Binding of cells of Staphylococcus aureus to fibronectin, which may represent a mechanism of host tissue adherence, involves a fibronectin-receptor protein present on the bacterial surface. Cloning of a gene coding for a staphylococcal fibronectin-binding protein and construction of a fusion protein with fibronectin-binding properties was previously reported from our laboratory. We have now sequenced the gene and deduced a primary sequence of the fibronectin-binding protein. The protein resembles other cell-wall-associated proteins on Gram-positive bacteria in that it (i) appears to be anchored in the cell membrane via its C-terminal end, (ii) contains a proline-rich repeating unit outside the membrane anchor, and (iii) contains a long (36-amino acid) signal sequence at the N terminus. The fibronectin-binding activity has been localized to a domain composed of a 38-amino acid unit repeated completely three times and partially a fourth time; the identity between the three 38-amino acid sequences varies from 42 to 87%. Three synthetic peptides mimicking the structure of each 38-amino acid unit were constructed. All three peptides interacted with fibronectin, as indicated by their ability to inhibit binding of fibronectin to staphylococcal cells, whereas an unrelated 37-amino acid peptide showed no inhibitory activity.

Real-time PCR was used to quantify populations of ammonia-oxidizing bacteria representing the ␤ subdivision of the class Proteobacteria in samples of arable soil, both nitrogen fertilized and unfertilized, from Mellby, Sweden. Primers and probes targeting a 16S ribosomal DNA region of the ammonia-oxidizing bacteria were designed and used. In the fertilized soil there were ϳ6.2 ؋ 10 7 ammonia-oxidizing bacteria per g of soil, three times more than the number of bacteria in the unfertilized soil. The lytic efficiency of bead beating in these soils was investigated by using populations of free or loosely attached bacteria, bacteria tightly bound to particles, and bacteria in nonfractionated samples. The shapes of the curves generated in these tests showed that the concentration of template DNA released at various times remained constant after 10 to 100 s of bead beating.Quantification of microbial populations is important in many aspects of microbial ecology. The development of molecular biological methods involving PCR has led to new techniques that are not limited by the culturability of the microorganisms. Since it is known that only a small proportion of the bacteria in soils can be cultivated under standard laboratory conditions, the PCR-based quantification methods have found many applications. Three PCR-based methods, limiting-dilution PCR (27, 31), kinetic PCR (3, 14), and competitive PCR (10, 11, 23), have been used for quantitative analysis of DNA having different origins. However, kinetic PCR and limitingdilution PCR often have the disadvantage of relying on endpoint measurements of the amount of DNA produced, which makes it difficult to deduce the initial concentration of template DNA. In competitive PCR it may be difficult to achieve the same affinity of the primers for the target and competitive molecules, which complicates quantification. A modified PCR technique, real-time PCR (13), measures the DNA concentration continuously during amplification, which enables the initial template concentration to be determined and the cell numbers to be more accurately deduced without the use of a competing molecule.Quantification of ammonia-oxidizing bacteria (AOB), which are responsible for the oxidation of ammonia to nitrite in the nitrification process, has been attempted by using several different methods. These include the most-probable-number technique (6,7,19), in situ hybridization (29), a competitive enzyme-linked immunosorbent assay using monoclonal antibodies (28), and competitive PCR (17, 26, 30) based on traditional methods of amplification. However, all of these methods have significant disadvantages. Thus, a reliable and reproducible method for quantifying AOB would be valuable for evaluating correlations between microbial activities and cell numbers, the effects of different treatments on cell density, and population changes in time and space. Determination of DNA concentrations with real-time PCR overcomes some of the problems associated with traditional PCR. The real-time PCR technique is based ...

The main recommendations according to current European case definitions for LB are as follows. Typical erythema migrans should be diagnosed clinically and does not require laboratory testing. The diagnosis of Lyme neuroborreliosis requires laboratory investigation of the spinal fluid including intrathecal antibody production, and the remaining disease manifestations require testing for serum antibodies to B. burgdorferi. Testing individuals with non-specific subjective symptoms is not recommended, because of a low positive predictive value.

Using consensus regions in gene sequences encoding the two forms of nitrite reductase (Nir), a key enzyme in the denitrification pathway, we designed two sets of PCR primers to amplifycd 1- and Cu-nir. The primers were evaluated by screening defined denitrifying strains, denitrifying isolates from wastewater treatment plants, and extracts from activated sludge. Sequence relationships ofnir genes were also established. Thecd 1 primers were designed to amplify a 778 to 799-bp region of cd1-nir in the six published sequences. Likewise, the Cu primers amplified a 473-bp region in seven of the eight published Cu-nir sequences. Together, the two sets of PCR primers amplified nir genes in nine species within four genera, as well as in four of the seven sludge isolates. The primers did not amplify genes of nondenitrifying strains. The Cu primers amplified the expected fragment in all 13 sludge samples, but cd1-nir fragments were only obtained in five samples. PCR products of the expected sizes were verified as nir genes after hybridization to DNA probes, except in one case. The sequenced nir fragments were related to other nir sequences, demonstrating that the primers amplified the correct gene. The selected primer sites for Cu-nir were conserved, while broad-range primers targeting conserved regions of cd1-nir seem to be difficult to find. We also report on the existence of Cu-nir in Paracoccus denitrificans Pd1222.

Norovirus (NoV) is a leading cause of acute gastroenteritis and is often spread via wastewater contamination. Little is known about how the wastewater treatment process affects norovirus, and which factors influence virus concentrations. To investigate this, we collected wastewater samples monthly during one year at eight different key sites at the municipal wastewater treatment plant in Gothenburg, Sweden. Virus particles were concentrated using ultracentrifugation, viral RNA was subsequently extracted, and transformed into cDNA by reverse transcription. The quantification was performed with real-time PCR assays for NoV genogroups I (GGI) and II (GGII), respectively. We found seasonal changes of NoV genogroups, with the highest concentration of NoV GGII during the winter months, and the highest concentration of NoV GGI during the summer months. Virus transmission in wastewater was more stable for NoV GGI, with NoV GGII demonstrating larger seasonal peaks. Virus reduction took place at similar rates in the primary settling, and in the activated sludge in combination with the secondary settling. Different physicochemical parameters and incoming virus concentrations were correlated to reduction of NoV between different treatment sites.This study gives new information about NoV transmission and virus reduction in a wastewater treatment plant.