Search PubMed⌕ Search

Biomedical subjects

B C Millar

Publications and source records attributed to B C Millar.

At least 55 records · Page 3Linked to original sources
PubMed2003

Biohazards and ecotoxicological considerations of landspreading of spent compost wastes.

Spent mushroom compost (SMC) is a major waste of the mushroom industry with low economic value. SMC arises after mushroom production in phase II compost (pIIC), predominantly comprising straw and chicken litter as principal raw ingredients. The majority of SMC waste is disposed off by application to agricultural land. It is an attractive proposition for utilising SMC as soil inorganic fertiliser supplementation. However, there is limited data available as to the consequences of this method of disposal either in terms of microbiological loading of food-borne pathogens and those of concern to mushroom industry itself. The resulting imbalance of the natural flora of the agricultural land has not been properly audited. This study aims to initially examine SMC for prevalence of faecal bacterial pathogens including Campylobacter spp., Salmonella spp. and Listeria monocytogenes that may arise from chicken litter. At another level, it aims to ascertain the pathogenic bacteria (Pseudomonas syringae, pv phaseolicola or tolasii) and fungal populations (Trichoderma, Verticillium species) originating mainly from the straw component of the SMC, which are of concern to the mushroom industry. Lastly, the study would also qualitatively identify the diversity of bacterial populations within SMC. This was largely accomplished through employment of rDNA, PCR and direct sequencing strategies on the culturable microflora. However, for specific mushroom pathogens, nucleic acids (DNA or RNA) were directly extracted from composts before subjecting to sequence analysis. In accordance with the current legislation (ABP 02/02, Animal By Products wastes disposal EC No. 1774/2002), it is imperative to regulate the farm wastes carrying residues from animal sources including SMC before they are regarded safe for land spreading operations. The ecological microbe-microbe and plant-microbe interactions that potentially occur between the native bacterial soil flora and those added annually (approximately 10(18) cells) needs to be reviewed with caution. The above study highlights the ecological consequences involved in the disposal of SMC wastes on agricultural land and its implications for plant, animal and human health.

Agaricales↗
PubMed2002

Molecular diversity of Campylobacter coli and C. jejuni isolated from pigs at slaughter by flaA-RFLP analysis and ribotyping.

A population of porcine isolates of Camplobacter jejuni (n = 11) and C. coli (n = 17) were examined for genotypic relatedness employing ribotyping, as well as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the flagellin (fla)A gene locus. PCR was employed to amplify a 533 bp fragment from the flaA gene, including the previously described short variable region (SVR), employing the novel primers, A2 and Al and successfully generated this amplicon for all wild-type strains examined (n = 28) of both C. jejuni and C. coli, as well as with both type strains, i.e. C. jejuni NCTC 11351 and C. coli NCTC 11366. Individual genotypes were assigned to each isolate typed employing the four typing methods (flaA-RFLP(Hae) III, flaA-RFLP(Pst) I ribotyping(Hae) III and ribotyping(Pst) I) and were assigned an arbitrary genotype code in ascending alphabetical order in comparison with a database of established genotypes for each of the methods employed. This study showed that several flaA-RFLP and ribopatterns existed within C. jejuni and C. coli, and demonstrated a heterogeneous diversity of strains occurring in the pigs examined. Ribotyping of strains with 16S and 23S rRNA with Pst I and Hae III digested chromosomal DNA allowed subdivision of strains into nine and eight groups, respectively. RFLP analyses with Pst I and Hae III digests probed with the flaA gene probe allowed subdivision of strains into eight and eleven subtypes, respectively. Employment of RFLP with the flaA nucleic acid probe and Hae III digests produced the greatest amount of variation of any genotyping scheme employed. Although there was a high degree of variability demonstrated by both typing methods, most isolates ( > 60%) clustered into four main genotypes, i.e. genotypes A-D. FlaA-PCR-RFLP typing demonstrated that the majority of isolates, 67.9 and 60.7%, were included in these four main genotypes for Pst I and Hae III restriction digests, respectively, although there was a high prevalence (7/11; 63.6%) of fla(Hae) III genotype A occurring within the C. jejuni isolates. Likewise, ribotyping studies demonstrated that most isolates were clustered into these four main genotypes, accounting for 81.5 and 60.7% of isolates for Pst I and Hae III restriction digests, respectively. This may indicate that the clonal population of campylobacters within this pig population is largely composed of persistent and dominant types, with a smaller number of hypervariable subtypes. Such data may useful in determining epidemiological routes of transmission of campylobacters from animal to animal, as well as helping to identify virulence determinants in persistent subtype populations.

Animals↗
PubMed2002

Cloning and sequencing of 16S rDNA and 16S-23S rDNA internal spacer region (ISR) from urease-positive thermophilic Campylobacter (UPTC).

AIMS: To clone and sequence the 16S rDNA and 16S-23S rDNA internal spacer region (ISR) from urease-positive thermophilic Campylobacter (UPTC). METHODS AND RESULTS: The primer sets for 16S rDNA and 16S-23S rDNA ISR amplified almost the full length of 16S rDNA and 16S-23S rDNA ISR. About 1500 bp for 16S rDNA and about 720 bp for 16S-23S rDNA ISR of the rrn operon of four strains of UPTC were identified after molecular cloning and sequencing. CONCLUSIONS: The four strains and CCUG18267 of UPTC showed approximately 99% sequence homology of 16S rDNA to each other, 96-97% to Camp. coli, 97-98% to Camp. jejuni and 97-98% to Camp. lari. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time, the nucleotide sequence of 16S-23S rDNA ISR of UPTC has been analysed. The sequence of ISR was almost identical among the four strains of UPTC. It is interesting that the UPTC intercistronic tRNAs demonstrated an order of tRNA of 5'-16S-tRNAAla-tRNAIle-23S-3' in the organisms.

Campylobacter↗
PubMed2002

Genotypic subgrouping of clinical isolates of Candida albicans and Candida dubliniensis by 25S intron analysis.

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.

Candida↗
PubMed2002

flaA-like sequences containing internal termination codons (TAG) in urease-positive thermophilic Campylobacter isolated in Japan.

AIMS: To demonstrate two flaA-like sequences containing two internal termination codons (TAG) in two Japanese strains of urease-positive thermophilic Campylobacter (UPTC). METHODS AND RESULTS: A primer pair of A1 and A2, which ought to generate a product of approx. 1700 bp of the flaA gene for Campylobacter jejuni, was used to amplify products of approx. 1450 bp for two Japanese strains of UPTC, CF89-12 and CF89-14. After molecular cloning and sequencing, the nucleotide sequences of the amplicons from the two strains were found to be 1461 bp in length and to have nucleotide sequence differences in relation to each other at four nucleotide positions, respectively. CONCLUSIONS: Nucleotide and amino acid sequence alignment and homology analysis demonstrated that the polymerase chain reaction (PCR) amplicons from the two Japanese strains have approx. 83% nucleotide and 80% amino acid sequence homology to the possible open reading frame of the flaA gene of UPTC NCTC 12892. SIGNIFICANCE AND IMPACT OF THE STUDY: Surprisingly, both PCR amplicons from the Japanese UPTC have two internal termination codons (TAG) at nucleotide positions from 775 to 777 and 817 to 819, respectively.

Amino Acid Sequence↗
PubMed2002

Mycobacterium triplex pulmonary infection in an immunocompetent patient.

We present the case of a 47-year-old immunocompetent patient with clinical evidence of pulmonary mycobacterial disease which was found to be due to Mycobacterium triplex. This novel organism is an uncommon, emerging, pathogen for which few reports of clinical infection exist in the medical literature.

Bronchoalveolar Lavage Fluid↗
PubMed2002

Trends in the epidemiology of Candida bloodstream infections in Northern Ireland between January 1984 and December 2000.

OBJECTIVE: To describe the epidemiology of Candida bloodstream infections (BSI) in Northern Ireland. METHODS: Retrospective collation of data relating to all clinically significant BSI in a university teaching hospital, which had been recorded prospectively, between 1984 and 2000. RESULTS: One hundred and forty five episodes of candidaemia occurred in 144 patients (of mean age 56.6 years). The contribution of Candida spp. towards all significant BSI increased from 2.0% to 2.5%. C. albicans was the most frequently isolated species, however, its incidence fell from 70% to 53% during the study period. The greatest increase in incidence was seen with C. glabrata which was the most common non-albicans species. Twenty-nine per cent of isolates occurred in patients from an intensive care unit and, surprisingly, a further 25.5% occurred in patients from a surgical service. CONCLUSION: There appears to be several subtle differences in the epidemiology of candidal BSI between Northern Ireland and other countries.

Adolescent↗
PubMed2002

Comparison of API20C with molecular identification of Candida spp isolated from bloodstream infections.

A study was carried out to compare the API20C technology with polymerase chain reaction amplification and direct sequencing of the short internal transcribed spacer region 2 (ITS2) for the identification of 58 isolates of invasive candida species obtained from patients with bloodstream infections over the seven year period 1994 to 2000. Overall, there was only one disagreement between the phenotypic and genotypic identification, where the API scheme identified the isolate as C albicans but the molecular method identified it as C dubliniensis. This study demonstrated that the API20C method is useful in the identification of Candida spp isolated from blood culture and that molecular methods do not enhance identifications made using the API20C scheme. However, for correct reporting of C dubliniensis, an emerging bloodborne pathogen, it is recommended that all isolates identified as C albicans by the API20C scheme are further examined phenotypically and/or genotypically.

Candida↗
PubMed2002

Misidentification of a genomovar of Burkholderia cepacia by recA restriction fragment length polymorphism.

An 8 year old girl with cystic fibrosis presented with a pulmonary exacerbation from which Burkholderia cepacia was cultured. Subsequent polymerase chain reaction restriction fragment length polymorphism analysis of the recA gene suggested the presence of B cepacia Genomovar V (Burkholderia vietnamiensis); however, on subsequent sequence typing, this isolate was confirmed as B cepacia Genomovar IIIb. This report outlines the potential difficulties in the correct characterisation of the various genomovars within the B cepacia complex of organisms, which has particularly important implications for patient segregation and infection control.

Bacterial Typing Techniques↗
PubMed2002

Incidence of Pseudomonas aeruginosa in recreational and hydrotherapy pools.

Pseudomonas aeruginosa remains an important agent of opportunistic infection in patients, particularly those with respiratory complications and burns. One natural niche of this organism is water and water-associated facilities, hence the aim of this study was to examine specimens from recreational and hydrotherapy pools in Northern Ireland over a two-year period. Water specimens (n = 3,510) were obtained from three amenity categories, namely, 13 hydrotherapy pools (specimen number [n] = 323), 51 Jacuzzis/spas (n = 1,397) and 68 swimming pools (n = 1,790). Specimens (100 ml) were filtered through a cellulose acetate (0.45 micron pore size) gridded filter and the membrane was placed on Pseudomonas CFC agar (Oxoid CM559 + SR103) and incubated at 37 degrees C for 48 +/- 2 h. Colonies that clearly showed pyocyanin production or met other identification criteria were considered P. aeruginosa. Of the amenities examined 4/13 hydrotherapy pools (30.8%), 37/51 Jacuzzis/spas (72.5%) and 26/68 swimming pools (38.2%) were positive for P. aeruginosa. The most heavily contaminated amenity category was the Jacuzzi/spa, where 34.7% and 12% of private and public sites respectively were positive for P. aeruginosa at a level of greater than 1,000 cfu 100 ml-1. Approximately twice as many samples were positive in private Jacuzzis/spas compared to publicly operated facilities. There was a similar trend with respect to public and private hydrotherapy pools, though bacterial counts did not exceed 1,000 cfu 100 ml-1. Recreational and therapeutic amenities involving the use of water may be a potential source of P. aeruginosa for susceptible patient groups, including patients with cystic fibrosis and bronchiectasis. This may vary depending on amenity type and public/private ownership of such amenities.

Humans↗
PubMed2001

PCR-IMS detection and molecular typing of Cryptosporidium parvum recovered from a recreational river source and an associated mussel (Mytilus edulis) bed in Northern Ireland.

PCR-IMS was used to detect Cryptosporidium spp. in environmental water samples in Northern Ireland which had previously tested negative by a conventional IFA staining method. Oocysts of C. parvum detected in river water and final treated sewage effluent collected from various sites along the river Lagan were identified as genotype 2 (animal origin) based on polymorphisms observed at the thrombospondin related adhesion protein gene locus. Similarly, genotype 1 (human origin) oocysts of C. parvum were detected in the marine filter feeder mussel, Mytilus edulis, collected from the shores of Belfast Lough. Detection of the human genotype of Cryptosporidium in mussels destined for human consumption identifies the organism's serious potential as a foodborne pathogen. This work highlights the possible value of monitoring filter feeder systems, in conjunction with specific molecular epidemiological tools, as an alternative monitoring system for the parasite within the aquatic environment.

Animals↗
PubMed2001

A rapid molecular assay for the detection of antibiotic resistance determinants in causal agents of infective endocarditis.

AIMS: To develop and employ a PCR amplification system, directly from clinical specimens, for the rapid molecular detection of common antimicrobial resistance genes for streptococci, staphylococci and enterococci organisms causing infective endocarditis (IE). METHODS AND RESULTS: Eleven antibiotic resistance genes were targeted by PCR along with four identification-related loci. Blood culture and heart valve material from staphylococcal endocarditis patients were directly examined for methicillin resistance. PCR conditions were optimized for the following antibiotic resistance loci: staphylococci (mecA, aacA-aphD), streptococci (PBP 1A, PBP 2B, gyrB, parE) and enterococci (vanA, vanB, vanC-1, vanC-2, aacA-aphD, aphA3). The presence of methicillin resistance was confirmed in one of the eight IE patients examined. CONCLUSION: This study presents a PCR amplification system for the detection of antibiotic resistance genes. Detection of such genes may indicate susceptibility of the causal agents of IE to commonly prescribed antimicrobial agents. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid detection of antibiotic resistant organisms may reduce the use of inappropriate antibiotic agents or enable the use of the most appropriate combinations of antibiotics, other than those that would normally be prescribed empirically for IE. Such a method may be particularly valuable in cases of culture-negative endocarditis. Detection of antibiotic resistance genes by molecular-based techniques, namely PCR, will allow more directed antibiotic therapy and may also provide opportunities for earlier identification of resistant organisms.

Drug Resistance, Microbial↗
PubMed2001

Occurrence and molecular genotyping of Cryptosporidium spp. in surface waters in Northern Ireland.

AIMS: To investigate the incidence and genotype of Cryptosporidium parvum oocysts in drinking water sources in Northern Ireland for the period 1996-1999, and to compare conventional and molecular methods of detection. METHODS AND RESULTS: Four hundred and seventy-four waters were investigated by conventional methods, namely immuno-fluorescent antibody detection (IFA; 380) and immuno-magnetic separation-IFA (IMS-IFA; 94), of which 14/474 (3%) were positive. Two hundred and fourteen samples (214/474) were also investigated by PCR techniques, targeting both the 18S rRNA and TRAP-C2 genes, of which 11/214 (5.1%) were positive. These 11 samples were classified as genotype II following sequence analysis of the TRAP-C2 amplicon. CONCLUSIONS: This study demonstrated the low incidence of oocysts of C. parvum in water sources in Northern Ireland. SIGNIFICANCE AND IMPACT OF THE STUDY: Such molecular-based techniques offer a number of advantages over conventional detection methodologies, namely greater sensitivity and specificity as well as the ability to provide accurate genotyping data rapidly, which may be valuable in directing operational management in potential outbreak situations.

Animals↗
PubMed2001

recA genotyping of Salmonella enteritidis phage type 4 isolates by restriction fragment length polymorphism analysis.

AIMS: To subtype Salmonella enteritidis phage type 4 isolates by using recA genotyping. METHODS AND RESULTS: Random amplified polymorphic DNA analysis using a primer ERIC2 of 76 isolates of Salmonella enteritidis phage type 4 obtained in Northern Ireland in 1998 and in 1999 demonstrated the presence of five genotypes. Restriction fragment length polymorphism analysis, using a degenerate primer pair designed to amplify a segment (about 640 bp in length) of the recA gene from several members of the Enterobacteriaceae with restriction enzymes, HhaI and Sau3AI, showed that the resulting fragments could differentiate the isolates into three groups, respectively. CONCLUSION: recA gene amplification and HhaI and Sau3AI restriction digestion was demonstrated to increase the differentiating power between isolates of Salmonella enteritidis phage type 4 by combining the patterns of the random amplified polymorphic DNA analysis procedure using a primer ERIC2. SIGNIFICANCE AND IMPACT OF THE STUDY: A novel restriction fragment length polymorphism assay for isolates of Salmonella enteritidis phage type 4, based on the amplification of the recA gene was attained and its comparison and its combination with random amplified polymorphic DNA analysis was provided.

Bacterial Proteins↗
PubMed2001

Rapid characterization of the genomovars of the Burkholderia cepacia complex by PCR-single-stranded conformational polymorphism (PCR-SSCP) analysis.

Four polymerase chain reaction (PCR) primer pairs (A-D), specific for the Burkholderia cepacia complex of organisms were designed to encompass the entire gene (approximately 1300 bp) from sequence alignments of the rec A operon of B. cepacia. Genomic bacterial DNA from type strains and wild-type B. cepacia complex isolates of previously determined genomovar status was amplified employing these four primer pairs, as well as a fifth primer set (E) already published. Primer sets B, C and E were successful in obtaining a PCR amplicon of correct estimated size of 598, 1107 and 1043 bp, respectively. Subsequent single-stranded conformational polymorphism (SSCP) analysis of PCR amplicons demonstrated unique profiles for the five genomovar types for primer pairs B and E, but was unable to differentiate distinguishable profile types for primer pair C. SSCP analysis was demonstrated to be simple, rapid and cost effective and may prove a useful method of genomovar typing of B. cepacia complex organisms from cystic fibrosis patients in busy diagnostic laboratories.

Burkholderia cepacia↗