Application of denaturing gradient gel electrophoresis (DGGE) for assessing fecal pollution sources at a recreational beach.

We investigated the effectiveness of Escherichia coli community fingerprinting for identifying fecal pollution sources impacting a recreational beach. E. coli in water collected from the beach, nearby creek and storm sewer outfall were enumerated using membrane filtration, while E. coli communities were characterized following polymerase chain reaction analysis and denaturing gradient gel electrophoresis (DGGE) fingerprinting. Analysis of E. coli densities to determine the contributions of the creek and storm sewer during dry weather was inconclusive. However, DGGE fingerprinting indicated that the creek E. coli communities had a greater impact on the beach community composition (80-95% similarity), than on storm sewer communities (41-64%). Following rainfall events, E. coli communities in the creek were at least 93% similar to those at the beach, while the similarity of the outfall and beach communities varied from 72 to 96%. Furthermore, E. coli communities at the beach were more similar to creek communities than to storm sewer communities after the first 2 h and 48 h following the onset of rainfall, and of comparable similarity following 24 h of rainfall, suggesting transient contributions from the storm sewer. DGGE analysis of E. coli communities provided evidence that the creek was a consistent source of E. coli to the beach, while the storm sewer was a transient source.

Persistence of mixed staphylococci assemblages following disinfection of hospital room surfaces.

The distribution of staphylococcal assemblages on surfaces in hospital rooms was assessed before and after daily disinfection with quaternary ammonia products. DNA was extracted from enrichment cultures of bacteria, which were swabbed from each of nine surface types, and subjected to analysis by staphylococci-specific, denaturing gradient gel electrophoresis. A genetic marker for Staphylococcus epidermidis/kloosii was detected on all surface types before and after cleaning, whereas markers for Staphylococcus aureus and Staphylococcus lugdunensis were detected on five surface types. Overall, genetic makers for several staphylococci known to colonize and infect humans remained ubiquitous in each room following daily disinfection practices.

Detection and differentiation of staphylococcal contamination of clinical surfaces using denaturing gradient gel electrophoresis.

The detection and identification of staphylococci from the environment of at-risk patients can be an important step in determining the role of the environment in hospital-acquired infections. Current methods that are used to identify these pathogens are either limited in their capabilities, expensive and/or labour intensive. We developed a denaturing gradient gel electrophoresis (DGGE) analysis protocol for the detection and identification of staphylococci that takes advantage of species-specific polymorphisms in the gene that encodes elongation factor Tu (tuf). The protocol was optimised by performing polymerase chain reaction (PCR)-DGGE analysis on DNA isolated from pure cultures of 27 different staphylococcal species. This resulted in the separation of the PCR products into 19 different band positions, including unique positions for important species such as Staphylococcus aureus, S. hominis, S. lugdunensis, S. warneri, S. capitis, S. caprae and S. saprophyticus. Application of the method was demonstrated by swabbing 15 clinical surfaces in an isolation room occupied by a patient before and after routine cleaning. PCR-DGGE analysis of tuf showed that despite cleaning efforts, the surfaces remained contaminated with several species of staphylococci, including S. aureus, S. epidermidis, S. lugdunensis, S. hominis, S. haemolyticus and S. simulans. We conclude that DGGE of tuf represents a promising technique for the detection, characterisation and monitoring of mixed assemblages of staphylococci in the healthcare environment.