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B C Millar

Publications and source records attributed to B C Millar.

At least 37 records · Page 2Linked to original sources
PubMed2004

Current trends in the molecular diagnosis of infective endocarditis.

The introduction of molecular methods into biomedical science, particularly nucleic acid amplification techniques including the polymerase chain reaction, has significantly improved the diagnosis of several diseases. Likewise, the adoption of such molecular techniques to aid in the detection and identification of causal organisms in patients with infective endocarditis (IE) has been particularly beneficial in cases of difficult, atypical or culture-negative IE. Several different molecular approaches have been suggested for the diagnosis of IE, including variations in the type of cardiological specimens examined, nucleic acid extraction, gene target and molecular platform, each presenting their own advantages and disadvantages. This review examines the molecular approach to the detection and identification of causal agents of IE and provides details and a discussion of the application of such methods, particularly those implemented over the last 7 years.

Bacteria↗
PubMed2004

Nucleotide sequence analysis of the recA gene and discrimination of the three isolates of urease-positive thermophilic Campylobacter (UPTC) isolated from seagulls (Larus spp.) in Northern Ireland.

Nucleotide sequencing after TA cloning of the amplicon of the almost-full length recA gene from three strains of UPTC (A1, A2, and A3) isolated from seagulls in Northern Ireland, the phenotypical and genotypical characteristics of which have been demonstrated to be indistinguishable, clarified nucleotide differences at three nucleotide positions among the three strains. In conclusion, the nucleotide sequences of the recA gene were found to discriminate among the three strains of UPTC, A1, A2, and A3, which are indistinguishable phenotypically and genotypically. Thus, the present study strongly suggests that nucleotide sequence data of the amplicon of a suitable gene or region could aid in discriminating among isolates of the UPTC group, which are indistinguishable phenotypically and genotypically.

Animals↗
PubMed2004

Employment of 16 S rDNA gene sequencing techniques to identify culturable environmental eubacteria in a tertiary referral hospital.

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 labour-intensive, 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.

Actinomycetales↗
PubMed2004

Identification of novel eubacteria from spent mushroom compost (SMC) waste by DNA sequence typing: ecological considerations of disposal on agricultural land.

A small study was undertaken to examine the microbiological characteristics of spent mushroom compost (SMC), which is the major waste by-product of the mushroom industry and which is regularly disposed off by application to agricultural land. The primary aim of this study was to examine SMC for the presence of faecal bacterial pathogens, including Campylobacter spp., Salmonella spp. and Listeria monocytogenes. Secondly it was desirable to quantify bacterial and fungal populations within SMC, and also qualitatively identify the diversity of bacterial populations within SMC, through employment of rDNA PCR and direct sequencing techniques on the culturable microflora. Conventional microbiological analyses of SMC material (n=30) from six commercial operations in both Northern Ireland and the Republic of Ireland, failed to detect Salmonella spp, Listeria spp. or Campylobacter spp. in any of the SMC material examined. Total aerobic plate counts gave a mean count of log10 7.01 colony forming units (cfu) per gram SMC material (range: log10 6.53-7.52 cfu/g). Fungal counts gave a mean count of log(10) 4.57 cfu per gram SMC material (range: log10 3.93-4.98 cfu/g). From a total of greater than 50 colony picks, a total of 12 bacterial morphotypes were identified and were further examined by employment of partial 16S rRNA gene amplification and sequencing techniques, yielding several genera and species, including Bacillus licheniformis, Bacillus subtilis, Klebsiella/Enterobacter sp. Microbacterium sp. Paenibacillus lentimorbus, Pseudomonas mevalonii, Sphingobacterium multivorum and Stenotrophomonas sp. This is the first preliminary report on the microbial diversity of SMC waste and demonstrates the presence of several species that have not been previously described in SMC, in addition to two potentially novel species within the genera Microbacterium and Stenotrophomonas. It is thereby important to examine the ecological microbe-microbe and plant-microbe interactions that are occurring between the native bacterial soil flora and those added annually (theoretically estimated at approximately 10(18) cells) through the application of SMC. Such studies would be beneficial in helping to ascertain the ecological consequences involved in the disposal of SMC waste on agricultural land.

Agaricales↗
PubMed2004

Determination of verocytotoxin and eae gene loci by multiplex PCR in Escherichia coli O157:H7 isolated from human faeces in Northern Ireland: a four-year study of trends, 1997-2000.

This study aims to determine the distribution and frequency of verocytotoxin genes in human faecal clinical isolates of Escherichia coli O157 in Northern Ireland during the period 1997-2000, using a special four-target multiplex polymerase chain reaction (PCR) assay. One hundred and thirty two isolates of E. coli O157:H7 cultured during the four-year period (1997 [n=28]; 1998 [n=25]); 1999 (n=43); 2000 [n=36]), representing approximately 79% of total E. coli O157 laboratory isolations throughout N. Ireland, are examined for the presence of verocytotoxin gene loci (VT1, VT2 and eae) using a multiplex PCR assay. These isolates originate from the four Regional Area Health Boards that constitute the healthcare system in N. Ireland as follows: Eastern (53.8%; n=71), Northern (34.1%; n=45), Western (6.8%; n=9) and Southern (5.3%; n=7). Results showed that over 80% of these isolates possessed the VT2 and eae gene loci, with the remainder being predominantly VT1-, VT2- and eae-positive. None possessed the VT1 gene locus alone. Development and adoption of this simple four-target (three virulence and one control gene loci) multiplex PCR assay and subsequent recording of resulting verocytotoxin-typing data in a database, permitted local, rapid determination of carriage of known molecular virulence determinants of E. coli O157 isolates, which may aid in outbreak-related epidemiological investigations or other longitudinal studies.

Adolescent↗
PubMed2004

Culture-negative Bartonella endocarditis in a patient with renal failure: the value of molecular methods in diagnosis.

Members of the genus Bartonella are increasingly recognised as a cause of culture-negative endocarditis, particularly in those patients with underlying risk factors (e.g., homelessness and alcoholism (B. quintana) or valvulopathy and cat ownership (B. henselae). The aortic and mitral-valves are most commonly involved. Here, a case is reported of culture-negative right-sided endocarditis, without any of the above risk factors, due to Bartonella sp. in a 69-year-old man who presented with acute renal failure. The diagnosis was made using a broad-range 16S rRNA polymerase chain reaction (PCR) technique and direct automated sequencing on a peripheral blood sample, which was subsequently confirmed serologically. A review of the literature on Bartonella endocarditis is also presented. Molecular laboratory methods using peripheral blood or blood cultures may be very useful in the diagnosis of causal agents in culture-negative endocarditis and add further support to the recently inclusion of molecular (PCR) diagnosis, as a major Duke's criterion, for the diagnosis of infective endocarditis.

Acute Kidney Injury↗
PubMed2003

Employment of broad-range 16S rRNA PCR to detect aetiological agents of infection from clinical specimens in patients with acute meningitis--rapid separation of 16S rRNA PCR amplicons without the need for cloning.

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.

Acinetobacter↗
PubMed2003

Development of a Gram-negative selective agar (GNSA) for the detection of Gram-negative microflora in sputa in patients with cystic fibrosis.

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 (30 g), bacteriological agar no.1 (10 g), yeast extract (5 g), crystal violet (2 mg), nisin (48 mg), novobiocin (5 mg), cycloheximide (100 mg), amphotericin (2 mg) 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 x 102 CFU ml-1 sputum Pseudomonas aeruginosa, 2.38 x 102 CFU ml-1 sputum Burkholderia cepacia genomovar IIIb and 6.70 x 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.

Adult↗
PubMed2003

Novel employment of lactate dehydrogenase release from porcine aortic endothelial cells (PAEC) as a quantitative marker of cytotoxic activity in thermophilic Campylobacter spp. from human faecal isolates, poultry and environmental sources.

The aim of this study was to employ a novel cytotoxicity assay based on primary porcine aortic endothelial cells in combination with a lactate dehydrogenase release assay to quantitatively determine differences in cytotoxin production between Campylobacter jejuni, C. coli, C. lari and urease-positive thermophilic campylobacters (UPTC), isolated from human faeces, animals and environmental sources. Campylobacter isolates totalling 34 and comprising of C. jejuni (n = 24) C. coli (n = 5) and UPTC (n = 4) and C. lari (n = 1) were analysed. The cytotoxic response ranged from 32.15 to 64.47% and 33.08 to 59.41%, for C. jejuni from chicken and human isolates, respectively and there was no statistically significant difference (P > 0.05) in cytotoxic response between C. jejuni isolated from humans and chicken isolates (50.78% versus 50.55% cytotoxicity, respectively). However, there was a difference in response between C. jejuni and C. coli isolated from chickens (50.78% versus 33.22% cytotoxicity, respectively). The greatest cytotoxic response was obtained with the UPTC group of organisms examined (n = 4 isolates) (mean cytotoxic response = 57.11% cytotoxicity. Employment of this cytotoxin assay may help identify virulent strains in poultry that could potentially proceed to cause clinical problems for humans and thus intervention measures targeted at the reduction or elimination of such specific strains, may be sought.

Animals↗
PubMed2003

Haemophilus segnis: a rare cause of endocarditis.

This report presents a case of endocarditis due to Haemophilus segnis, which represents a speciation difficulty for the routine laboratory. In this study, a molecular approach provided speciation, which was confirmed phenotypically by a reference laboratory. The use of molecular genotypic analysis is an additional strategy in the investigation of endocarditis. It has applications not only in isolate identification but also in primary detection of infection, particularly in patients whose blood is culture negative by conventional methodologies.

Base Sequence↗
PubMed2003

Prevalence of bacterial faecal pathogens in separated and unseparated stored pig slurry.

AIMS: To examine the prevalence and diversity of bacterial faecal pathogens in unseparated slurry, separated solids and liquid fractions from a commercial pig farm. METHODS: A total of 43 stored slurry specimens originating from a fattening house over the period February-April 2002 were analysed, consisting of unseparated (n = 14) slurry, separated solids (n = 16) and separated liquid (n = 13). Specimens were examined for the presence of five bacterial pathogens including Salmonella spp., Shigella spp., Campylobacter spp., Escherichia coli O157 and Yersinia enterocolitica. Selective enrichment and plating methods were employed for detection of Salmonella spp. and Campylobacter spp. and conventional selective plating techniques for the remaining genera. Antibiogram profiles to 12 antibiotic agents were obtained for all Salmonella isolates obtained. RESULTS: Salmonella spp. were identified in all components of the slurry specimens, whereas Campylobacter spp. was only recovered from the unseparated and separated liquid fractions. In both cases, the separated liquid fraction had the highest prevalence of pathogens and the separated solid fraction had the lowest prevalence. None of the slurry specimens examined were positive for E. coli O157:H7, Shigella spp. or Y. enterocolitica. Twenty-nine isolates of Salmonella were recovered from the slurry specimens, comprising seven serovars, of which Salmonella manhattan was the most prevalent, accounting for over half [15 of 29 (51.7%)] of all Salmonella isolates. Salmonella anatum, Salm. derby, Salm. give, Salm. heidelberg, Salm. simi and Salm. stanley serovars were also recovered. All Salmonella isolates were sensitive to ampicillin, augmentin (amoxicillin/clavulanic acid), chloramphenicol, ciprofloxacin, gentamicin, kanamycin and trimethoprim, but has variable resistance to tetracycline (100%), sulphonamides (84.6%), furazolidone (38.5%), nalidixic acid (15.4%) and streptomycin (15.4%). The majority (57.7%) of isolates displayed antibiotic resistance to at least two antibiotic agents, followed by 34.6% of isolates being resistant to three agents and the remainder (7.7%) being resistant to four antibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated a marked reduction in the prevalence of Campylobacter and Salmonella in the solids component of separated pig slurry. The adoption of control processes such as aeration of slurry prior to its spread onto agricultural land and newer approaches to pathogen reduction should be investigated, to reduce the transmission of pathogens from pig slurry to the environment.

Animal Husbandry↗
PubMed2003

Structural analysis and genetic variation of the 16S-23S rDNA internal spacer region from Micrococcus luteus strains.

AIMS: To clone and sequence the 16S-23S ribosomal DNA (rDNA) internal spacer region (ISR) from Micrococcus luteus. METHODS AND RESULTS: The primer pair for 16S-23S rDNA ISR amplified a fragment of about 850 bp in length for two strains, JCM3347 and JCM3348 and a fragment of about 790 bp for a strain, ATCC9341. After sequencing the ISRs were identified by the comparison of the ISRs and the flanking regions of ISR. CONCLUSIONS: Although the sequence difference of the ISR occurred at only one position between the two JCM strains, the highly variable length (440 and 370 bp) and sequence similarity (about 40%) were demonstrated between the ISRs of the two JCM strains and a ATCC strain. SIGNIFICANCE AND IMPACT OF THE STUDY: A CCTCCT sequence was first detected at the 3'-end of the 16S rDNA of the three strains. Moreover, highly similar sequence to the 21-bp region containing a putative rRNA processing site was observed in the ISR of the three strains. Interestingly, no intercistronic tRNAs were demonstrated in the ISRs from the three strains.

Base Sequence↗