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The present study had three goals: (i) to evaluate the relative quantities of aerosolized Bacillus atrophaeus spores deposited on the vertical, horizontal top, and horizontal bottom surfaces in a chamber; (ii) to assess the relative recoveries of the aerosolized spores from glass and stainless steel surfaces with a polyester swab and a macrofoam sponge wipe; and (iii) to estimate the relative recovery efficiencies of aerosolized B. atrophaeus spores and Pantoea agglomerans using a foam spatula at several different bacterial loads by aerosol distribution on glass surfaces. The majority of spores were collected from the bottom horizontal surface regardless of which swab type and extraction protocol were used. Swabbing with a macrofoam sponge wipe was more efficient in recovering spores from surfaces contaminated with high bioaerosol concentrations than swabbing with a polyester swab. B. atrophaeus spores and P. agglomerans culturable cells were detected on glass surfaces using foam spatulas when the theoretical surface bacterial loads were 2.88 ؋ 10 4 CFU and 8.09 ؋ 10 6 CFU per 100-cm 2 area, respectively. The median recovery efficiency from the surfaces using foam spatulas was equal to 9.9% for B. atrophaeus spores when the recovery was calculated relative to the theoretical surface spore load. Using a foam spatula permits reliable sampling of spores on the bioaerosol-exposed surfaces in a wide measuring range. The culturable P. agglomerans cells were recovered with a median efficiency of 0.001%, but staining the swab extracts with fluorescent dyes allowed us to observe that the viable cell numbers were higher by 1.83 log units than culturable organisms. However, additional work is needed to improve the analysis of the foam extracts in order to decrease the limit of detection of Bacillus spores and Gram-negative bacteria on contaminated surfaces.Surface sampling is performed on a frequent basis in all situations where clean environment monitoring is needed, e.g., in health care facilities and in the pharmaceutical industry and food industry. An anthrax bioterrorist event in the fall of 2001 has emphasized the importance of efficient sampling methods for detection of pathogenic microorganisms on surfaces within intentionally contaminated locations (22). Unfortunately, our knowledge on the most effective sampling methodology as well as the level of confidence we may have in the results obtained by wiping, swabbing, and other sample collection strategies is still limited (1). Moreover, in most of the studies performed so far, bacteria and/or spores were collected from test samples or coupons of various materials, inoculated with a suspension of microorganisms that had been placed and spread over the surface, and then dried (14, 15). This may not mimic the true situation of surface contamination by a pathogen that has been intentionally released. Edmonds et al. (12) recently reported lower swabbing efficiencies of different types of swab materials used for sampling glass, polycarbonate, and vinyl surfaces contaminat...

Aims:  To develop a multiplex PCR assay for the detection of Salmonella enterica serovar Enteritidis in human faeces. Methods and Results:  A total of 54 Salmonella strains representing 19 serovars and non‐Salmonella strains representing 11 different genera were used. Five primer pairs were employed in the assay. Three of them targeted to the genes hilA, spvA and invA that encode virulence‐associated factors. A fourth primer pair amplified a fragment of a unique sequence within S. enterica serovar Enteritidis genomes. An internal amplification control (a fragment of a conservative sequence within the 16S rRNA genes) was targeted by a fifth primer pair. The assay produced two or three amplicons from the invA, hilA and 16S rRNA genes for 19 Salmonella serovars. All Salmonella and non‐Salmonella strains yielded a band of an internal amplification control. For S. enterica serovar Typhimurium, four products (the fourth from the spvA gene), and for S. enterica serovar Enteritidis five amplicons (the fifth from the sdf gene) were observed. S. enterica serovar Enteritidis was cultured from three of 71 rectal swabs from diarrhoeal patients. Five specific amplicons were generated with the multiplex PCR assay only from culture‐positive faecal samples. Conclusion:  The multiplex PCR assay specifically detects S. enterica serovar Enteritidis. Significance and Impact of the Study:  This is a novel multiplex PCR assay, which contains an internal amplification control and enables concurrent survey for Salmonella virulence genes.

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A family of phenotypically and biologically different transplantable hamster melanomas was derived from a single tumor more than 40 yr ago. In this work, we were seeking the differences between the abilities of the cells from two biologically heterogeneous (melanotic and amelanotic) members of this family to undergo spontaneous or camptothecin-induced apoptosis. We studied these differences by looking at three important features of the apoptotic process, i.e. binding of annexin V, DNA fragmentation and caspase-3 activity. Of these, annexin binding and DNA fragmentation were more pronounced in the parental, melanotic line while the activity of caspase-3 was stronger in the amelanotic tumor cells. We concluded that a spontaneous alteration of the original, melanotic melanoma line into an amelanotic one, associated with more aggressive tumor progression, was accompanied by significant decrease in ability to undergo spontaneous and camptothecin-induced apoptosis, and that apoptosis of these two cell types may not depend on the activity of caspase-3.

Objectives: Changes in the sensitivity of two lines of transplantable melanoma cells to the antiproliferative activity of interleukin 6 (IL-6), oncostatin M (OSM), tumor necrosis factor-α (TNF-α) during melanoma progression were the subject of this study. We were looking for a correlation between these changes and the ability of melanoma cells to undergo spontaneous apoptosis. Methods: The influence of exogenous cytokines on the proliferation of melanoma cells was measured by colorimetric methods with MTT and apoptosis of these cells was estimated by staining with annexin V/propidium iodide, measurement of DNA degradation and cell cycle analysis. Results: It was observed that during melanoma progression the sensitivity of those two melanoma line cells to the antiproliferative effect of IL-6 and OSM did not change, while a spontaneous alteration of the melanotic line into an amelanotic one seemed to be accompanied by resistance of the amelanotic melanoma cells to the antiproliferative activity of TNF-α. Simultaneously, we observed a decreased ability of amelanotic melanoma cells (in comparison with the native line) to undergo spontaneous apoptosis. Conclusions: The observed resistance to the TNF-α antiproliferative effect which appears during melanoma progression seems to be correlated with a lower ability of the amelanotic melanoma cells to undergo spontaneous apoptosis.

In the study described here we investigated the possibility of an association between the aggressiveness of melanoma and multidrug resistance phenotype by analyzing the expression and activity of P-glycoprotein (Pgp) in two genetically related transplantable hamster melanomas--a melanotic (Ma) and an amelanotic (Ab) form --which differed in aggressiveness and metastatic potential. Flow cytometric analysis of Pgp activity (using a verapamil-sensitive rhodamine R123 exclusion test) as well as Western blotting of cellular lysates showed its preferential (although not very marked) expression in the Ab melanoma cells. The Ab melanoma cells also exhibited a higher proportion of tumor-infiltrating lymphocytes (TIL), mostly of T cell phenotype, that may have reflected a higher immunogenicity of the tumor. In conclusion, Pgp activity appeared to be associated with less-differentiated more aggressively metastasizing melanoma (the Ab variant) although its role in maintaining this phenotype remains to be established.