Article

Fungi in bottled water: A case study of a production plant

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Abstract

A one-year fungal survey of a water bottling plant was conducted in order to evaluate the incidence and fluctuations of the mycobiota. The dominant fungal genera in order of highest numbers isolated were Penicillium, Cladosporium and Trichoderma followed by Aspergillus, Paecilomyces, and others. As expected, the highest number of isolates were collected during the warmer months, particularly May and June. Indeed during these two months there were more fungi present in the water, indicating that during those times of the year when fungal contamination is high, 0.4 mm filters should be changed on a more regular basis. In order to assess whether contamination was single or multi-loci, molecular methods based on the PCR were used for Penicillium brevicompactum. Overall, fungal contamination arose from multiple sources. Some P. brevicompactum strains were very "alike" and were detected during different sampling times, indicating that they were endemic to the plant. There was no evidence to suggest that fungi detected in the source water passed through to other parts of the plant. However, there was evidence that fungal strains isolated from the water filter were detected elsewhere in the factory, confirming the need to change filters more regularly during periods of high fungal contamination. In order to improve quality control a HACCP programme was implemented and Best Practice Guidelines introduced.

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The reproducibility of the generation of random amplified polymorphic DNA fragments from three commonly used thermal cyclers was determined using identical assay conditions. In all cases, different results were obtained from the three instruments. Variation in the length of the primer (20 nt or 10 nt) did not have any effect on the reproducibility of the assays from the three machines tested. A DNA concentration of 1 ng generated poorly staining DNA fragments whereas concentrations between 10 ng and 100 ng gave similar banding patterns when using the same thermal cycler. Low concentrations of primer (0.05 microM) did not produce any detectable DNA fragments. Increased primer concentrations of 0.25 microM or higher generated intensely staining DNA fragments, and concentrations above 0.5 microM did not improve the clarity of the banding patterns but did direct the synthesis of increasing amounts of very short DNA fragments. Surprisingly, the 20 nt-long primer was able to direct the synthesis of more DNA fragments than a primer of only 10 nt long.
Article
Evolutionary conservation of an interspersed repetitive DNA sequence, BOX, from Streptococcus pneumoniae was investigated to explore the mosaic nature of these elements. BOX elements consist of various combinations of three subunits, boxA, boxB, and boxC. Eight oligonucleotide probes were designed based on consensus DNA sequences of boxA, boxB, and boxC subunits. DNA hybridization studies and PCR using these probes/primers demonstrate that oligonucleotide sequences within the boxA subunit appear to be conserved among diverse bacterial species. The boxB and boxC subunits show only limited, if any, sequence conservation in bacteria other than S. pneumoniae. Intact BOX elements with boxA, boxB, and boxC subunits were only present in high copy number in pneumococcal strains. This pattern of differential conservation lends support to the modular nature of BOX repetitive elements in that boxA-like subsequences are effectively independent of boxB-like or boxC-like subunits in bacteria other than S. pneumoniae. Furthermore, dendrograms derived from repetitive sequence-based PCR (rep-PCR) fingerprints of S. pneumoniae isolates using the BOXA1R primer yielded clustering patterns that were similar to those obtained previously by other methods, suggesting that these repetitive sequence-based DNA fingerprints represent intrinsic properties of an S. pneumoniae strain's genome. Our results indicate widespread conservation of boxA-like subsequences in the bacterial kingdom, lend support to the mosaic nature of BOX in S. pneumoniae, and demonstrate the utility of boxA-based primers for rep-PCR fingerprinting of many microorganisms.
The microbiology of water Part 1: Drinking water, Report on Public Health and Medical Subjects No.71, Methods for the examination of waters and associated materials. London, Her Majesty's Stationery Office
  • Anonymous
Anonymous. The microbiology of water. Part 1: Drinking water, Report on Public Health and Medical Subjects No.71, Methods for the examination of waters and associated materials. London, Her Majesty's Stationery Office, 1994. References
Report DW-01/F for UK Water Industry Research Limited. Egham, International Mycological Institute
  • J Kelley
  • G Hall
  • Rrm Paterson
Methods for the examination of waters and associated materials. London, Her Majesty's Stationery Office
  • Anonymous