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

BACKGROUND: Pseudomonas 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. METHODS: Adult 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 (oprL) gene locus and (ii) the exotoxin A (ETA) gene locus. RESULTS: By 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 oprL 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 oprL 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. CONCLUSIONS: This study indicates that molecular detection of PA in sputum employing the oprL 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

Molecular characterization of the sequences of the 16S-23S rDNA internal spacer region (ISR) from isolates of Taylorella asinigenitalis

<p>Abstract</p> <p>Background</p> <p>Sequence information on the 16S-23S rDNA internal spacer region (ISR) exhibits a large degree of sequence and length variation at both the genus and species levels. A primer pair for the amplification of 16S-23S rDNA ISR generated three amplicons for each of isolates of <it>Taylorella asinigenitalis </it>(UCD-1^T, UK-1 and UK-2).</p> <p>Findings</p> <p>Following TA cloning and sequencing, the three isolates of <it>T. asinigenitalis </it>were demonstrated to possess three ISR units of different lengths. Although the three corresponding ISRs (A, B and C) were identified to be identical to each other (UK-1 and UK-2 isolates), the ISRs shared approximately 95.3–98.9% nucleotide sequence similarities between the UCD-1^Tand UK-1/-2 isolates. A typical order of two intercistronic tRNA genes (5'-tRNA^Ile-tRNA^Ala-3') with the different nucleotide spacers [44 through 51 base pairs (bp)] in length was identified among the isolates. The consensus sequences of the antiterminators of <b>boxB </b>and <b>boxA </b>were also identified in all ISRs. Thus, three ISRs were identified for each isolate, and therefore, at least three distinctly different ribosomal RNA operons were suggested to occur in the genome of <it>T. asinigenitalis</it>. This was also confirmed by Southern hybridization procedure.</p> <p>Conclusion</p> <p>The present study represents a dendrogram constructed based on the nucleotide sequence data of 16S-23S rDNA ISR for <it>T. asinigenitalis</it>, which may aid in the phylogenetic positioning of <it>T. asinigenitalis </it>within the genus <it>Taylorella</it>, and in the molecular discrimination of <it>T. asinigenitalis</it>.</p

Antimicrobial resistance to 14 antimicrobials in marine coastal waters around Northern Ireland: Use of the novel Relative Resistance Index as a marker of ecological status

Relatively little work has been published on the incidence of antibiotic resistance (ABR) in the marine microbiological environment, which is of importance to animal (fish, mammals, birds) health, zoonotic transmission, distribution of ABR bacteria with oceanic drift, and ultimately human health. A study was performed to determine the diversity of total ABR (intrinsic and acquired resistance) in marine bacteria in shallow coastal waters surrounding Northern Ireland through the use of a novel Relative Resistance Index (RRI) as a surrogate marker for ecological change, particularly in comparing marine water in commercial versus non-commercial sites. Total antibiotic resistance was observed to varying degrees in all marine water specimens and specific resistance levels were as follows, in order of diminishing antibacterial effectiveness: fluoroquinolones \u3e rifampicin \u3e polymyxin \u3e tetracycline \u3e sulphamethoxazole/trimethoprim \u3e third generation cephalosporin and streptomycin \u3e carbapenem \u3e macrolide \u3e clindamycin \u3e vancomycin \u3e fucidic acid \u3e penicillin. None of the sampling sites contained endogenous bacteria that were resistant to ciprofloxacin, while nearly all (19 of 20 sites; 95%) contained bacteria that were resistant to penicillin. Commercial sites had a higher mean RRI score of 6.57±3.58 than non-commercial sites (RRI = 4.08 ± 2.02), which was statistically significant (p = 0.037), indicating that bacteria isolated from seawater in commercial coastal harbors had a higher frequency of antibiotic resistance than non-commercial sources. This novel RRI marker may be useful in assessing ecological change in marine water environments. In conclusion, this study demonstrated that there can be a high level of total ABR (intrinsic and acquired) in bacterial populations in marine water environments, which are multi- and pan-resistant to up to 11 major classes of antibiotics simultaneously. Ecological studies are urgently needed to help define the fate of ABR marine bacteria in their natural environment and their ability to act as reservoirs and donors of ABR to pathogenic bacteria, many of which transiently inhabit the natural environment

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 (TM) 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

Epidemiological study of E. coli O157:H7 isolated in Northern Ireland using pulsed-field gel electrophoresis (PFGE)

In Northern Ireland over the last 7 years, there is a mean of 41.9 laboratory reports per annum of human gastrointestinal infection (range 19-54) caused by Escherichia coli O157:H7. In the preceding years 1992-1996, reports were 5.4 per annum, whereas in 1997-2000, reports increased from 30 to 54 per annum. This high level has continued on an annual basis to date. The aim of this study was therefore to retrospectively examine this period of exponential increase in reports to help ascertain the genetic relatedness of strains employing pulsed-field gel electrophoresis (PFGE), as no data on the molecular epidemiology of E. coli O157:H7 in Northern Ireland has yet been published. Clinical isolates (n=84) were PFGE typed employing Xba I digestion and resulting band profiles demonstrated the presence of 13, 9 and 16 clonal types, for 1997, 1998 and 1999, respectively. In 1998, five clonal types remained from 1997 with the introduction of 4 new clonal types, whereas in 1999, 10 new clonal types were observed, accounting for over half (58%) of the E. coli O157 isolates for that year. These data suggest that, unlike gastrointestinal infections due to thermophilic campylobacters, there was considerable genetic evolution of PFGE clonal types of E. coli O157, through the displacement and emergence of genotypes. Further studies are now required to find the environmental reservoirs of these common clonal types of clinical E. coli O157:H7 in Northern Ireland to help define sources and routes of transmission of this infection locally

A two-step species-specific 16S rRNA PCR assay for the detection of Taylorella equigenitalis in horses

<p/> <p>A two-step PCR assay was developed for the molecular detection of Taylorella equigenitalis, a Gram-negative genital bacterial pathogen in horses. Two specific oligonucleotide primers (TE16SrRNABCHf [25mer] and TE16SrRNABCHr [29mer]) were designed from multiple alignments of the 16S rRNA gene loci of several closely related taxa, including T. asinigenitalis. Subsequent enhanced surveillance of 250 Thoroughbred animals failed to detect the presence of this organism directly from clinical swabs taken from the genital tract of mares and stallions. Such a molecular approach offers a sensitive and specific alternative to conventional culture techniques, and has the potential to lead to improved diagnosis and subsequent management of horses involved in breeding programmes.</p