A simplified low-cost approach to antibiotic resistance profiling for faecal source tracking.

Antibiotic resistance profiling (ARP) is a potentially useful method for distinguishing faecal bacteria according to host source. This phenotypic approach has cost benefits over genotypic methods, but existing protocols are time-consuming and manual data handling is open to human error. A simplified, low-cost approach to the ARP technique was developed that used automated data recording techniques combined with simple statistical analyses to compare isolates of the genus Enterococcus from various faecal sources. An initial battery of 21 antibiotics (at up to four concentrations) was chosen for source discrimination. Images of growth or non-growth in microplate wells were stored as bitmaps and converted to binary data to form a database of known antibiotic resistance profiles. Discriminant function analysis (DFA) showed that the average rate of isolates correctly classified by the database (wastewater vs non-wastewater) was 86%. Once the more discriminating antibiotics and their concentrations had been identified, it was possible to reduce the number of tests from 80 to 18 whilst increasing the number of correctly classified human isolates. ARP could offer a low-cost and rapid means of identifying sources of faecal pollution. As such, the technique may be of particular benefit to developing countries, where water quality may have a significant impact on health and where cost is a major factor when choosing environmental management technology.

Phenotypic population characteristics of the enterococci in wastewater and animal faeces: implications for the new European directive on the quality of bathing waters.

Faecal pollution of recreational bathing waters may derive from point sources of various wastewaters or from more diffuse sources such as run-off of agricultural wastes. The paper describes the application of population similarity studies to the enterococcal flora of various animal faeces and municipal wastewaters as a means of distinguishing human from animal faecal material. A simplified phenotypic testing technique (PhenePlate, PhP) was used to study the fermentation kinetics of eleven carbohydrates by all bacterial isolates. Enterococcal isolates (1,766) from six sources were investigated. Enterococcal population diversity (measured as Simpson's Diversity Index) in wastewater samples was high (mean D(i) = 0.95) compared with those of non-human faeces. The mean diversity of isolates in seabird faeces was 0.72, in sheep and donkey faeces 0.44, in dog faeces 0.42 and in cattle faeces 0.32. Analysis of population similarity coefficients demonstrated that faeces from sheep and cattle showed the greatest similarity (S(p) = 0.72). Sheep and cattle faeces demonstrated a low similarity to municipal wastewater samples. This would suggest that population similarity studies might be a useful tool for distinguishing the relative contributions of municipal wastewater and agricultural run-off to bathing water pollution. The PhP procedure identified a specific PhP type that appears to have high specificity to non-human faeces. It may, therefore, represent an important tool in source tracking. Additional phenotypic and genotypic analysis of PhP types that demonstrate a high degree of source specificity is required. The benefits and limitations of the use of population similarity studies to distinguish pollution sources are discussed in comparison with other source tracking approaches and the implications of these developments for future European Union legislation on the quality of bathing waters are discussed.

Characterization of the nucleoside triphosphate phosphohydrolase I gene from the Choristoneura fumiferana entomopoxvirus.

Poxviruses carry the enzyme, nucleoside triphosphate phosphohydrolase I (NPH I), required for early viral transcription in the cytoplasm of infected cells. The gene (nph I) encoding this enzyme from Choristoneura fumiferana entomopoxvirus (CfEPV) has been located in the viral genome, cloned and characterized. It has an open reading frame of 1941 nucleotides, potentially encoding a protein with a predicted molecular mass of 76.04 kDa and a pI of 8.83. It has a TAAATG motif where the trinucleotide ATG represents the translational start signal an AT-rich (88%) sequence and an early transcription termination signal (TTTTTAT) upstream of the ATG codon. Northern blot analysis of mRNA from infected larvae showed that a single 4.0 kb transcript which appeared late at day 20 post infection (p.i.) and its transcription continued till day 37 p.i.. Primer extension experiments suggested that the main transcripts started at 15 bases upstream of AUG codon. NPH I homologues have been found in the genomes of other entomopoxviruses and vertebrate poxviruses. Alignment of their amino acid sequences suggested three conserved domains, two of which are considered as ATP binding domains. The most similar homologue is from the closely related entomopoxvirus. Choristoneura biennis EPV (CbEPV) where 98.2% of nucleotide and 97.2% of amino acid identities are observed, respectively. A single nucleotide difference in CfEPV nph I was sufficient to distinguish it from CbEPV by PCR amplification and digestion with a restriction enzyme.

Duodenal brush-border mucosal glucose transport and enzyme activities in aging man and effect of bacterial contamination of the small intestine.

Duodenal biopsies were collected from 38 subjects (24 female and 14 male) ranging in age from 55 to 91 years. Evidence of bacterial contamination of the small bowel (BCSB) was sought at the same time by bacterial culture of duodenal aspirates and by hydrogen and [14C]glycocholic acid breath tests; subjects were considered to be positive for BCSB if any one of the three tests was abnormal. Biopsies were analyzed for six brush-border membrane enzyme activities: maltase, sucrase, lactase, alkaline phosphatase, leucine aminopeptidase, and alpha-glucosidase. Analysis of covariance with age as the covariate indicated no significant effect of age on the specific activities of these enzymes. Mucosal Na(+)-dependent glucose transport was quantified in brush-border membrane vesicles prepared from the biopsies. In all groups, glucose transport at 20-30 sec was greater (ranging from mean values of 2.45 to 3.66 times) than at 45 min, consistent with Na(+)-coupled glucose transport, and no significant effect of age was observed. BCSB had no significant effect on specific activities of any of the duodenal mucosal hydrolases but was associated with reduced (P = 0.05) brush-border glucose transport. None of the variables studied was significantly affected by the gender of subjects. In conclusion, these biochemical data do not support the contention that reduced capacity for carbohydrate absorption in the elderly is explained by reductions in duodenal brush-border mucosal disaccharidase activities or glucose transport.