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.

This study investigated the various commercially available kits and 'in-house' methods to extract DNA from Gramnegative and Gram-positive bacteria, yeast and fungal agents in commonly employed blood culture material. The main methods investigated were as follows; Qiagen QIAmp Blood kit, Roche high PCR template preparation kit, Puregene DNA extraction kit, boiling, glass beads/sonication and wash/ alkali/heat lysis. The results indicated that a simple wash/alkali/heat lysis method was the most sensitive, reproducible, simple and cost-effective extraction method. This was the only method which removed any PCR inhibitors and inherent DNA which existed in virgin BacT/Alert aerobic, anaerobic and paediatric blood culture material. Contaminating microbial DNA from Lactococcus lactis or Bacillus coagulans was identified in all batches of BacT/Alert

Mushroom worker's lung (MWL) is a hypersensitivity pneumonitis or allergic alveolitis caused by a type III IgG-mediated immunopathogenic inflammatory reaction in the host due to the inhalation of several thermophilic organisms, including Thermoactinomyces spp. It is difficult to distinguish phenotypically the eight species of this genus; therefore, this study sought to develop an improved molecular means of identifying Thermoactinomyces spp. associated with MWL by partial 16S rDNA PCR amplification and direct sequencing. Hypervariable regions within the 16S rRNA gene, which could be employed as signature sequences of the eight individual species, were identified and employed with highly conserved flanking primers to allow initial PCR amplification, before direct DNA sequencing of the 16S rDNA amplicons. A novel 24-mer 16S rDNA oligonucleotide upstream primer was designed from in silico alignments of all Thermoactinomyces spp. and was employed in combination with downstream (reverse) 16S rDNA primers. This permitted the successful identification of all four isolates associated with mushroom workers' lung. The method may be useful in the identification of Thermoactinomyces spp. associated with allergic alveolitis or pneumonitis associated with occupational exposure in agricultural and horticultural environments.

Optimum detection of the Burkholderia cepacia complex (BCC) from sputum of patients with cystic fibrosis (CF) is essential in preventing patient-to-patient transmission of this organism. The aim of this study was to develop an improved PCR assay with reference to sensitivity for the direct detection of BCC organisms from CF sputum employing the recA locus. The sensitivity results of three recA PCR assays were compared using various combinations of previously published primers. These included (i) a single-round approach using the primer set BCR1/BCR2, yielding a 1036-bp product, (ii) a single-round approach using the primer set BCR1/Mr, yielding a 465-bp product, and (iii) a semi-nested PCR (SN-PCR) approach using the primer set BCR1/BCR2 followed by BCR1/Mr. The sensitivity of these assays were determined by spiking B. cepacia-free sputum with known numbers of four strains of BCC, namely, genomovar II [B. multivorans] (C1576), genomovar IIIa (C5424, C6433) and genomovar IIIb (C1394). Following optimization, the chosen assay was performed on 14 patients. Employment of the singleround assay with BCR1/BCR2 was the least sensitive with a detection threshold of 107 cfu/g sputum for GIIIa and GIIIb, and 108 cfu/g sputum for GII. Sensitivity was improved by targeting the smaller amplification region of the recA locus (465 bp) employing the BCR1/Mr primer pair, in combination with a single-round approach, whereby the detection threshold was improved by 1 log for each genomovar. Employment of the semi-nested assay demonstrated optimum sensitivity, whereby the detection threshold increased to 101 and 102 cfu/g sputum for genomovar IIIa/IIIb and genomovar II, respectively. Subsequent genomovar characterisation can be performed by sequencing of the PCR amplicon without the need for culture which may be beneficial in patients in the initial stages of colonisation or who are transiently colonised and who may be culture-negative for BCC.

J. E. MOORE, J. XU, B.C. MILLAR, J. COURTNEY AND J. S. ELBORN. 2003. Aims: To develop a selective agar medium to help detect and quantify Gram-negative flora in the sputum of patients with cystic fibrosis (CF). Methods and Results: A novel Gram-negative Selective Agar (GNSA) medium was developed consisting of tryptone soya broth (30g), bacteriological agar no.1 (10g), yeast extract (5g), crystal violet (2mg), nisin (48mg), novobiocin (5mg), cycloheximide (100mg), amphotericin (2mg) and double distilled water (1 l), for the selective culture of all Gram-negative flora from the sputum of patients with CF. GNSA was able to support the proliferation of all 34 Gram-negative organisms examined, including 23 species most commonly associated with CF, but was unable to support the growth of the 12 Gram-positive or seven fungal organisms examined. Sensitivity studies demonstrated that the GNSA medium was able to detect not less than 1•50×102 CFU ml-1 sputum Pseudomonas aeruginosa, 2•38×102 CFU ml)1 sputum Burkholderia cepacia genomovar IIIb and 6•70×103 CFU ml-1 sputum Stenotrophomonas maltophilia. A comparison of the microbial flora detected in the sputa of 12 adult CF patients by employment of routine bacteriological agar media and GNSA, demonstrated that GNSA was able to detect all Gram-negative organisms cultured by routine media, but had the advantage of detecting Alcaligenes xylosoxidans in two CF patients, whom had no previous history of Gram-negative infection. Conclusions: GNSA was unable to support the proliferation of any Gram-positive organism or yeast/fungi, but was successful in supporting the growth of all Gram-negative organisms challenged. Significance and Impact of the Study: Employment of this medium coupled with semi-automated technology may aid in helping to efficiently determine Gram-negative loading of respiratory secretions, particularly in response to antibiotic intervention.