Department of Microbiology,1 BioTechnology Institute,2 Department of Soil, Water, and Climate, University of Minnesota, St. Paul, Minnesota 551083
Received 22 November 2005/ Accepted 29 March 2006
The contamination of waterways with fecal material is a persistent threat to public health. Identification of the sources of fecal contamination is a vital component for abatement strategies and for determination of total maximum daily loads. While phenotypic and genotypic techniques have been used to determine potential sources of fecal bacteria in surface waters, most methods require construction of large known-source libraries, and they often fail to adequately differentiate among environmental isolates originating from different animal sources. In this study, we used pooled genomic tester and driver DNAs in suppression subtractive hybridizations to enrich for host source-specific DNA markers for Escherichia coli originating from locally isolated geese. Seven markers were identified. When used as probes in colony hybridization studies, the combined marker DNAs identified 76% of the goose isolates tested and cross-hybridized, on average, with 5% of the human E. coli strains and with less than 10% of the strains obtained from other animal hosts. In addition, the combined probes identified 73% of the duck isolates examined, suggesting that they may be useful for determining the contribution of waterfowl to fecal contamination. However, the hybridization probes reacted mainly with E. coli isolates obtained from geese in the upper midwestern United States, indicating that there is regional specificity of the markers identified. Coupled with high-throughput, automated macro- and microarray screening, these markers may provide a quantitative, cost-effective, and accurate library-independent method for determining the sources of genetically diverse E. coli strains for use in source-tracking studies. However, future efforts to generate DNA markers specific for E. coli must include isolates obtained from geographically diverse animal hosts.
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Applied and Environmental Microbiology, June 2006, p.
4028-4035, Vol. 72, No. 6
0099-2240/06/$08.00+0 doi:10.1128/AEM.00034-06
Copyright
© 2006, American
Society for Microbiology. All Rights Reserved.
Ecology of Antibiotic Resistance Genes: Characterization
of Enterococci from Houseflies Collected in Food Settings
Lilia Macovei and Ludek Zurek*
Department of Entomology, Kansas State University, Manhattan, Kansas 66506
Received 5 January 2006/ Accepted 29 March 2006
In this project, enterococci from the digestive tracts of 260 houseflies (Musca domestica L.) collected from five restaurants were characterized. Houseflies frequently (97% of the flies were positive) carried enterococci (mean, 3.1 x 103 CFU/fly). Using multiplex PCR, 205 of 355 randomly selected enterococcal isolates were identified and characterized. The majority of these isolates were Enterococcus faecalis (88.2%); in addition, 6.8% were E. faecium, and 4.9% were E. casseliflavus. E. faecalis isolates were phenotypically resistant to tetracycline (66.3%), erythromycin (23.8%), streptomycin (11.6%), ciprofloxacin (9.9%), and kanamycin (8.3%). Tetracycline resistance in E. faecalis was encoded by tet(M) (65.8%), tet(O) (1.7%), and tet(W) (0.8%). The majority (78.3%) of the erythromycin-resistant E. faecalis isolates carried erm(B). The conjugative transposon Tn916 and members of the Tn916/Tn1545 family were detected in 30.2% and 34.6% of the identified isolates, respectively. E. faecalis carried virulence genes, including a gelatinase gene (gelE; 70.7%), an aggregation substance gene (asa1; 33.2%), an enterococcus surface protein gene (esp; 8.8%), and a cytolysin gene (cylA; 8.8%). Phenotypic assays showed that 91.4% of the isolates with the gelE gene were gelatinolytic and that 46.7% of the isolates with the asa1 gene aggregated. All isolates with the cylA gene were hemolytic on human blood. This study showed that houseflies in food-handling and -serving facilities carry antibiotic-resistant and potentially virulent enterococci that have the capacity for horizontal transfer of antibiotic resistance genes to other bacteria.
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Applied and Environmental Microbiology, June 2006, p.
4200-4206, Vol. 72, No. 6
0099-2240/06/$08.00+0 doi:10.1128/AEM.00137-06
Copyright
© 2006, American
Society for Microbiology. All Rights Reserved.
A Virulence and Antimicrobial Resistance DNA Microarray
Detects a High Frequency of Virulence Genes in Escherichia coli
Isolates from Great Lakes Recreational Waters
Katia Hamelin,1,2 Guillaume Bruant,3
Abdel El-Shaarawi,4 Stephen Hill,4 Thomas A. Edge,4
Sadjia Bekal,5 John Morris Fairbrother,3 Josée Harel,3
Christine Maynard,1 Luke Masson,1 and Roland Brousseau1,2*
Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Ave., Montreal, Quebec, Canada H4P 2R2,1 Département de Microbiologie et Immunologie, Université de Montréal, 2900 Edouard Montpetit Blvd., Montréal, Québec, Canada H3T 1J4,2 Groupe de Recherche en Maladies Infectieuses du Porc, Faculté de Médecine Vétérinaire, Université de Montréal, 3200 Sicotte Str., Saint-Hyacinthe, Québec, Canada J2S 7C6,3 National Water Research Institute, Environment Canada, 867 Lakeshore Road, Burlington, Ontario, Canada L7R 4A6,4 Laboratoire de Santé Publique du Québec, 20045 Chemin Sainte-Marie, Sainte-Anne-de-Bellevue, Québec, Canada H9X 3R55
Received 18 January 2006/ Accepted 5 April 2006
Escherichia coli is generally described as a commensal species with occasional pathogenic strains. Due to technological limitations, there is currently little information concerning the prevalence of pathogenic E. coli strains in the environment. For the first time, using a DNA microarray capable of detecting all currently described virulence genes and commonly found antimicrobial resistance genes, a survey of environmental E. coli isolates from recreational waters was carried out. A high proportion (29%) of 308 isolates from a beach site in the Great Lakes carried a pathotype set of virulence-related genes, and 14% carried antimicrobial resistance genes, findings consistent with a potential risk for public health. The results also showed that another 8% of the isolates had unusual virulence gene combinations that would be missed by conventional screening. This new application of a DNA microarray to environmental waters will likely have an important impact on public health, epidemiology, and microbial ecology in the future.
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Applied and Environmental Microbiology, June 2006, p.
4214-4224, Vol. 72, No. 6
0099-2240/06/$08.00+0 doi:10.1128/AEM.01036-05
Copyright
© 2006, American
Society for Microbiology. All Rights Reserved.
Development of Bacteroides 16S rRNA Gene TaqMan-Based
Real-Time PCR Assays for Estimation of Total, Human, and Bovine Fecal Pollution
in Water
Alice Layton,1,2* Larry McKay,1,3
Dan Williams,1 Victoria Garrett,1 Randall Gentry,1,4
and Gary Sayler1,2
Center for Environmental Biotechnology,1 Department of Microbiology,2 Department of Earth and Planetary Sciences,3 Department of Civil and Environmental Engineering, The University of Tennessee, Knoxville, Tennessee 379964
Received 5 May 2005/ Accepted 10 April 2006
Bacteroides species are promising indicators for differentiating livestock and human fecal contamination in water because of their high concentration in feces and potential host specificity. In this study, a real-time PCR assay was designed to target Bacteroides species (AllBac) present in human, cattle, and equine feces. Direct PCR amplification (without DNA extraction) using the AllBac assay was tested on feces diluted in water. Fecal concentrations and threshold cycle were linearly correlated, indicating that the AllBac assay can be used to estimate the total amount of fecal contamination in water. Real-time PCR assays were also designed for bovine-associated (BoBac) and human-associated (HuBac) Bacteroides 16S rRNA genes. Assay specificities were tested using human, bovine, swine, canine, and equine fecal samples. The BoBac assay was specific for bovine fecal samples (100% true-positive identification; 0% false-positive identification). The HuBac assay had a 100% true-positive identification, but it also had a 32% false-positive rate with potential for cross-amplification with swine feces. The assays were tested using creek water samples from three different watersheds. Creek water did not inhibit PCR, and results from the AllBac assay were correlated with those from Escherichia coli concentrations (r2 = 0.85). The percentage of feces attributable to bovine and human sources was determined for each sample by comparing the values obtained from the BoBac and HuBac assays with that from the AllBac assay. These results suggest that real-time PCR assays without DNA extraction can be used to quantify fecal concentrations and provide preliminary fecal source identification in watersheds.
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Murulee Byappanahalli, Ph. D.
Research Investigator
U.S. Geological Survey, Great Lakes Science Center, and
University of Michigan,
School of Natural Resources & Environment, Ann Arbor, Michigan
Lake Michigan Ecological Research Station,
1100 N. Mineral Springs Road
Porter, Indiana 46304
Phone: (219) 926-8336 ext. 421
Fax: (219) 929-5792
E-mail: byappan@usgs.gov