Environmental relative moldiness index and associations with home characteristics and infant wheeze.

Possible relationships between mold contamination, as described by the Environmental Relative Moldiness Index (ERMI), home characteristics, and the development of wheeze in the first year of life were evaluated among a cohort of urban infants (n = 103) in Syracuse, New York. Pregnant women with a history of asthma were recruited in 2001-2002 for the "Assessment of Urban Dwellings for Indoor Toxics" (AUDIT) study. When the infants were approximately 3 months of age, a home inspection was carried out and indoor environmental samples collected, including vacuumed house dust. ERMI levels in the Syracuse cohort homes were higher than the U.S. average, with an overall mean of 11.4. ERMI levels were significantly higher in homes with visible water problems (p = 0.023) and visible mold (p = 0.023). ERMI levels in apartments were significantly lower than the values measured in houses (p = 0.0003). While infants experiencing wheeze (38%) tended to live in homes with higher ERMI values than those without wheeze (ERMI values of 12.3 and 10.9, respectively), the differences did not reach statistical significance. A subset analysis limited to infants with living room samples who remained in the same home during the study (n = 25) was suggestive of an association between higher ERMI values and wheeze (p = 0.10). In summary, the ERMI is a standardized metric which allows for comparison of moldiness levels in homes across studies and regions in the United States. ERMI levels in Syracuse homes were skewed to the high end of the national scale. Higher ERMI levels were indicators of water problems, mold, and type of housing.

Predicting virulence of Aeromonas isolates based on changes in transcription of c-jun and c-fos in human tissue culture cells.

AIMS: To screen for the virulence potential of Aeromonas isolates based on the change in regulation of c-jun and c-fos in the human intestinal tissue culture cell line Caco-2. METHODS AND RESULTS: Aeromonas cells were added to Caco-2 cells at a ratio of approx. 1 : 1. After 1-, 2- and 3-h incubation at 37 degrees C, mRNA was extracted from the cells and gene expression of two host genes, c-jun and c-fos, quantified. Aeromonas isolates which were pathogenic in the neonatal mouse model demonstrated up-regulation of c-jun and c-fos compared to avirulent isolates. CONCLUSIONS: Human cell culture results showed that c-jun and c-fos were predictive of Aeromonas virulence. SIGNIFICANCE AND IMPACT OF THE STUDY: An Aeromonas relative virulence scale is proposed for use in the testing of Aeromonas drinking water isolates.

Opportunistic Aspergillus pathogens measured in home and hospital tap water by quantitative PCR (QPCR).

Opportunistic fungal pathogens are a concern because of the increasing number of immunocompromised patients. The goal of this research was to test a simple extraction method and rapid quantitative PCR (QPCR) measurement of the occurrence of potential pathogens, Aspergillus fumigatus, A. flavus, A. terreus and A. niger, in home tap water and a hospital water supply. Water samples were taken from the kitchen tap in the homes of 60 patients who were diagnosed with legionellosis. Water samples were also taken from three locations in a hospital that generated all of its hot water by flash heating. Opportunistic infectious agents Aspergillus fumigatus, A. flavus, A. terreus and A. niger were measured using QPCR. Aspergillus terreus DNA was found in 16.7% and A. fumigatus DNA in 1.7% of the samples taken from the kitchen tap. None of the Aspergillus species were found in any of the hospital water samples.The development of a simple DNA extraction method along with QPCR analysis is suitable for rapid screening of tap water for opportunistic fungal pathogens. This simple method can be used to obtain pathogen occurrence results in about 3 h, instead of waiting days to weeks for culture data. Obtaining pathogen occurrence data in a timely manner could promote the elimination of the pathogens from the water supply of immunocompromised patients.

Monitoring Aspergillus species by quantitative PCR during construction of a multi-storey hospital building.

During the enlargement of an existing hospital, quantitative polymerase chain reaction (PCR) was used to monitor Aspergillus spp. populations within the construction site. The rapid availability of results meant that the construction schedule was largely uninterrupted, while assuring that the new construction was free from contamination by the targeted Aspergillus spp.

Quantification of Stachybotrys chartarum conidia in indoor dust using real time, fluorescent probe-based detection of PCR products.

Analyses of fungal spores or conidia in indoor dust samples can be useful for determining the contamination status of building interiors and in signaling instances where potentially harmful exposures of building occupants to these organisms may exist. A recently developed method for the quantification of Stachybotrys chartarum conidia, using real-time, fluorescence probe--based detection of PCR products (TaqMan system) was employed to analyze indoor dust samples for this toxigenic fungal species. Dust samples ofup to 10 mg were found to be amenable to DNA extraction and analysis. Quantitative estimates of S. chartarum conidia in composite dust samples, containing a four-log range of these cells, were within 25 -- 104% of the expected quantities in 95% of analyses performed by the method. Calibrator samples containing known numbers of S. chartarum conidia were used as standards for quantification. Conidia of an arbitrarily selected strain of Geotrichum candidum were added in equal numbers to both dust and calibrator samples before DNA extraction. Partial corrections for reductions in overall DNA yields from the dust samples compared to the calibrator samples were obtained by comparative analyses of rDNA sequence yields from these reference conidia in the two types of samples. Dust samples from two contaminated homes were determined to contain greater than 10(3) S. chartarum conidia per milligram in collection areas near the sites of contamination and greater than 10(2) conidia per milligram in several areas removed from these sites in analyses performed by the method. These measurements were within the predicted range of agreement with results obtained by direct microscopic enumeration of presumptive Stachybotrys conidia in the same samples.

Evaluating virulence of waterborne and clinical Aeromonas isolates using gene expression and mortality in neonatal mice followed by assessing cell culture's ability to predict virulence based on transcriptional response.

AIMS: To assess the virulence of Aeromonas spp. using two models, a neonatal mouse assay and a mouse intestinal cell culture. METHODS AND RESULTS: After artificial infection with a variety of Aeromonas spp., mRNA extracts from the two models were processed and hydridized to murine microarrays to determine host gene response. Definition of virulence was determined based on host mRNA production in murine neonatal intestinal tissue and mortality of infected animals. Infections of mouse intestinal cell cultures were then performed to determine whether this simpler model system's mRNA responses correlated to neonatal results and therefore be predictive of virulence of Aeromonas spp. Virulent aeromonads up-regulated transcripts in both models including multiple host defense gene products (chemokines, regulation of transcription and apoptosis and cell signalling). Avirulent species exhibited little or no host response in neonates. Mortality results correlated well with both bacterial dose and average fold change of up-regulated transcripts in the neonatal mice. CONCLUSIONS: Cell culture results were less discriminating but showed promise as potentially being able to be predictive of virulence. Jun oncogene up-regulation in murine cell culture is potentially predictive of Aeromonas virulence. SIGNIFICANCE AND IMPACT OF THE STUDY: Having the ability to determine virulence of waterborne pathogens quickly would potentially assist public health officials to rapidly assess exposure risks.

Comparison of populations of mould species in homes in the UK and USA using mould-specific quantitative PCR.

AIMS: To compare the populations of 81 mould species in homes in the USA and UK using mould-specific quantitative polymerase chain reaction (MSQPCR) technology. METHODS AND RESULTS: Dust samples were obtained from randomly selected homes in the UK (n=11). The mould populations in British homes were compared with those found in typical homes (no visible mould) in the USA (in the state of Ohio, n=45). Only 13 of 81 species screened showed significantly different concentrations in these two sets of home. CONCLUSIONS: Although only a small survey, the results suggest that typical mould profiles in the USA (Ohio) and British homes are very similar. Analysis of 26 mould indicator species revealed that the British homes fell into two clusters, tentatively identified as 'atypical' and 'typical' mould conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: MSQPCR analysis of dust samples can provide an objective measure of indoor moulds which could lead to better management of their health effects.

Production of Pili (Fimbriae) by Pseudomonas fluorescens and Correlation with Attachment to Corn Roots.

Pseudomonas fluorescens isolates 13525 and 2-79 were grown in Luria broth and low-nutrient medium (LNM). Pililike fibrils were very rarely produced in Luria broth but were abundantly produced in LNM. In LNM the pili were peritrichously distributed and had diameters ranging from 3 to 8 nm. Pili were purified from strain 2-79, and the pilin subunit was found to have a molecular weight of about 34,000. Strain 2-79 produced two colony types on Luria agar, nonmucoidal and mucoidal. Cells in LNM cultures of the nonmucoidal colony type were highly piliated, and cells from the mucoidal type were nearly devoid of pili. The presence of pili on nonmucoidal isolate 2-79 was quantitatively correlated with hydrophobic attachment to polystyrene, hemagglutination, and attachment to corn roots.

Role of Pili (Fimbriae) in Attachment of Bradyrhizobium japonicum to Soybean Roots.

Pili (fimbriae) were observed on cells of each of the five strains of Bradyrhizobium japonicum and the one strain of Rhizobium trifolii examined. Pili on B. japonicum were about 4 nm in diameter and polarly expressed. Piliated cells were estimated by transmission electron microscopy and hydrophobic attachment to polystyrene to constitute only a small percentage of the total population. The proportion of piliated cells in these populations was dependent on culture age in some strains. Piliated B. japonicum cells were selectively and quantitatively removed from suspension when cultures were incubated with either soybean roots or hydrophobic plastic surfaces, indicating that pili were involved in the attachment of the bacteria to these surfaces. Pili from B. japonicum 110 ARS were purified and found to have a subunit molecular weight of approximately 21,000. Treatment of B. japonicum suspensions with antiserum against the isolated pili reduced attachment to soybean roots by about 90% and nodulation by about 80%. Pili appear to be important mediators of attachment of B. japonicum to soybean roots under the conditions examined.

Transposon Mutants of Bradyrhizobium japonicum Altered in Attachment to Host Roots.

Transposon mutants of Bradyrhizobium japonicum 110 ARS were produced and screened for changes in attachment ability. Mutant CFK4 produced twice as many piliated cells, attached in 2.5-fold-higher numbers to soybean root segments, and colonized roots in about 2-fold-higher numbers than did the parental strain, 110 ARS. Mutants CFK35 and CFK38 were reduced in their attachment about 2-fold and 3.5-fold, respectively. This corresponded to reductions in piliated cells in their populations, reduced reaction with anti-pilus antiserum, and reduced hydrophobic attachment. Mutants CFK4 and CFK38 nodulated soybeans at about the same level as the parent strain, but CFK35 induced only pseudonodules. Two-dimensional gel analyses of the proteins from the mutants showed relatively few changes in proteins.

Quantitative measurement of Stachybotrys chartarum conidia using real time detection of PCR products with the TaqMan(TM)fluorogenic probe system.

The occurrence of Stachybotrys chartarum in indoor environments has been associated with a number of human health concerns, including fatal pulmonary haemosiderosis in infants. Currently used culture-based and microscopic methods of fungal species identification are poorly suited to providing quick and accurate estimates of airborne human exposures to the toxin containing conidia of this organism. In this study, real-time polymerase chain reaction (PCR) product analysis using the TaqManU fluorogenic probe system and an Applied Biosystems PrismS model 7700 sequence detection instrument (model 7700) was applied to the specific detection of S. chartarum ribosomal DNA (rDNA) sequences. Based upon this assay and a recently reported comparative cycle threshold method for quantifying target DNA sequences using data from the model 7700, a simple method for the direct quantification of S. chartarum conidia was developed. In analyses of samples containing several different strains and from two to over 2x10(5)cells, this method consistently provided quantitative estimates of S. chartarum conidia that were within a one-fold range (50-200%) of those determined on the basis of direct microscopic counts in a haemocytometer. The method showed a similar level of agreement with direct counting in the quantification of S. chartarum conidia in air samples collected from several contaminated homes.

Quantification of siderophore and hemolysin from Stachybotrys chartarum strains, including a strain isolated from the lung of a child with pulmonary hemorrhage and hemosiderosis.

A strain of Stachybotrys chartarum was recently isolated from the lung of a pulmonary hemorrhage and hemosiderosis (PH) patient in Texas (designated the Houston strain). This is the first time that S. chartarum has been isolated from the lung of a PH patient. In this study, the Houston strain and 10 strains of S. chartarum isolated from case (n = 5) or control (n = 5) homes in Cleveland were analyzed for hemolytic activity, siderophore production, and relatedness as measured by random amplified polymorphic DNA analysis.

Hemolysis, toxicity, and randomly amplified polymorphic DNA analysis of Stachybotrys chartarum strains.

Stachybotrys chartarum is an indoor air, toxigenic fungus that has been associated with a number of human and veterinary health problems. Most notable among these has been a cluster of idiopathic pulmonary hemorrhage cases that were observed in the Cleveland, Ohio, area. In this study, 16 strains of S. chartarum isolated from case (n = 8) or control (n = 8) homes in Cleveland and 12 non-Cleveland strains from diverse geographic locations were analyzed for hemolytic activity, conidial toxicity, and randomly amplified polymorphic DNA banding patterns. In tests for hemolytic activity, strains were grown at 23 degrees C on wet wallboard pieces for an 8-week test period. Conidia from these wallboard pieces were subcultured on sheep's blood agar once a week over this period and examined for growth and clearing of the medium at 37 or 23 degrees C. Five of the Cleveland strains (all from case homes) showed hemolytic activity at 37 degrees C throughout the 8-week test compared to 3 of the non-Cleveland strains. Five of the Cleveland strains, compared to two of the non-Cleveland strains, produced highly toxic conidia (>90 microgram of T2 toxin equivalents per g [wet weight] of conidia) after 10 and 30 days of growth on wet wallboard. Only 3 of the 28 strains examined both were consistently hemolytic and produced highly toxic conidia. Each of these strains was isolated from a house in Cleveland where an infant had idiopathic pulmonary hemorrhage.

Initial characterization of the hemolysin stachylysin from Stachybotrys chartarum.

Stachybotrys chartarum is a toxigenic fungus that has been associated with human health concerns, including pulmonary hemorrhage and hemosiderosis. This fungus produces a hemolysin, stachylysin, which in its apparent monomeric form has a molecular mass of 11,920 Da as determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry. However, it appears to form polydispersed aggregates, which confounds understanding of the actual hemolytically active form. Exhaustive dialysis or heat treatment at 60 degrees C for 30 min inactivated stachylysin. Stachylysin is composed of about 40% nonpolar amino acids and contains two cysteine residues. Purified stachylysin required more than 6 h to begin lysing sheep erythrocytes, but by 48 h, lysis was complete. Stachylysin also formed pores in sheep erythrocyte membranes.

Evaluation of infrared spectroscopy as a bacterial identification method.

A study motivated by the recent revival of interest in the use of IR spectroscopy to identify bacteria is reported. A library of FT-IR spectra of dried bacterial films was complied using 16 different strains. A test set was compiled from spectra of the same strains grown several months later. The test set was quantitatively compared with the library on the basis of spectral similarity in the region 980-1190 cm-1. Six of the strains in the test set were not matched with the correct strain in the library despite efforts to reproduce the conditions under which cells were grown and prepared. The results suggest that reproducibility of the bacterial spectra is a potential difficulty that must be addressed by any attempts to develop FT-IR spectroscopy as a bacterial identification method.