Summary Universal or 'broad-range' eubacterial polymerase chain reaction (PCR) was performed on 53 isolates from environmental water-associated sites in a haematology unit (N=22), and the outer surfaces of cleaning lotion containers sited throughout a tertiary referral hospital (N=31). 16 S rDNA PCR was performed using two sets of universal primers, including the novel reverse primer, XB4, to generate a composite amplicon of 1068 bp, which was sequenced to obtain each isolate's identity. Sequence analysis was able to identify 51 isolates. Most (75% from the haematology unit and 81% from cleaner containers) were Gram-positive. Nine different genera were identified from the haematology unit and 13 from the cleaning lotion containers. This study provides the first reports of Terrabacter spp. And Brachybacterium paraconglomeratum isolated from a hospital environment. As unusual and difficult-to-identify environmental organisms are unlikely to be clinically significant, and molecular identification is costly and labourintensive, we recommend that molecular methods are only used as an adjunct to first-line phenotypic identification schemes where a definitive identification is required. Where molecular identification methods are justified, partial 16 S rDNA PCR and sequencing employing the novel universal primer XB4, is a valuable and reliable technique.

During October 1999 to November 2000, 98 blood culture specimens from the same number of febrile episodes originating from 49 patients with haematological malignancies were examined for the presence of eubacteria and fungi based on 16S rRNA gene and the 5.8, 18 and 28S rRNA combined with in vitro PCR amplification and sequencing, in addition to conventional blood culture laboratory techniques. Nineteen of the samples were associated with positive blood cultures. Eubacterial (16S rRNA) PCR detected bacterial DNA in 26 febrile episodes, i.e. in an additional seven febrile episodes than blood-culture alone. The species identified by partial 16S Rrna gene sequencing were as follows Staphylococcus spp (n=6), Staphylococcus epidermidis (n=5), Acinetobacter spp (n=5), Escherichia coli (n=2), Enterobacter agglomerans (n=2), Campylobacter spp (n=1), Citrobacter spp (n=1), Corynebacterium spp (n=1), Enterobacter faecium (n=1), Ralstonia spp (n=1), Acidovorax spp. (n=1) and Stenotrophomonas maltophilia (n=1). Gram-positive bacteria were found in 12/27 (44.6%) and Gram-negative bacteria were found in 15/27 (55.6%). After optimization of a PCR-based fungal detection method, none of the febrile episodes were shown to be attributable to fungi. The results of this study suggest that fungi are not common causal agents of febrile episodes in patients with a haematological malignancy at this centre and that molecular techniques can augment cultural methods in the diagnosis of causal agents of bacteraemia in patients, so that appropriate antibiotic regimens may be commenced in patients with culture-negative episodes of infection.

Bacterial culture was allowed to dry to completeness on Columbia agar base with defibrinated horse blood. Following 6 months storage at room temperature, microbial DNA was extracted and successfully amplified by PCR. This storage technique has the advantage over other methods of not requiring (i) a DNA extraction protocol prior to storage and (ii) refrigeration and/or freezing. This technique maybe useful in the transportation of bacterial genomic DNA in nonviable cells as well as reliable method for the storage of DNA in underdeveloped countries.

The aim of this investigation was to identify the bacterial microflora in monetary coinage from 17 countries by the employment of PCR sequenced-based molecular identification of rDNA from bacterial cultures. Silver, bronze and other alloy coins (approx. 300g) from 17 currencies were enriched individually by culturing aerobically in tryptone soya broth for 72h at 30oC. Following this, 20μl broth was inoculated onto Columbia blood agar supplemented with 5% [v/v] defibrinated horse blood for 72h at 30oC and resulting colonies were purified by further subculture, as detailed above for a further 72h. All colonies were identified by initial PCR amplification of a partial region of the 16S rRNA gene locus, which was then sequenced and the sequence aligned using the BLASTn algorithm. Twenty five isolates were obtained from the coinage, and of these, 25/25 (100%) were Gram positive and the most prevalent genus observed was Bacillus [B. megaterium, B. lentus, B. litoralis, B. subtilis, B. circulans and other Bacillus spp.] accounting for 10/25 (40%) isolates and was isolated from 10/17 (58.8%) countries. This was followed by Staphylococcus spp. [Staph. aureus, Staph. epidermidis, Staph. hominis, Staph. schleiferi], which accounted for 7/25 (28%) of isolates and was isolated from 7/17 (41.2%) countries. Given the organisms identified in this study, it is not believed that monetary coinage presents any additional risk to public health, however we support the principles of basic hygiene in terms of proper hand washing, avoidance of handling money when working with food, wounds and skin lesions. In conclusion, this study demonstrated that money from 17 countries was contaminated by environmental Gram positive flora, in particular Bacillus spp. and that universal 16S rDNA-PCR approach coupled with automated direct sequencing provides a rapid means of identifying the contaminant organisms present.