B. Cherie Millar

Molecular characterisation of the faecal microbiota in patients with type II diabetes.

The investigation provides molecular analyses of the faecal microbiota in type 2 diabetic patients. In order to characterise the gut microbiota in diabetic patients and to assess whether there are changes in the diversity and similarity of gut microbiota in diabetic patients when compared with healthy individuals, bacterial DNAs from 16 type 2 diabetic patients and 12 healthy individuals were extracted from faecal samples and characterised by PCR-denaturing gradient gel electrophoresis (DGGE) with primers specifically targeting V3 region of the 16S rRNA gene, as well as been sequenced for excised gel bands. The counts of Bacteroides vulgatus, Clostridium leptum subgroup and Bifidobacterium genus were assessed using quantitative PCR. By comparing species diversity profiles of two groups, we observed that there were no significant differences between diabetic and healthy group, although a few diabetic individuals (D6, D8) exhibited a remarkable decrease in species profiles. As for the similarity index, it was lower in inter-group than that in intra-group, which showed that the composition of gut microbiota in diabetic group might be changed due to diabetes status. Sequencing results also revealed that bacterial composition of diabetic group was different from that of the healthy group. B. vulgatus and Bifidobacterium genus were low represented in the microbiota of diabetic group, and the significant decrease was observed for Bifidobacterium by real-time PCR. Taken together, in this work we observed the characterisation of gut microbiota in diabetic patients, which suggestes that the gut microbiota of diabetes patients have some changes associated with occurrence and development of diabetes.

Risk Assessment Models and Contamination Management: Implications for Broad-Range Ribosomal DNA PCR as a Diagnostic Tool in Medical Bacteriology

Molecular methods have now become established as accepted methods for the detection of causal agents of infection (viral, bacterial, fungal, and protozoal). In particular, the use of a combination of rRNA genes from bacteria, fungi, and protozoa, i.e., universal or broad-range targets, has become

Comparison of techniques to examine the diversity of fungi in adult patients with cystic fibrosis

This study compares conventional and molecular techniques for the detection of fungi in 77 adult cystic fibrosis (CF) patients. Three different methods were investigated, i.e., (1) conventional microbiological culture (including yeasts and filamentous fungi), (2) mycological culture with CF-derived fungal specific culture media, and (3) Non-culture and direct DNA extraction from patient sputa. Fungi isolated from environmental air samples of the CF unit were compared to fungi in sputa from CF patients. Fungi (n = 107) were detected in 14/77(18%) of patients by method 1, in 60/77 (78%) of patients by method 2 and with method 3, in 77/77(100%) of the patients. The majority of yeasts isolated were Candida albicans and C. dubliniensis. Exophiala (Wangiella) dermatitidis, Scedosporium apiospermum, Penicillium spp., Aspergillus fumigatus, and Aspergillus versicolor were also identified by sequence analysis of the rDNA short internal transcribed spacer (ITS2) region. Conventional laboratory analysis failed to detect fungi in 63 patients mainly due to overgrowth by Gram-negative organisms. Mycological culture with antibiotics dramatically increased the number of fungi that could be detected. Molecular techniques detected fungi such as Saccharomyces cerevisiae, Malassezia spp., Fuscoporia ferrea, Fusarium culmorum, Acremonium strictum, Thanatephorus cucumeris and Cladosporium spp. which were not found with other methods. This study demonstrates that several potentially important fungi may not be detected if mycological culture methods alone are used. A polyphasic approach employing both enhanced mycological culture with molecular detection will help determine the presence of fungi in the sputa of patients with CF and their healthcare environment.

Early detection of Pseudomonas aeruginosa - comparison of conventional versus molecular (PCR) detection directly from adult patients with cystic fibrosis (CF)

BackgroundPseudomonas aeruginosa (PA) is the most important bacterial pathogen in patients with cystic fibrosis (CF) patients. Currently, routine bacteriological culture on selective/non- selective culture media is the cornerstone of microbiological detection. The aim of this study was to compare isolation rates of PA by conventional culture and molecular (PCR) detection directly from sputum.MethodsAdult patients (n = 57) attending the regional adult CF centre in Northern Ireland, provided fresh sputum following airways clearance exercise. Following processing of the specimen with sputasol (1:1 vol), the specimen was examined for the presence of PA by plating onto a combination of culture media (Pseudomonas isolation agar, Blood agar & McConkey agar). In addition, from the same specimen, genomic bacterial DNA was extracted (1 ml) and was amplified employing two sequence-specific targets, namely (i) the outer membrane protein (opr L) gene locus and (ii) the exotoxin A (ETA) gene locus.ResultsBy sputum culture, there were 30 patients positive for PA, whereas by molecular techniques, there were 35 positive patients. In 39 patients (22 PA +ve & 17 PA -ve), there was complete agreement between molecular and conventional detection and with both PCR gene loci. The opr L locus was more sensitive than the ETA locus, as the former was positive in 10 more patients and there were no patients where the ETA was positive and the opr L target negative. Where a PCR +ve/culture -ve result was recorded (10 patients), we followed these patients and recorded that 5 of these patients converted to being culture-positive at times ranging from 4–17 months later, with a mean lag time of 4.5 months.ConclusionsThis study indicates that molecular detection of PA in sputum employing the opr L gene target, is a useful technique in the early detection of PA, gaining on average 4.5 months over conventional culture. It now remains to be established whether aggressive antibiotic intervention at this earlier stage, based on PCR detection, has any significant benefits on clinical outcome.

Antibacterial activity of honey against community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA)

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has now been described globally, as a clinically significant pathogen, particularly associated with skin and soft tissue infections, including abscesses, cellulitis and furunculosis. The recent emergence of CA-MRSA combined with its predominant presentation associated with skin and soft tissue infection, the previous literature indicating honey as an effective treatment of healthcare-associated HA-MRSA-related wound infection, as well as honeys ease of topical application, make the current study timely and of interest to healthcare practitioners involved with wound management. Although previous studies have examined the antimicrobial activity of honey against HA-MRSA, such data are limited regarding the activity of honey against this emerging type of MRSA. CA-MRSA (n=6 isolates), was examined for its susceptibility to natural honey (n=3 honey produced from bees in Northern Ireland and one commercial French honey). Results demonstrated that all honey was able to reduce the cultural count of all CA-MRSA from approximately 10(6) colony-forming units (cfus) (mean = 6.46 log10 cfu/g) to none detectable within 24h of co-culture of separate CA-MRSA organisms individually with all four-honey types examined. Subsequent non-selective enrichment of honey demonstrated that inoculated honey remained positive for CA-MRSA until 72h postinoculation, after which point no culturable organisms could be detected. This study demonstrated that, in vitro, these natural products had an antimicrobial activity against the CA-MRSA organisms tested. Further studies are now required to demonstrate if this antimicrobial activity has any clinical application.

18FDG-positron emission tomography (PET) has a role to play in the diagnosis and therapy of infective endocarditis and cardiac device infection.

In recent years, molecular imaging with positron emission tomography (PET) and more recently, PET coupled with computed tomography (CT) have made a valuable impact in various clinical arenas, primarily within the field of oncology, but also in cardiovascular medicine, particularly in detecting coronary artery disease and myocardial viability. More recently, PET imaging has been proven useful in the diagnosis and evaluation of inflammation and infection at different organ sites. Application of PET in the case of Infective Endocarditis (IE) is still in its infancy and indeed the value of this application in the detection of vegetations is debated primarily due to sensitivity issues of the tracer in cardiac tissue and small vegetations. Interestingly, however, reports are now emerging highlighting the role that this application has played in the diagnosis, assessment of complications such as emboli and metastatic infection and the monitoring of therapeutic treatment of IE. More recently, PET/CT has proven valuable in the diagnosis and assessment of cardiac implantable electronic device (CIED)-related infection and its use has highlighted the contribution that this imaging modality may play in assessing the need for surgery in patients with such infections. This article reviews the literature with regard to the potential value of 2-deoxy-2-[(18)F]fluoro-D-glucose ((18)FDG)-PET, as well as the pitfalls and limitations of this imaging modality, in relation to cardiac infection. The emerging role (18)FDG-PET/CT has in the diagnosis and monitoring of IE, particularly prosthetic valve IE and CIED-related infections should be considered, particularly in difficult cases.

Proteomics Analysis and Protein Expression during Sporozoite Excystation of Cryptosporidium parvum (Coccidia, Apicomplexa)

Cryptosporidiosis, caused by coccidian parasites of the genus Cryptosporidium, is a major cause of human gastrointestinal infections and poses a significant health risk especially to immunocompromised patients. Despite intensive efforts for more than 20 years, there is currently no effective drug treatment against these protozoa. This study examined the zoonotic species Cryptosporidium parvum at two important stages of its life cycle: the non-excysted (transmissive) and excysted (infective) forms. To increase our understanding of the molecular basis of sporozoite excystation, LC-MS/MS coupling with a stable isotope N-terminal labeling strategy using iTRAQ™ reagents was used on soluble fractions of both non-excysted and excysted sporozoites, i.e. sporozoites both inside and outside oocysts were examined. Sporozoites are the infective stage that penetrates small intestinal enterocytes. Also to increase our knowledge of the C. parvum proteome, shotgun sequencing was performed on insoluble fractions from both non-excysted and excysted sporozoites. In total 303 C. parvum proteins were identified, 56 of which, hitherto described as being only hypothetical proteins, are expressed in both excysted and non-excysted sporozoites. Importantly we demonstrated that the expression of 26 proteins increases significantly during excystation. These excystation-induced proteins included ribosomal proteins, metabolic enzymes, and heat shock proteins. Interestingly three Apicomplexa-specific proteins and five Cryptosporidium-specific proteins augmented in excysted invasive sporozoites. These eight proteins represent promising targets for developing vaccines or chemotherapies that could block parasite entry into host cells.

Prevalence and identity of Cryptosporidium spp. in pig slurry.

ABSTRACT Cryptosporidium spp. were detected in 25 of 56 pig slurry samples from 33 Irish farms by PCR and DNA sequencing. The organisms detected included C. suis, Cryptosporidium pig genotype II, and C. muris. We concluded that Cryptosporidium oocysts can persist in treated slurry and potentially contaminate surface water through improper discharge or uncontrolled runoff.

Fungal infections in patients with cystic fibrosis

Current US Cystic Fibrosis Foundation (CFF) Registry data indicates an approximate doubling of prevalence of fungi being detected in the sputum of patients with cystic fibrosis (CF), probably due to a better understanding of the disease and the way in which it is managed. The introduction of new antibiotics and the way in which they are used – in double or triple therapy and as well as in antibiotic cycling – has helped address the requirement to control chronic Gram-negative infections in CF. However, this has inadvertently created a niche for the colonization and proliferation of fungi in the CF lung. The ability to detect and isolate fungi in the sputum of CF patients is important due to the emerging significance of these organisms, particularly in relation to: (i) allergic bronchopulmonary aspergillosis (ABPA); (ii) post-transplant fungaemia; and (iii) chronic colonization/infection (bronchitis). Recent data has indicated that ABPA affects approximately 1–15% of CF patients and is associated with an accelerated decline in lung function. Most of the mycological aetiology of this condition is due to Aspergillus fumigatus and other Aspergillus spp, although other fungi have been reported. In addition, patients who have undergone lung transplantation are at increased risk of opportunistic fungal infection, such as invasive pulmonary aspergilloma and other invasive mycoses, due to the immunosuppression of the host. This review summarizes the important fungal pathogens in CF, namely Aspergillus spp., as well as the emerging fungi, including Scedosporium apiospermum and Exophiala dermatitidis and discusses the various conventional and molecular methods of detection and identification.

Asaia sp., an Unusual Spoilage Organism of Fruit-Flavored Bottled Water

ABSTRACT A gram-negative bacillus was isolated from a batch of fruit-flavored bottled water, which had spoiled as a result of bacterial overgrowth (>106 CFU/ml). The spoilage organism was extremely difficult to identify phenotypically and was poorly identified as Pasturella sp. (78.7% identification profile) employing the API 20NE identification scheme, which gave the profile 5040000. Molecular identification through PCR amplification of a partial region of the 16S rRNA gene followed by direct automated sequencing of the PCR amplicon allowed identification of the organism. Due to the sequence identity (100%) between the spoilage organism and a reference strain in GenBank, the spoilage isolate was considered to be an Asaia sp., a recently described genus and member of the acetic acid bacteria. This is the first report of Asaia sp. causing spoilage of a foodstuff and highlights the benefits of molecular identification techniques based on 16S rRNA gene sequences in the identification of unusual spoilage organisms.