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

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.

Edible seaweed (Palmaria palmata) has traditionally been consumed rawafter harvesting from coastal shorelines around Northern Ireland.To date, there have been no reports examining the microbiology of this material, hence itwas the aimof this study to detect the diversity of themicro£ora found on readytoeat produce. Conventionalmicrobiological analyses of the product failed to detect any gastrointestinal pathogens and gave a mean total count of 1•3×105 cfug-1. 16S rRNA sequencing of culturable bacteria identified the Sanguibacter/Oerskovia/Cellulomonas complex, the Clavibacter/Frigoribacterium/urtobacterium complex, Enterobacter agglomerans, Erwinia herbicola, Flavobacterium spp., Micrococcus lylae, Microbacterium spp., Corynebacterium spp., and Dietza maris. A comparison of growth of isolated environmental organisms was performed to ascertain the most appropriate artificial culture media to employ for their culture in vitro. Tryptone soya broth with yeast extract (TSBYE) and brainfiheart infusion brothwith yeast extract (BHIYE) may be employed as suitable basal broth media for the laboratory culture of these organisms.This is the ¢rst preliminary report on the microbial diversity of edible seaweed and demonstrated the presence of several halophilic genera and species in fresh ready-to-eat edible seaweed from Northern Ireland. Although no gastrointestinal pathogens were cultured from this material, a larger study requiring examination of seasonal e¡ects, quality of marine water and e¡ect of drying on faecal pathogens, is required to support a functional HACCP-based approach to ensuring safety of this product.

J. XU, B.C. MILLAR, J. E. MOORE, K. MURPHY, H. WEBB, A. J. FOX, M. CAFFERKEY AND M. J. CROWE. 2003. Aims: The aim of this study was to develop a polyacrylamide gel electrophoresis (PAGE) method for the rapid separation of 16S rRNA PCR amplicons from aetiological agents of acute meningitis. Methods and Results: Blood samples from 40 patients with suspected acute meningococcal meningitis were examined for the presence of causal agents, including Neisseria meningitidis employing two methods: (i) broad-range 16S rRNA PCR in conjunction with PAGE and automated sequencing and (ii) species-specific PCR employing ABI TaqMan technology for N. meningitidis. Analysis of clinical specimens employing 16S rRNA PCR yielded 33/40 (82•5%) positive for the presence of bacterial DNA. Species-specific PCR yielded 30/40 (75%) clinical specimens positive for N. meningitidis. Prior to separation by PAGE, only 6/33 (18•2%) amplicons were able to be identified by sequence analysis, the remaining amplicons (n=27) did not yield an identification due to the presence of mixed 16S rRNA PCR amplicons. Following separation, amplicons were re-amplified and sequenced, yielding 24/27 (88•9%) positive for N. meningitidis and three specimens positive for Acinetobacter sp., Staphylococcus aureus and Streptococcus pneumoniae. One specimen was positive for both N. meningitidis and Streptococcus spp. and another specimen was positive for N. meningitidis and Pseudomonas sp., by broad-range PCR. Seven clinical specimens were negative for N. meningitidis and other eubacteria using both detection techniques. Conclusions: Clinical specimens including blood and cerebrospinal fluid from patients with suspected acute bacterial meningitis, may become contaminated with commensal skin flora, resulting in difficulties in downstream sequencing of pathogen plus contaminant DNA. This study allows for the rapid separation of amplified pathogen　from contaminant DNA. Significance and Impact of Study: This study demonstrated the usefulness of the rapid separation of multiple 16S rRNA PCR amplicons using a combination of PAGE and automated sequencing, without the need of cloning. Adoption of this technique is therefore proposed when trying to rapidly identify pathogens in clinical specimens employing broad-range 16S rRNA PCR.

B.C. MILLAR, J. E. MOORE, J. XU, M. J. WALKER, S. HEDDERWICK AND R. MCMULLAN. 2002. Aims: To determine the frequency, distribution and association of genotypes of Candida albicans and C. dubliniensis in invasive and noninvasive clinical isolates. Methods: Twenty-one invasive and 18 noninvasive isolates were examined by PCR amplification of a transposable intron region in the 25S rRNA gene. Isolates were genotyped following analysis of the size of resulting DNA amplicons. The isolates could be subdivided into four genotypes (A-D). Results: There was no significant difference between the frequency and genotype distribution of the invasive and noninvasive Candida isolates. Impact of the Study: Therapeutic prophylaxis against candidal infections remains an area of controversy. Any diagnostic markers that reflect the potential of isolates to become invasive should be fully explored, so that more focused antifungal intervention should be targeted at these patients with these potential invasive markers. This study demonstrated that analysis of the transposable intron region in the 25S rRNA gene may be useful in helping to differentiate C. albicans from C. dubliniensis isolates, without the need for sequence analysis, which may not be readily available at primary diagnostic laboratories. However, employment of this genotypic assay is not a suitable locus to determine invasiveness and other more reliable markers of invasiveness should be sought.

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.

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.

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.

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.