the resulting mutations included targeted substitutions and near-targeted single base additions, as well as the T deletions. Replication efficiency was 29% in these conditions. SOS induction therefore leads not only to an enhanced capacity to replicate damaged DNA but also to a marked change in mutation frequency and spectrum.

Mutation frequency and spectrum resulting from a single abasic site in a single-stranded vector.

We have investigated the mutagenic properties of an abasic site in DNA by transfecting SOS-induced and uninduced cells of E. coli with a single-stranded M13mp7-based vector that carries a single example of this lesion at one or other of two unique and adjacent sites. Random samples of progeny phage were sequenced to determine the nature of the replication events that occurred at and around these locations. 5% to 7% of the vectors could be replicated in SOS-induced cells, but only 0.1% to 0.7% of them gave plaques in the absence of SOS induction. In SOS-induced cells, 93% and 96% of the phage replicated resulted from the insertion of a nucleotide opposite the abasic site, while the remainder resulted from a targeted omission of a single nucleotide. At one of the sites, nucleotide insertions were 54% dAMP, 25% dTMP, 20% dGMP and 1% dCMP. At the other site they were 80% dAMP, 4% dTMP, 15% dGMP and 1% dCMP. The sequence variation in all but two of the 204 sequences analyzed was restricted to the abasic site itself. In the remaining two, a change at the abasic site was accompanied by a mutation at an immediately flanking nucleotide.

Mutational analysis of the lac regulatory region: second-site changes that activate mutant promoters.

Second-site mutations that restored activity to severe lacP1 down-promoter mutants were isolated. This was accomplished by using a bacteriophage f1 vector containing a fusion of the mutant E. coli lac promoters with the structural gene for chloramphenicol acetyltransferase (CAT), so that a system was provided for selecting phage revertants (or pseudorevertants) that conferred resistance of phage-infected cells to chloramphenicol. Among the second-site changes that relieved defects in mutant lac promoters, the only one that restored lacP1 activity was a T----G substitution at position -14, a weakly conserved site in E. coli promoters. Three other sequence changes, G----A at -2, A----T at +1, and C----A at +10, activated nascent promoters in the lac regulatory region. The nascent promoters conformed to the consensus rule, that activity is gained by sequence changes toward homology with consensus sequences at the -35 and -10 regions of the promoter. However, the relative activities of some promoters cannot be explained solely by consideration of their conserved sequence elements.

Frequency and spectrum of mutations produced by a single cis-syn thymine-thymine cyclobutane dimer in a single-stranded vector.

We have constructed a single-stranded vector that contains a uniquely located cis-syn T-T cyclobutane dimer by ligating a synthetic oligomer containing this UV photoproduct into M13mp7 viral DNA linearized with EcoRI. In the absence of SOS induction, transfection of a uvrA6 mutant of Escherichia coli with this vector gave very few progeny plaques, and the data imply that a single dimer blocks replication in at least 99.5% of the molecules. In vitro photoreactivation completely abolished this inhibition. Transfection of cells irradiated with UV at 4 J.m-2 to induce the SOS response gave 27% of the number of plaques found with a dimer-free control. Nucleotide sequence analysis of 529 progeny phage showed that translesion synthesis was usually accurate: the normal sequence was found in 93% of them. Where mutations occurred, all were targeted single-nucleotide substitutions, with approximately 90% being targeted at the 3' nucleotide of the lesion: of a total of 26 mutations, 15 were 3' T----A, 8 were 3' T----C, and 3 were 5' T----C. No T----G mutations were found. In addition to these results with the normal construct, data were also obtained from vectors in which the M13mp7 cloning site, which forms a hairpin in single-stranded DNA, was present 4 nucleotides on the 3' side of the T-T dimer. These hairpin-containing vectors gave a very similar mutation frequency (8% versus 7%) but altered mutation spectrum: all 12 mutations detected were 3' T----A transversions, a difference from the previous set of data that is significant (P = 0.03).

Ultraviolet light induces different spectra of lacI sequence changes in vegetative and conjugating cells of Escherichia coli.

We have analyzed the nucleotide sequence changes responsible for mutations from lacIs to lacI- induced in ultraviolet light-irradiated, excision-deficient cells. Irradiated cells were either used as donors in the conjugational transfer of an F' lacIs plasmid to SOS-induced, excision-deficient recipients or allowed to continue vegetative growth. Although the types and proportions of premutagenic lesions are likely to have been very similar in these two circumstances, analysis of the sequence data shows that different spectra of mutations were induced. In vegetative cells there were about equal numbers of transitions and transversions, but transitions outnumbered transversions by about three to one in exconjugants. About 90% of the single nucleotide substitutions could be assigned to a bipyrimidine target sequence in both sets of data, but they differed with respect to the location of the substitution: more or less equal numbers were found at the 3' and 5' sites of the probable bipyrimidine target in vegetative cells, but in exconjugants over 80% were at the 3' site. It is also possible that mutations were targeted more commonly at T-C sequences in exconjugants than in vegetative cells, but the evidence for this is less secure. We conclude that these results reflect some dissimilarity between vegetative cells and exconjugants in the way damaged DNA is replicated or lesions tolerated, but the particular features of these processes responsible for the different mutational spectra have not yet been identified.

UmuC function is not essential for the production of all targeted lacI mutations induced by ultraviolet light.

Up to a quarter or more of the normal yield of lacI- mutations could be induced by ultraviolet light in a uvrA6 umuC122:: Tn5 strain if they were detected by plating on 5-bromo-4-chloro-3-indolyl-beta-D-galactoside medium, where all surviving cells can form colonies. Using phenyl beta-D-galactoside selection, which curtails post-irradiation growth, only low yields of mutations were induced. Nucleotide sequence analysis of 134 spontaneous and 145 ultraviolet light-induced mutations shows that broadly similar kinds of mutations were induced in the umuC mutant and its uvrA6 umuC+ counterpart. In particular, these data offer no reason for believing that most of the mutations induced in the umuC mutant were other than normal, targeted events. We conclude that UmuC function, rather than being essential, facilitates recovery and specifically, following the model of Bridges & Woodgate, that it facilitates the prompt resumption of chain elongation.