A pan-European ring trial to validate an International Standard for detection of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus in seafoods.

Globally, vibrios represent an important and well-established group of bacterial foodborne pathogens. The European Commission (EC) mandated the Comite de European Normalisation (CEN) to undertake work to provide validation data for 15 methods in microbiology to support EC legislation. As part of this mandated work programme, merging of ISO/TS 21872-1:2007, which specifies a horizontal method for the detection of V. parahaemolyticus and V. cholerae, and ISO/TS 21872-2:2007, a similar horizontal method for the detection of potentially pathogenic vibrios other than V. cholerae and V. parahaemolyticus was proposed. Both parts of ISO/TS 21872 utilized classical culture-based isolation techniques coupled with biochemical confirmation steps. The work also considered simplification of the biochemical confirmation steps. In addition, because of advances in molecular based methods for identification of human pathogenic Vibrio spp. classical and real-time PCR options were also included within the scope of the validation. These considerations formed the basis of a multi-laboratory validation study with the aim of improving the precision of this ISO technical specification and providing a single ISO standard method to enable detection of these important foodborne Vibrio spp.. To achieve this aim, an international validation study involving 13 laboratories from 9 countries in Europe was conducted in 2013. The results of this validation have enabled integration of the two existing technical specifications targeting the detection of the major foodborne Vibrio spp., simplification of the suite of recommended biochemical identification tests and the introduction of molecular procedures that provide both species level identification and discrimination of putatively pathogenic strains of V. parahaemolyticus by the determination of the presence of theromostable direct and direct related haemolysins. The method performance characteristics generated in this have been included in revised international standard, ISO 21872:2017, published in July 2017.

Multiplex PCR for detection of virulence markers of Vibrio vulnificus.

UNLABELLED: Vibrio vulnificus is a Gram-negative pathogen found in coastal and estuarine waters worldwide that can cause life threatening diseases. Characterization of the vcg (virulence correlated gene) or 16S rRNA alleles is used to distinguish virulent (clinical (C)-type) from presumably avirulent (environmental (E)-type) strains. However, some studies reported a significant number of clinical strains belonging to the E-type. In recent years more potential virulence markers have been identified, that are useful for the identification of potentially pathogenic isolates of the E-type. In this study, we successfully combined detection of pathogenicity region XII, nanA and a mannitol fermentation operon with the virulence associated alleles of the 16S rRNA and vcg genes in one multiplex PCR. Additionally, toxR primers for species confirmation and internal amplification control were included. Validation of multiplex amplification was performed with a total of 132 bacterial strains, including V. vulnificus (n = 71), other Vibrionaceae (n = 50) and non-Vibrio isolates (n = 11). Multiplex PCR showed reliable amplification of four of the five virulence markers with a high sensitivity and specificity. Amplification of the 16S rRNA type B allele was not completely reliable with conventional PCR assays, however, the positive predictive value of this marker was 100 %. SIGNIFICANCE AND IMPACT OF THE STUDY: A multiplex PCR for simultaneous detection and characterization of potentially virulent strains of Vibrio vulnificus was developed and validated. Monitoring programs will benefit from this cost and time effective method when screening large strain collections. Application of the multiplex PCR simplifies determination of risks emanating from V. vulnificus in recreational waters or mussel primary production.

Genetic and phenotypic analysis of Vibrio cholerae non-O1, non-O139 isolated from German and Austrian patients.

Vibrio cholerae belonging to the non-O1, non-O139 serogroups are present in the coastal waters of Germany and in some German and Austrian lakes. These bacteria can cause gastroenteritis and extraintestinal infections, and are transmitted through contaminated food and water. However, non-O1, non-O139 V. cholerae infections are rare in Germany. We studied 18 strains from German and Austrian patients with diarrhea or local infections for their virulence-associated genotype and phenotype to assess their potential for infectivity in anticipation of possible climatic changes that could enhance the transmission of these pathogens. The strains were examined for the presence of genes encoding cholera toxin and toxin-coregulated pilus (TCP), as well as other virulence-associated factors or markers, including hemolysins, repeats-in-toxin (RTX) toxins, Vibrio seventh pandemic islands VSP-1 and VSP-2, and the type III secretion system (TTSS). Phenotypic assays for hemolysin activity, serum resistance, and biofilm formation were also performed. A dendrogram generated by incorporating the results of these analyses revealed genetic differences of the strains correlating with their clinical origin. Non-O1, non-O139 strains from diarrheal patients possessed the TTSS and/or the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin, which were not found in the strains from ear or wound infections. Routine matrix-assisted laser desorption/ionization (MALDI-TOF) mass spectrometry (MS) analysis of all strains provided reliable identification of the species but failed to differentiate between strains or clusters. The results of this study indicate the need for continued surveillance of V. cholerae non-O1, non-O139 in Germany, in view of the predicted increase in the prevalence of Vibrio spp. due to the rise in surface water temperatures.

[Vibrio infections from food and sea water. Introducing the "VibrioNet"]. / Vibrio-Infektionen durch Lebensmittel und Meerwasser. Das Netzwerk "VibrioNet" stellt sich vor.

Vibrio is a genus of bacteria present in surface and coastal waters as well as in marine organisms worldwide. In many countries, pathogenic Vibrio species are a main cause of bacterial diarrhea, which may result from comsumption of contaminated seafood and fish products or from drinking contaminated water. Vibrio infections may also gain in importance in our regions due to global warming and the increase in the world trade of seafood. The research network "VibrioNet" studies pathogenic Vibrios in the marine environment and in seafood consumed by humans as a potential, new emerging zoonotic agent. An assessment of the risk arising from pathogenic non-cholera-vibrios in central Europe is the target of a multidisciplinary research effort. The research network will be strengthened by cooperations with international partners from countries in which Vibrio infections play a major role (Bangladesh, Chile, India, Thailand, and Vietnam).

Characterization of a Yersinia enterocolitica biotype 1A strain harbouring an ail gene.

AIMS: The chromosomal ail gene (attachment and invasion locus) is commonly used as target gene for the detection of pathogenic Y. enterocolitica strains in food testing. The ail PCR does not detect strains of biotype 1A (BT1A), which are regarded as non-pathogenic because BT1A strains lack the virulence plasmid and chromosomally encoded virulence genes. In some recent reports, however, BT1A strains were discovered that harboured the ail gene. We isolated an ail-positive strain and characterized this strain with phenotypic and genotypic methods to study its possible relation to pathogenic Y. enterocolitica strains. METHODS AND RESULTS: The ail region of the BT1A strain was sequenced and compared with the corresponding region of nonpathogenic BT1A strains and pathogenic strains. Pulsed field gel electrophoresis (PFGE) analysis was applied revealing no similarity of the PFGE pattern of this strain to the patterns of pathogenic strains. Virulence-gene-based PCR analyses showed the strain to be positive for ystB, but negative for virulence genes ystA, virF and yadA. Whole-cell MALDI-TOF MS combined with a shrinkage discriminant analysis approach was applied and clearly classified the ail-positive biotype 1A strain within the cluster of BT1A strains. CONCLUSIONS: PCR detection of ail sequences in food matrices should be followed by the isolation of the responsible strain and its characterization using phenotypic or genotypic methods. SIGNIFICANCE AND IMPACT OF THE STUDY: The ail gene may be present in Y. enterocolitica BT1A strains, which are commonly considered as nonpathogenic. Efficient methods such as PCR typing of other virulence genes or rapid MALDI-TOF MS-based bacterial profiling allow a more comprehensive assessment of the pathogenicity potential of Yersinia strains.

Rapid identification and characterization of Vibrio species using whole-cell MALDI-TOF mass spectrometry.

AIMS: Vibrio identification by means of traditional microbiological methods is time consuming because of the many biochemical tests that have to be performed to distinguish closely related species. This work aimed at evaluating the use of MALDI-TOF mass spectrometry for the rapid identification of Vibrio (V.) spp. as an advantageous application to rapidly discriminate the most important Vibrio spp. and distinguish Vibrio spp. from closely related bacterial species like Photobacterium damselae and Grimontia hollisae and other aquatic bacteria like Aeromonas spp. METHODS AND RESULTS: Starting from sub-colony amounts of pure cultures grown on agar plates, a very simple sample preparation procedure was established and combined with a rapid and automated measurement protocol that allowed species identification within minutes. Closely related species like Vibrio alginolyticus and Vibrio parahaemolyticus or Vibrio cholerae and Vibrio mimicus could thus be differentiated by defining signatures of species-identifying biomarker ions (SIBIs). As a reference method for species designation and for determination of relationships between strains with molecular markers, partial rpoB gene sequencing was applied. CONCLUSIONS: The MALDI-TOF MS-based method as well as the rpoB sequence-based approach for Vibrio identification described in this study produced comparable classification results. The construction of phylogenetic trees from MALDI-TOF MS and rpoB sequences revealed a very good congruence of both methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that whole-cell MALDI-TOF MS-based proteometric characterization represents a powerful tool for rapid and accurate classification and identification of Vibrio spp. and related species.

Studies of the efficacy of Enterocoliticin, a phage-tail like bacteriocin, as antimicrobial agent against Yersinia enterocolitica serotype O3 in a cell culture system and in mice.

The efficacy of enterocoliticin, a phage tail-like bacteriocin, as antimicrobial compound against infections with pathogenic Yersinia enterocolitica serotype O3 strains was assessed. In cell cultures, which were infected with the Y. enterocolitica strains 13 169 or 6471/76, bactericidal activity of enterocoliticin was found for bacteria adhering to the surface of eukaryotic cells, whereas bacteria, which had invaded the eukaryotic cells, were not accessible to the bacteriocin. The interaction of enterocoliticin with Y. enterocolitica was further examined in animals. Female BALB/c mice were experimentally infected with the two Y. enterocolitica strains and enterocoliticin was applied as antimicrobial compound by the oral route. Experimental variations concerning the infectious doses of the Y. enterocolitica strains and the time points of application of the bacteriocin were investigated. The increase of the Yersinia CFU titre in animals was retarded at time points shortly after the application of enterocoliticin indicating that the particles were effective on recently introduced Yersinia. The repeated application of enterocoliticin, however, did not prevent the colonization of the gastrointestinal tract by Yersinia.

[The relationship of plasmids from environmental Yersinia isolates and the virulence plasmid of enteropathogenic Yersinia strains]. / Untersuchungen zur Verwandtschaft der Plasmide aus Yersinia-Umweltisolaten und dem Virulenzplasmid enteropathogener Yersinien.

The human pathogenic strains of Yersinia harbour a conserved plasmid carrying the Yop virulon. The virulence plasmid of Yersinia enterocolitica strains belonging to the serogroups O:3 and O:9 were used as probes to detect homologous sequences in plasmids of "avirulent" Yersinia strains. "Avirulent" Yersinia strains (Y. enterocolitica biogroup 1A, Y. intermedia, Y. kristensenii and Y. frederiksenii) lack the virulence plasmid. They are widely distributed in the environment and can frequently be isolated from clinical samples. Hybridisation experiments revealed a number of common genetic elements of the virulence plasmid and the plasmids of "avirulent" Yersinia strains. These elements were identified as genes involved in plasmid replication, as an endonuclease gene and as mobile genetic elements. However, none of the plasmid encoded virulence genes was present in the plasmids of "avirulent" Yersinia strains. The frequent occurrence and the possible etiological relevance of "avirulent" isolates will be discussed.

Natural history of hay fever and pollen sensitization, and doctors' diagnosis of hay fever and pollen asthma in German schoolchildren.

BACKGROUND: In order to prevent pollen asthma by immunotherapy it is mandatory to know the best time to initiate it. Children with hay fever complaints are at considerable risk of developing pollen asthma. Population-based data on their natural history is urgently needed. METHODS: A longitudinal cohort study was conducted over four years in six rural towns in Baden-Württemberg, Germany. A questionnaire with questions taken from the International Study of Asthma and Allergies in childhood (ISAAC) was filled in every spring and autumn. Hay fever complaints, asthma defining symptoms and new doctors' diagnosis of hay fever and asthma were recorded. Additionally a skin prick test with pollen allergens was performed every autumn. RESULTS: In 1996, 19.7% of 1101 elementary school children (age: 8.1-9.9 years (5-95%)) were found to be sensitized to pollen and 8.7% had already been diagnosed as having hay fever. In a pooled analysis of 2478 children-summers, children with positive pollen sensitization had a significantly higher risk of developing hay fever symptoms (2.63; 2.17-3.10 odds ratio (OR); 95% confidence interval (CI)) and of being diagnosed as suffering from hay fever (7.88; 4.70-13.20). Furthermore, although their OR for the development of asthma symptoms during the pollen season was 3.88 (2.48-6.07 CI), it was only 0.69 (0.24-2.01 CI) for doctors' diagnosis of pollen asthma. CONCLUSION: Children of elementary school age with pollen sensitization and a history of hay fever are at considerable risk of getting pollen asthma, but they are not quickly diagnosed as such. Specific immunotherapy might be a means of preventing asthma completely in such a situation. Our data helps to estimate the sample size for intervention studies of this kind.

Characterization of enterocoliticin, a phage tail-like bacteriocin, and its effect on pathogenic Yersinia enterocolitica strains.

Yersinia enterocolitica 29930 (biogroup 1A; serogroup O:7,8) produces a bacteriocin, designated enterocoliticin, that shows inhibitory activity against enteropathogenic strains of Y. enterocolitica belonging to serogroups O:3, O:5,27 and O:9. Enterocoliticin was purified, and electron micrographs of enterocoliticin preparations revealed the presence of phage tail-like particles. The particles did not contain nucleic acids and showed contraction upon contact with susceptible bacteria. Enterocoliticin addition to logarithmic-phase cultures of susceptible bacterial strains led to a rapid dose-dependent reduction in CFU. Calorimetric measurements of the heat output of cultures of sensitive bacteria showed a complete loss of cellular metabolic activity immediately upon addition of enterocoliticin. Furthermore, a dose-dependent efflux of K(+) ions into the medium was determined, indicating that enterocoliticin has channel-forming activity.

Characterization of a Shiga toxin-encoding temperate bacteriophage of Shigella sonnei.

A Shiga toxin (Stx)-encoding temperate bacteriophage of Shigella sonnei strain CB7888 was investigated for its morphology, DNA similarity, host range, and lysogenization in Shigella and Escherichia coli strains. Phage 7888 formed plaques on a broad spectrum of Shigella strains belonging to different species and serotypes, including Stx-producing Shigella dysenteriae type 1. With E. coli, only strains with rough lipopolysaccharide were sensitive to this phage. The phage integrated into the genome of nontoxigenic S. sonnei and laboratory E. coli K-12 strains, which became Stx positive upon lysogenization. Moreover, phage 7888 is capable of transducing chromosomal genes in E. coli K-12. The relationships of phage 7888 with the E. coli Stx1-producing phage H-19B and the E. coli Stx2-producing phage 933W were investigated by DNA cross-hybridization of phage genomes and by nucleotide sequencing of an 8,053-bp DNA region of the phage 7888 genome flanking the stx genes. By these methods, a high similarity was found between phages 7888 and 933W. Much less similarity was found between phages H-19B and 7888. As in the other Stx phages, a regulatory region involved in Q-dependent expression is found upstream of stxA and stxB (stx gene) in phage 7888. The morphology of phage 7888 was similar to that of phage 933W, which shows a hexagonal head and a short tail. Our findings demonstrate that stx genes are naturally transferable and are expressed in strains of S. sonnei, which points to the continuous evolution of human-pathogenic Shigella by horizontal gene transfer.

Bile salt stimulates intestinal epithelial cell migration through TGFbeta after wounding.

BACKGROUND: In addition to aiding in the digestion of fats, luminal bile salts have been shown to modulate gastrointestinal epithelial growth, differentiation, and other functions. We hypothesized that bile acids could modulate the intestinal mucosal repair process of restitution. We investigated the effect of the bile salt taurodeoxycholic acid on epithelial migration and identified a role for TGFbeta, a widely expressed cytokine in the intestinal villus, in this repair process. METHODS: Using a well-established model of epithelial restitution, IEC-6 cells were plated on 60-mm Matrigel-coated plastic dishes and grown to confluence. The epithelium was wounded by scraping with a 6-mm-wide blade to create a smooth denuded edge and cell migration was measured 8 h later. Cells were grown in control DMEM with 5% FBS with or without 0.01-2 mM taurodeoxycholic acid (TDCA). In parallel experiments, cells were harvested for Northern analysis of TGFbeta and GAPDH expression; [3H]thymidine uptake was used to measure proliferation. Anti-TGFbeta antibody was added to cells grown in the presence of 0.05 mM TDCA and migration was measured at 8 h. RESULTS: TDCA at physiologic luminal concentrations augments IEC-6 cell migration, with a maximal effect at 0.05 mM. TDCA inhibited proliferation at these concentrations. TGFbeta expression increased in response to bile acid, while wounding had less of an effect on TGFbeta expression. Blockade of TGFbeta function with TGFbeta antibody eliminated the effect of bile on cell migration. CONCLUSIONS: Bile acid at physiologic concentrations augments small intestinal epithelial cell migration. The process is dependent on TGFbeta and is independent of cell division. The data further support a role for bile acids and TGFbeta in differentiated intestinal cell function and in preservation of an intact mucosa.

La operación de Hartmann en el Hospital de Temuco / Hartmannïs surgery at the Temuco hospital

El objetivo del trabajo es conocer en nuestro medio las principales indicaciones de la operación de Hartmann, su morbilidad y mortalidad y analizar si ha habido una sobreutilización de este procedimiento quirúrgico. Se estudió una serie de 60 pacientes, 35 hombres y 25 mujeres intervenidos en eI Hospital Regional de Temuco entre 1994 y 1999, cuyas edades fluctuaron entre 26 y 99 años, con un promedio de 62,2 años. Las principales indicaciones de la cirugía fueron: cáncer rectocolónico complicado (43,2 por ciento), vólvulo de sigmoides complicado (20 por ciento), diverticulitis complicada (11,6 por ciento) y trauma (8,3 por ciento). En el 76 por ciento de los casos fue necesario efectuar resección de colon. En cuanto a morbilidad las complicaciones locales se presentaron en el 33,2 por ciento, sepsis en el 10 por ciento, infarto miocárdico en el 6,7 por ciento y complicaciones sépticas intraabdominales en un 5 por ciento que fueron resueltas quirúrgicamente. La mortalidad de la serie fue de un 18,3 por ciento

Ca2+-RhoA signaling pathway required for polyamine-dependent intestinal epithelial cell migration.

Expression of voltage-gated K(+) (Kv) channel genes is regulated by polyamines in intestinal epithelial cells (IEC-6 line), and Kv channel activity is involved in the regulation of cell migration during early restitution by controlling membrane potential (E(m)) and cytosolic free Ca2+ concentration ([Ca2+](cyt)). This study tests the hypothesis that RhoA of small GTPases is a downstream target of elevated ([Ca2+](cyt)) following activation of K(+) channels by increased polyamines in IEC-6 cells. Depletion of cellular polyamines by alpha-difluoromethylornithine (DFMO) reduced whole cell K+ currents [I(K(v))] through Kv channels and caused membrane depolarization, which was associated with decreases in ([Ca2+](cyt)), RhoA protein, and cell migration. Exogenous polyamine spermidine reversed the effects of DFMO on I(K(v)), E(m), ([Ca2+](cyt)), and RhoA protein and restored cell migration to normal. Elevation of ([Ca2+](cyt)) induced by the Ca2+ ionophore ionomycin increased RhoA protein synthesis and stimulated cell migration, while removal of extracellular Ca2+ decreased RhoA protein synthesis, reduced protein stability, and inhibited cell motility. Decreased RhoA activity due to Clostridium botulinum exoenzyme C(3) transferase inhibited formation of myosin II stress fibers and prevented restoration of cell migration by exogenous spermidine in polyamine-deficient cells. These findings suggest that polyamine-dependent cell migration is partially initiated by the formation of myosin II stress fibers as a result of Ca2+-activated RhoA activity.

Bronchial hyperresponsiveness to 4.5% hypertonic saline indicates a past history of asthma-like symptoms in children.

SUMMARY. To evaluate the importance of a past history of asthma-like symptoms over a period of 2 years and current bronchial hyperreactivity (BHR), 538 randomly selected schoolchildren, initially aged 7-8 years, were examined. At yearly intervals, three standardized questionnaires, including items from the ISAAC panel, were answered by parents. Following the last questionnaire, BHR to 4.5% hypertonic saline (HS) was recorded. In survey 1, lifetime prevalence of asthma was 4.9%. During the 12-month period, prevalence of wheeze and dyspnea ranged between 9.3 and 5.2% (Survey 1) and 5.9% and 4.4% (Survey 2). Among children with wheeze or dyspnea in Survey 3, BHR (defined as a fall of baseline FEV(1) > or = 15%) was significantly more frequent (50.0% and 60.7%, respectively) than among children without these symptoms (12.8%, P < 0.001, and 12.8%, P < 0.001, respectively). The negative predictive value of BHR to have neither wheeze nor dyspnea was about 88% and did not vary throughout the study (Survey 1, 87%; Survey 2, 88%; Survey 3, 88%). The relative risk of showing BHR was significantly increased in children with wheeze (survey 2, odds ratio (OR) 3.0 (95% confidence interval (CI) 1.0-8.7)) or dyspnea (Survey 1: OR 5.9 (95% CI 1.9-18.5), Survey 3: 5.2 (1.7-16.2), but not in children with dry cough or nocturnal cough (data not shown). Wheeze and dyspnea occurred repeatedly in the same individuals with BHR in a high percentage of children (83.3% and 76.5%, respectively). In conclusion, there is a strong association between recent and previous dyspnea and current BHR, and it indicates intraindividual persistence of symptom history.

Glypican-3 expression in Wilms tumor and hepatoblastoma.

BACKGROUND: Glypican-3 (GPC3) is a heparan sulfate proteoglycan. When it is disrupted, it causes the X-linked gigantism-overgrowth Simpson-Golabi-Behmel syndrome. Its involvement in growth control is consistent with recent reports that it can bind to growth factors, possibly including insulin-like growth factor 2. Further, it has been hypothesized that it may function as a tumor suppressor gene in breast and ovarian carcinomas and mesotheliomas. PATIENTS AND METHODS: RNA and protein were extracted from Wilms tumor and hepatoblastoma tissue samples and GPC3 levels were measured in these extracts by Northern blotting, reverse transcription polymerase chain reaction, and immunoblotting. RESULTS: In contrast to published results with carcinomas, high levels of GPC3 expression were found in Wilms tumor and hepatoblastoma. Low or undetectable expressions of this gene were found in normal tissue surrounding the tumor. CONCLUSIONS: Increased expression of GPC3 in Wilms tumor and hepatoblastoma suggests a growth-promoting or neutral activity for this gene product rather than a growth-suppressive effect.

Taxonomic studies of predatory bdellovibrios based on 16S rRNA analysis, ribotyping and the hit locus and characterization of isolates from the gut of animals.

The aim of our study was to obtain data for the molecular characterization of bdellovibrio bacteria, which were recently split into the genus Bdellovibrio and the newly designated genus Bacteriovorax. We determined the 16S rDNA sequences of five reference strains and performed a phylogenetic analysis including published 16S rRNA sequences of bdellovibrios. A comparison of the secondary structure showed significant differences in two regions of the 16S rRNAs of the species Bdellovibrio bacteriovorus, Bacteriovorax starrii, and Bacteriovorax stolpii. In addition, ribotyping techniques gave specific hybridization patterns and revealed that two rRNA operons are present in the investigated strains. A hybridization probe derived from the genetic locus hit, associated with the host independent (HI) phenotype of B. bacteriovorus, was found to be specific for this species. Sequence comparison of the hit locus revealed few base pair changes between host independent (HI) and host dependent (HD) strains. Ribotyping and hybridization experiments using the hit probe were applied to characterize bdellovibrio strains isolated from the gut of animals and humans and one isolate from sewage.

The relationship of eosinophil granule proteins to ions in the sputum of patients with cystic fibrosis.

Increased sputum levels of eosinophil granule proteins have been reported despite normal eosinophil numbers in peripheral blood and in the lung in cystic fibrosis (CF). Mechanisms of eosinophil priming and activation are still unclear in CF. In the present study we investigated whether ion concentrations in the sputa of CF patients are related to eosinophil activity. We assessed concentrations of eosinophil cationic protein (ECP), eosinophil protein X (EPX), major basic protein (MBP) and ions (Na+, Cl-, Ca2+, Mg2+) in sputum samples of 29 children with CF as well as in 10 controls with bronchial asthma. Patients with CF demonstrated significantly higher levels of ECP, Na+, Cl- and Ca2+ levels than asthmatics (P < 0.04, P < 0.0001, P < 0.0001, P < 0.02). No differences were seen between concentrations of EPX and Mg2+ in the two groups. In CF, eosinophil granule proteins correlated significantly with Ca2+ and Mg2+ concentrations (ECP, P < 0.0001, r = 0.65, P < 0.0001, r = 0.66; MBP, P < 0.03, r = 0.41, P < 0.03, r = 0.42), furthermore inversely with Cl- concentrations (ECP, P < 0. 0003, r = - 0.63; EPX, P < 0.02, r = - 0.45; MBP, P < 0.03, r = - 0. 41) but not with Na+ levels. ECP, Na+ and Cl- were also correlated with lung function variables (FVC, P < 0.04, r = - 0.38, P < 0.02, r = 0.44, P < 0.03, r = 0.41; FEV1, P < 0.007, r = - 0.49, P < 0.006, r = 0.5, P < 0.008, r = 0.48; MEF50, P < 0.003, r = - 0.54, NS, P < 0.03, r = 0.42; MEF25, P < 0.039, r = - 0.4, P < 0.005, r = 0.51, P < 0.05, r = 0.37). Our results demonstrated a significant relationship of eosinophil degranulation and ions in CF, indicating that ion composition in CF sputa may be at least partly be responsible for high levels of eosinophil products despite low eosinophil numbers.

Isolation of a new insertion element of Yersinia intermedia closely related to remnants of mobile genetic elements present on Yersinia plasmids harboring the Yop virulon.

A new insertion element present in two alleles, designated IS1635.1 and IS1635.2, was identified on a plasmid of a Yersinia intermedia strain by hybridization with the Yersinia enterocolitica pYV virulence plasmid. IS1635.1 and IS1635.2 are 861 bp long, carry imperfect inverted terminal repeats and possess a single open reading frame encoding a putative transposase of the IS6 family. A truncated IS1635 element is present immediately downstream of element IS1635.2. The capacity of the IS1635 elements to mediate transposition in Yersinia was demonstrated with a R6K-derived suicide vector, where a kanamycin resistance gene had been inserted between IS1635.1 and IS1635.2. Hybridization and sequence alignments showed that remnants of IS1635-like insertion elements harboring large deletions and point mutations are present on the Yop virulon harboring plasmids of pathogenic Yersinia strains. In a few cases, the IS1635 element has also been found on plasmids of apathogenic Yersinia strains.

Use of a plasmid of a yersinia enterocolitica biogroup 1A strain for the construction of cloning vectors.

A plasmid with a size of 2,682 base pairs isolated from the Yersinia enterocolitica biogroup 1A strain # 29807 was characterized in respect to its suitability as a basic replicon for cloning vectors. The copy number of the plasmid was determined to be approximately 14 copies per cell and it was shown to be compatible with vectors with an origin of replication derived from ColE1 and p115A. The replication region of the plasmid encodes a primer RNAI and countertranscript RNAII. Two vectors, pIV1 and pIV2, containing a kanamycin resistance gene and the lacZalpha fragment with the multiple cloning site of pBluescriptSK + were constructed. A mobilizable derivative was successfully introduced into different bacteria belonging to the family Enterobacteriacea. To prove the applicability of the novel vectors for cloning purposes, a 13 kb hemolysin operon of Escherichia coli was inserted into pIV1, and the resulting recombinant plasmid was stably maintained and expressed in E. coli and Y. enterocolitica.