Use of metagenomic microbial source tracking to investigate the source of a foodborne outbreak of cryptosporidiosis.

Cryptosporidium is a protozoan parasite of global public health importance that causes gastroenteritis in a variety of vertebrate hosts, with many human outbreaks reported yearly, often from ingestion of contaminated water or food. Despite the major public health implications, little is typically known about sources of contamination of disease outbreaks caused by Cryptosporidium. Here, we study a national foodborne outbreak resulted from infection with Cryptosporidium parvum via romaine lettuce, with the main goal to trace the source of the parasite. To do so, we combined traditional outbreak investigation methods with molecular detection and characterization methods (i.e. PCR based typing, amplicon and shotgun sequencing) of romaine lettuce samples collected at the same farm from which the contaminated food was produced. Using 18S rRNA typing, we detected C. parvum in two out of three lettuce samples, which was supported by detections in the metagenome analysis. Microbial source tracking analysis of the lettuce samples suggested sewage water as a likely source of the contamination, albeit with some uncertainty. In addition, the high degree of overlap in bacterial species content with a public human gut microbial database corroborated the source tracking results. The combination of traditional and molecular based methods applied here is a promising tool for future source tracking investigations of food- and waterborne outbreaks of Cryptosporidium spp. and can help to control and mitigate contamination risks.

Nitric oxide production in the sphenoidal sinus by the inducible and constitutive isozymes of nitric oxide synthase.

OBJECTIVE: To study the production of nitric oxide (NO), and the presence of different isoforms of the NO-synthesising enzyme, NO-synthase (NOS), in the paranasal sinus. MATERIALS AND METHODS: Ten patients, undergoing surgery for pituitary adenoma, were examined for the presence of NO gas in the sphenoidal and maxillary sinus. The distribution of different NOS isozymes in mucosal biopsies from sphenoid and maxillary sinus and ethmoidal cells was studied. RESULTS: The mean concentration of NO was 2575 ppb in the sphenoidal sinus and 6792 ppb in the maxillary sinus. Morphological analyses revealed intense NADPH-diaphorase staining throughout the epithelium. Immunoreactivity against NOS2 (inducible NOS) was observed in the apical cell layer but not of the basal layer. NOS1 (neuronal NOS)-immunoreactivity was mainly seen in the subapical part of the epithelium and NOS3 (endothelial NOS)-immunoreactivity was observed only in the most apical part of the epithelium. CONCLUSION: NO concentration in the sphenoidal sinus is about the same as in the nasal cavity and approximately half of the concentration found in the maxillary sinus. All of the three main different isozymes of NOS can be demonstrated in the mucosa of the sphenoidal and maxillary sinus and ethmoidal cells, NOS2 being the most abundant isoform.

Shotgun phage display cloning.

Shotgun phage display cloning is a useful tool for studying interactions between bacterial and host proteins. Libraries are constructed by cloning randomly fragmented prokaryotic DNA into phage mid-vectors. Theoretically, these libraries will consist of phages that together display all proteins encoded by the bacterial genome. Selecting a gene III-based library, made from Staphylococcus aureus DNA, against IgG and fibronectin resulted in 20-40% positive clones after two pannings. Increasing the number of fusion proteins per phage particle by using gene VIII-based display, increased the frequency of correct clones to 75-100%.

A primordial tRNA modification required for the evolution of life?

The evolution of reading frame maintenance must have been an early event, and presumably preceded the emergence of the three domains Archaea, Bacteria and Eukarya. Features evolved early in reading frame maintenance may still exist in present-day organisms. We show that one such feature may be the modified nucleoside 1-methylguanosine (m(1)G37), which prevents frameshifting and is present adjacent to and 3' of the anticodon (position 37) in the same subset of tRNAs from all organisms, including that with the smallest sequenced genome (Mycoplasma genitalium), and organelles. We have identified the genes encoding the enzyme tRNA(m(1)G37)methyltransferase from all three domains. We also show that they are orthologues, and suggest that they originated from a primordial gene. Lack of m(1)G37 severely impairs the growth of a bacterium and a eukaryote to a similar degree. Yeast tRNA(m(1)G37)methyltransferase also synthesizes 1-methylinosine and participates in the formation of the Y-base (yW). Our results suggest that m(1)G37 existed in tRNA before the divergence of the three domains, and that a tRNA(m(1)G37)methyltrans ferase is part of the minimal set of gene products required for life.

Expression of staphylococcal protein Sbi is induced by human IgG.

Protein Sbi is an IgG- and beta(2) glycoprotein I-binding protein on the surface of Staphylococcus aureus. In most strains, the amount of protein Sbi on the cell surface is very low under normal growth conditions. However, here we show that after growth in the presence of human serum, the amount is significantly increased. In S. aureus strain 8325-4, the observed increase is concentration-dependent and the highest level is found approximately 2 h after serum addition. The active molecule in serum was found to be IgG, which causes an increase of surface-located protein Sbi in S. aureus strain 8325-4, in the clinical isolates tested as well as in protein A-negative mutants. Thus, the results suggest that binding of IgG to protein Sbi upregulates protein Sbi synthesis.

Molecular identification of species from the Penicillium roqueforti group associated with spoiled animal feed.

The Penicillium roqueforti group has recently been split into three species, P. roqueforti, Penicillium carneum, and Penicillium paneum, on the basis of differences in ribosomal DNA sequences and secondary metabolite profiles. We reevaluated the taxonomic identity of 52 livestock feed isolates from Sweden, previously identified by morphology as P. roqueforti, by comparing the sequences of the ribosomal internal transcribed spacer region. Identities were confirmed with random amplified polymorphic DNA analysis and secondary metabolite profiles. Of these isolates, 48 were P. roqueforti, 2 were P. paneum, and 2 were Penicillium expansum. No P. carneum isolates were found. The three species produce different mycotoxins, but no obvious relationship between mold and animal disease was detected, based on medical records. P. roqueforti appears to dominate in silage, but the ecological and toxicological importance of P. carneum and P. paneum as feed spoilage fungi is not clear. This is the first report of P. expansum in silage.

Gene VIII-based, phage-display vectors for selection against complex mixtures of ligands.

Selection of shotgun phage-display libraries against complex mixtures of components, such as cells or sera, may result in a high number of nonspecifically binding phage. Consequently, correct interactions may be difficult to identify. To enable discrimination between faithful and nonspecific interactions, a set of eight different gene VIII-based, phage-display vectors were constructed. All vectors contain a "universal" screening tag positioned in such a way that it is only expressed when the inserted DNA encodes an open reading frame, which corrects a shift of reading frames in the vector. A Staphylococcus aureus shotgun phage-display library was made in a stoichiometric mixture of all vectors. After affinity-selection against IgG, one vector completely outcompeted the others. This vector contains the promoter and signal sequence from the gene encoding staphylococcal protein A and one suppressible stop codon immediately upstream of gene VIII. An increase in the frequency of clones expressing the affinity tag in all pannings correlated with selection for ligand-binding clones. This enables detection of putatively correct clones after selection of a shotgun phage-display library both against purified ligands and more complex materials like calf serum.

Shot-gun phage display mapping of two streptococcal cell-surface proteins.

We have used a phage display shot-gun cloning technique to map the binding domains in two cell surface proteins from animal group C streptococci. The proteins, MAG and ZAG, have affinity for alpha (2)-macroglobulin (alpha (2)M), serum albumin and IgG. In this work, parts of cloned i mag and zag genes were randomly cloned into a phagemid vector, and recombinant phages expressing alpha (2)-M- or albumin-binding activity were isolated through panning against immobilized alpha (2)M or albumin. Analysis of the clones revealed two distinct alpha (2)M-binding sites in protein MAG and two slightly overlapping binding sites in protein ZAG. The minimal albumin-binding domain in protein ZAG, as deduced from the affinity selected clones, consisted of 42 amino acids. These results show that the phage display shot-gun cloning is a rapid and convenient way to characterize the binding site(s) in receptor proteins without any prior knowledge of their number, size, and localization.

Fibronectin-binding protein of Streptococcus equi subsp. zooepidemicus.

By screening a genomic lambda library of Streptococcus equi subsp. zooepidemicus, we have cloned and sequenced a gene, termed fnz, encoding a fibronectin (Fn)-binding protein called FNZ. On the basis of the deduced amino acid sequence of FNZ, the mature protein has a molecular mass of approximately 61 kDa. Analysis of FNZ reveals a structural organization similar to that of other cell surface proteins from streptococci and staphylococci. The Fn-binding activity is localized to two domains in the C-terminal part of FNZ. One domain is composed of five repeats, which contain a motif similar to what has earlier been found in other Fn-binding proteins in streptococci and staphylococci. The first and second repeats are separated by a short stretch of amino acids, including the motif LAGESGET, which is an important part of the second Fn-binding domain. This motif is also present in an Fn-binding domain (UR) in protein F of Streptococcus pyogenes. A fusion protein covering the Fn-binding domain of FNZ inhibits the binding of the 29-kDa N-terminal fragment of Fn to cells of various streptococcal species as well as to Staphylococcus aureus.

Phage display shot-gun cloning of ligand-binding domains of prokaryotic receptors approaches 100% correct clones.

We recently presented an application of the phage display technique enabling cloning of DNA encoding ligand-binding domain(s) of prokaryotic receptors directly from chromosomal DNA. Here we show that the use of a gene VIII-based, instead of a gene III-based, phagemid vector system results in a much more efficient selection for phage displaying a binding capacity. A phagemid library was made by insertion of randomly fragmented chromosomal DNA from Staphylococcus aureus strain 8325-4 into gene VIII in the constructed phagemid vector pG8H6. The library, which in theory should express parts of all proteins encoded by the bacterial genome, was affinity panned against the ligands IgG, fibronectin and fibrinogen, respectively. After a second panning against the same ligand, a significant increase in the number of eluted phagemid particles was observed, and 75%-100% of randomly picked clones contained inserts derived from genes encoding proteins with a binding affinity for the respective ligand. The results show that this technique can be used for cloning prokaryotic receptor genes without any prior knowledge of the receptor, thus eliminating the need for probes in the identification of receptor genes.

Comparison of some criteria for diagnosing a myocardial infarction after aorto-coronary bypass grafting (CABG).

The incidence of myocardial infarction during a 1-year follow-up period after coronary bypass surgery (CABG), i.e. a recent myocardial infarction (RMI), was studied in 86 patients. Different criteria for the diagnosis of a RMI were compared. Clinical observation, including ECG and serum enzyme analysis, diagnosed RMI in 8% of patients. Specific ECG changes indicating RMI (ECGsp) occurred in 12% of cases, and if less specific ECG changes were also taken into account (ECGsp+nonsp) RMI was found in 30% of cases. Asynergy, detected by two-plane ventriculography, indicated RMI in 24% of the patients, and was probably the most valid of the criteria examined. The differences in diagnostic accuracy of the various criteria highlight the importance of defining diagnostic criteria.

Comparative antibacterial activity of L-695,256, a carbapenem active against methicillin-resistant staphylococci.

The activity of a new prototype carbapenem, L-695,256, against clinical isolates of gram-positive and gram-negative aerobes was studied in vitro by agar dilution. L-695,256 was highly active against methicillin-resistant and -susceptible isolates of staphylococci (MICs, 0.016 to 2 micrograms/ml) and against penicillin-resistant pneumococci (MICs, 0.016 to 0.064 micrograms/ml), irrespective of penicillin susceptibility. Activity against members of the family Enterobacteriaceae was less than that of imipenem, while Proteus mirabilis and Morganella morganii were more susceptible to L-695,256.

Cloning of ligand-binding domains of bacterial receptors by phage display.

We present an application of the phage display technique which makes it possible, through affinity selection, to clone the part of a prokaryotic receptor gene that encodes the ligand-binding domain. A phage display library was constructed by insertion of randomly fragmented chromosomal DNA from Staphylococcus aureus strain 8325-4 into the phagemid vector pHEN1. Domains of the genes encoding staphylococcal protein A and fibronectin binding proteins were isolated from the library by affinity panning of the phage against the immobilized ligands. Approximately 1%-10% of the eluted phage encoded polypeptides that specifically bound the respective ligand. Nucleotide sequences of the isolated clones were in agreement with earlier known sequences of domains encoding the IgG and fibronectin-binding proteins. In addition, a second, so far unknown, nucleotide sequence encoding an IgG-binding polypeptide was identified.

Salmonella isolated from animals and feedstuffs in Sweden during 1988-1992.

The present paper surveys the number of Salmonella isolations in animals and feedstuffs in Sweden during 1988-1992. It is the eighth in a series of reports published by the National Veterinary Institute (NVI) since 1949. During the period referred to, 602 outbreaks of Salmonella were reported in animals, both domestic and wild. Compared with the previous 5-year period there was a 20% reduction in the number of outbreaks (760). Fifty-six different serotypes were reported, 19 of which had never been isolated in any animal in Sweden previously. A temporary increase in the number of outbreaks in poultry was seen in 1991 following an extended sampling before slaughter of layers. A remarkably high prevalence (38%) of Salmonella was observed in snakes in the wild. In 1990, the end-point testing of feeds was replaced by an approach based on HACCP (Hazard Analysis Critical Control Point) principles for the monitoring of feed mills. Significantly higher number of Salmonella positive samples were found by using this technique compared with the previous analysis of finished feed. It is concluded that the adopted Salmonella control program has contributed to a reduced number of Salmonella outbreaks in animals in Sweden.

Effects of digitalis, anti-anginal drugs and arterial hypertension on the relationships between exercise ECG and angiocardiographic variables and on the diagnostic accuracy of exercise ECG in patients with coronary heart disease.

We have previously found a statistically significant correlation between some exercise ECG variables and angiocardiographic scores used to evaluate the extent and type of coronary heart disease (CHD). In the present study we examined the effects of digitalis, beta-adrenergic blockers, slow release nitrates, calcium channel blockers, presence of arterial hypertension (AHT), and angiocardiographic changes on the exercise ECG variables. The effects of drugs and AHT were small as compared to the effects of the angiocardiographically detected pathological changes caused by the CHD. Sensitivity and specificity of the exercise ECG ST criteria in identifying patients with an angiocardiographic criterion indicating coronary insufficiency were not much different in the whole group and in the subsets with AHT or medication with digitalis and anti-anginal drugs.

Risk indicators of ischemic heart disease among male professional drivers in Sweden.

Possible risk indicators of ischemic heart disease relevant to the occupation of professional driving were identified in a cohort of 440 professional drivers and 1000 referents from the Swedish countries of Västerbotten and Norrbotten. The subjects were randomly selected. Data on cardiovascular risk indicators were collected from questionnaires, blood pressure measurements, serum lipid levels, height, and weight. The results showed that significantly more drivers than referents were overweight, smokers, and shift workers; were sedentary in their leisure time; and had a work situation characterized by high demands, low decision latitude, and low social support. There were no significant differences concerning blood pressure and serum lipid levels. The odds ratio for having a high score on a cardiovascular risk index was 3.18 (95% confidence interval 2.41-4.20) for the drivers when they were compared with the referents. When adjusted for age, heredity, shift work, educational level, marital status, and working class, the odds ratio was 2.34 (95% confidence interval 1.70-3.21).

Relationships between exercise ECG and angiocardiographic indices of coronary insufficiency and myocardial fibrosis in coronary heart disease.

Patients with coronary heart disease were examined with exercise ECG and angiocardiography. Maximum work capacity expressed as a percentage of the predicted normal exercise tolerance (Wmax%) was significantly associated with the angiocardiographic score of the myocardial mass subserved by obstructed coronary arteries (MCOS). Variables related to myocardial fibrosis (MF) such as post infarction ECG signs, the left ventricular wall motion score (LVMS) and the ejection fraction of the left ventricle (LVEF) correlated significantly as did variables related to reversible myocardial ischaemia or coronary insufficiency (CI), such as ST depression during exercise (STdepr), ST/W and ST/HR indices, effort angina (EA/W) index, the extent of collaterals (CollS), and 'MCOS-LVMS'. MF variables correlated weakly with CI variables. Wmax% covariated with the variables related to both CI and MF, and most closely with MCOS. Discrepancies between results of exercise ECG and angiocardiography have to some extent been overcome by comparing appropriate parameters.

Acute haemodynamic effects and pharmacokinetics of ramipril in patients with heart failure. A placebo controlled three-dose study.

The aim of the present study was primarily to evaluate the haemodynamic effects of the ACE-inhibitor ramipril which is active via its metabolite ramiprilat. Ramipril 1.25, 2.5 and 5 mg and placebo was administered orally to 4 groups of 12 patients with heart failure (NYHA III) in a double-blind randomised, parallel study. Haemodynamics were monitored for 24 h and blood was sampled and urine collected for up to 96 h. In the placebo-treated group the cardiac index (CI) was significantly increased (15.8%) and right atrial pressure decreased (26.6%). Ramipril 1.25 mg had insignificant haemodynamic effects compared to placebo and the 2.5 mg dose had significant effects on some haemodynamic variables. Ramipril 5 mg had pronounced and sustained effects on pulmonary artery pressure, which fell by 43.7%, and pulmonary capillary wedge pressure (PCWP; -59.1%); systemic vascular resistance was also decreased 21%. A significant effect on CI was only seen after 2.5 mg ramipril (+7.4%). The mean maximal degree of ACE inhibition was 73.2, 90.4 and 98.5%, respectively, after the three doses of ramipril. Complete inhibition of ACE-activity was seen at a mean plasma concentration of ramiprilat of 4.7 ng.ml-1. The degree of inhibition declined with a half life of about 75 h. There was a significant relation between the degree of ACE-inhibition and change in PCWP but not with the change in SVR. Ramipril was mainly eliminated in the form of ramiprilat and inactive metabolites.

Specificity of oligonucleotide probes complementary to evolutionarily variable regions of 16S rRNA from Mycoplasma hyopneumoniae and Mycoplasma hyorhinis.

Mycoplasma is the common name for the smallest free-living microorganisms, the Mollicutes. Mycoplasma hyopneumoniae is of great importance in veterinary medicine, causing enzootic pneumonia in pigs. M hyorhinis can cause polyserositis and may cause pneumonia in piglets. Oligonucleotides complementary to variable regions of 16S rRNA from these mycoplasmas were designed and used as probes for detection and identification of these mycoplasmas. The probe complementary to 16S rRNA of M hyorhinis gave a very weak cross-hybridisation with M hyosynoviae in filter hybridisation experiments, but not with any of the other porcine mycoplasmas tested. Three oligonucleotide probes complementary to M hyopneumoniae 16S rRNA were tested. One of the probes (Mhp6/30) was found to be specific to M hyopneumoniae, but the other two gave cross-hybridisation with M flocculare. Using the Mhp6/30 probe in direct filter hybridisation experiments, it proved possible to detect M hyopneumoniae in lung biopsies from experimentally infected pigs.

Mortality in circulatory diseases, especially ischaemic heart disease, among Swedish professional drivers: a retrospective cohort study.

The aim of the investigation was to study mortality from circulatory diseases, especially ischaemic heart disease (IHD), in a cohort of Swedish professional drivers. The cohort included 1,731 male members of the Swedish Transport Workers Union. During the follow-up period, 1974-1985, 123 drivers died. Information concerning the cause of death was acquired from the Cause-of-Death Register. Standardized Mortality Ratio (SMR) for circulatory diseases and ischaemic heart disease were significantly higher among professional drivers (SMR = 127 and 138, respectively) than in the reference group consisting of Swedish males.