tir- and stx-positive Escherichia coli in stream waters in a metropolitan area

Diarrheagenic Escherichia coli, which may include the enteropathogenic E. coli and the enterohemorrhagic E. coli, are a significant cause of diarrheal disease among infants and children in both developing and developed areas. Disease outbreaks related to freshwater exposure have been documented, but the presence of these organisms in the urban aquatic environment is not well characterized. From April 2002 through April 2004 we conducted weekly surveys of streams in the metropolitan Baltimore, Md., area for the prevalence of potentially pathogenic E. coli by using PCR assays targeting the tir and stx(1) and stx(2) genes. Coliforms testing positive for the presence of the tir gene were cultured from 653 of 1,218 samples (53%), with a greater prevalence associated with urban, polluted streams than in suburban and forested watershed streams. Polluted urban streams were also more likely to test positive for the presence of one of the stx genes. Sequence analysis of the tir amplicon, as well as the entire tir gene from three isolates, indicated that the pathogenic E. coli present in the stream waters has a high degree of sequence homology with the E. coli O157:H7 serotype. Our data indicate that pathogenic E. coli are continually deposited into a variety of stream habitats and suggest that this organism may be a permanent member of the gastrointestinal microflora of humans and animals in the metropolitan Baltimore area.     

Ubiquity and Persistence of Escherichia coli in a Midwestern Coastal Stream

Dunes Creek, a small Lake Michigan coastal stream that drains sandy aquifers and wetlands of Indiana Dunes, has chronically elevated Escherichia coli levels along the bathing beach near its outfall. This study sought to understand the sources of E. coli in Dunes Creek's central branch. A systematic survey of random and fixed sampling points of water and sediment was conducted over 3 years. E. coli concentrations in Dunes Creek and beach water were significantly correlated. Weekly monitoring at 14 stations during 1999 and 2000 indicated chronic loading of E. coli throughout the stream. Significant correlations between E. coli numbers in stream water and stream sediment, submerged sediment and margin, and margin and 1 m from shore were found. Median E. coli counts were highest in stream sediments, followed by bank sediments, sediments along spring margins, stream water, and isolated pools; in forest soils, E. coli counts were more variable and relatively lower. Sediment moisture was significantly correlated with E. coli counts. Direct fecal input inadequately explains the widespread and consistent occurrence of E. coli in the Dunes Creek watershed; long-term survival or multiplication or both seem likely. The authors conclude that (i) E. coli is ubiquitous and persistent throughout the Dunes Creek basin, (ii) E. coli occurrence and distribution in riparian sediments help account for the continuous loading of the bacteria in Dunes Creek, and (iii) ditching of the stream, increased drainage, and subsequent loss of wetlands may account for the chronically high E. coli levels observed.     

Extraintestinal Pathogenic and Antimicrobial-Resistant Escherichia coli Contamination of 56 Public Restrooms in the Greater Minneapolis-St. Paul Metropolitan Area

How extraintestinal pathogenic Escherichia coli (ExPEC) and antimicrobial-resistant E. coli disseminate through the population is undefined. We studied public restrooms for contamination with E. coli and ExPEC in relation to source and extensively characterized the E. coli isolates. For this, we cultured 1,120 environmental samples from 56 public restrooms in 33 establishments (obtained from 10 cities in the greater Minneapolis-St. Paul, MN, metropolitan area in 2003) for E. coli and compared ecological data with culture results. Isolates underwent virulence genotyping, phylotyping, clonal typing, pulsed-field gel electrophoresis (PFGE), and disk diffusion antimicrobial susceptibility testing. Overall, 168 samples (15% from 89% of restrooms) fluoresced, indicating presumptive E. coli: 25 samples (2.2% from 32% of restrooms) yielded E. coli isolates, and 10 samples (0.9% from 16% of restrooms) contained ExPEC. Restroom category and cleanliness level significantly predicted only fluorescence, gender predicted fluorescence and E. coli, and feces-like material and toilet-associated sites predicted all three endpoints. Of the 25 E. coli isolates, 7 (28%) were from phylogenetic group B2(virulence-associated), and 8 (32%) were ExPEC. ExPEC isolates more commonly represented group B2 (50% versus 18%) and had significantly higher virulence gene scores than non-ExPEC isolates. Six isolates (24%) exhibited ≥3-class antibiotic resistance, 10 (40%) represented classic human-associated sequence types, and one closely resembled reference human clinical isolates by pulsed-field gel electrophoresis. Thus, E. coli, ExPEC, and antimicrobial-resistant E. coli sporadically contaminate public restrooms, in ways corresponding with restroom characteristics and within-restroom sites. Such restroom-source E. coli strains likely reflect human fecal contamination, may pose a health threat, and may contribute to population-wide dissemination of such strains.     

Survival of Escherichia coli in Stream Water in Relation to Carbon Dioxide and Plant Photosynthesis

During a study of the survival of Escherichia coli in a chalk stream and in the laboratory, the organism ceased to grow in filtered stream water if air free of atmospheric carbon dioxide was passed over the culture. Fewer E. coli cells grew with the alga Stichococcus bacillaris in the light than in the dark. In the stream, low numbers of the organism were associated with plant blooms (macrophytes and microphytes). It is suggested that the numbers of E. coli in natural waters may sometimes be determined by the content of dissolved carbon dioxide which itself must be affected by the volume of photosynthesizing green cells.     

Antibiotic-Resistant Escherichia coli in Wastewaters, Surface Waters, and Oysters from an Urban Riverine System

The antibiotic resistance (AR) patterns of 462 Escherichia coli isolates from wastewater, surface waters, and oysters were determined. Rates of AR and multiple-AR among isolates from surface water sites adjacent to wastewater treatment plant (WWTP) discharge sites were significantly higher (P < 0.05) than those among other isolates, whereas the rate of AR among isolates from oysters exposed to WWTP discharges was low (<10%).     

Evaluation of an Inexpensive Growth Medium for Direct Detection of Escherichia coli in Temperate and Sub-Tropical Waters

The cost and complexity of traditional methods for the detection of faecal indicator bacteria, including E. coli, hinder widespread monitoring of drinking water quality, especially in low-income countries and outside controlled laboratory settings. In these settings the problem is exacerbated by the lack of inexpensive media for the detection of E. coli in drinking water. We developed a new low-cost growth medium, aquatest (AT), and validated its use for the direct detection of E. coli in temperate and sub-tropical drinking waters using IDEXX Quanti-Tray^®. AT was compared with IDEXX Colilert-18^® and either EC-MUG or MLSB for detecting low levels of E. coli from water samples from temperate (n = 140; Bristol, UK) and subtropical regions (n = 50, Pretoria/Tshwane, South Africa). Confirmatory testing (n = 418 and 588, respectively) and the comparison of quantitative results were used to assess performance. Sensitivity of AT was higher than Colilert-18^® for water samples in the UK [98.0% vs. 86.9%; p<0.0001] and South Africa [99.5% vs. 93.2%; p = 0.0030]. There was no significant difference in specificity, which was high for both media (>95% in both settings). Quantitative results were comparable and within expected limits. AT is reliable and accurate for the detection of E. coli in temperate and subtropical drinking water. The composition of the new medium is reported herein and can be used freely.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> Diversity in Livestock Manures and Agriculturally Impacted Stream Waters

Escherichia coli (E. coli) isolate diversity enhances the likelihood of survival, spread, and/or transmission of the organism among environments. Understanding the ecology of this important organism is requisite for development of more accurate protocols for monitoring and regulatory purposes. In this study, E. coli diversity, gene profiles and transport properties of isolates from different livestock and water sources were evaluated. Strain diversity was evaluated by BOX-PCR, phylotyping, and profiling for 15 genes associated with adhesion, toxin production, iron acquisition or capsular synthesis. Attachment efficiencies were calculated for 17 isolates following transport through saturated porous media. Richness of genotype profiles for livestock isolates was relatively low (25, 12, and 11 for swine, poultry and dairy, respectively) compared to those from stream-water (115 and 126 from dry or wet weather events, respectively). Attachment efficiencies varied by an order of magnitude (0.039–0.44) and the isolate with the highest attachment efficiency possessed the largest suite of targeted genes including those for adherence (iha, agn43, and fimH), surface exclusion (traT) and the siderophore iroN _E.coli . Variation in E. coli isolates based on temporal and ecological source was found to translate to equally broad ranges in transport efficiency underscoring the large degree of genotypic and phenotypic variation that exists among E. coli isolates. The impact of this diversity on genetic exchange and the concomitant effect on the organisms’ fate and transport under in situ environmental conditions warrant further investigation. These factors also require careful consideration for purposes of modeling, source tracking, and risk assessment.     

A Survey of Escherichia coli and Salmonella in the Hyporheic Zone of a Subtropical Stream: Their Bacteriological,Physicochemical and Environmental Relationships

The Hyporheic Zone is among the most important interstitial freshwater habitats, but the relationship between biotic and abiotic factors in this zone remains under-explored. Enterobacteria were expected to be present, but no specific studies had ever confirmed this prediction. The aim of this study was, therefore, to evaluate the total coliforms, Escherichia coli and Salmonella spp. in hyporheic water and to determine the relationship of the physical, chemical and environmental factors at different depths in a rainforest stream. To this end, thirty-six water samples were collected at three depths in sites located in the first, second and third orders in diverse substrates. The total coliforms, Escherichia coli and Salmonella sp. were evaluated in terms of their CFU/ml. In the interstitial samples, coliforms were detected in 100% of the samples. The total coliform counts had higher values at intermediate depths, while E. coli and Salmonella spp. instead had higher values at intermediate and large depths, often reaching or exceeding the values of the surface samples. Our results revealed that Salmonella spp. and the coliforms have different microhabitat preferences. Salmonella spp. and coliform species prefer deposition areas, such as lateral sides of pools, curves and bars, but they have a tendency to distribute into different depths, likely due to temperature differences. Salmonella spp. prefer compact substrata, with fewer fluids passing through and with upwelling areas with lower oxygen inflow. The coliform species showed the opposite preference. Our results suggest that bacterial variation is related to environmental factors and physical-chemical parameters within the HZ and may play a key role in the microbial diversity and distribution in these ecosystems.     

Escherichia coli as a bioreporter in ecotoxicology

Ecotoxicological assessment relies to a large extent on the information gathered with surrogate species and the extrapolation of test results across species and different levels of biological organisation. Bacteria have long been used as a bioreporter for genotoxic testing and general toxicity. Today, it is clear that bacteria have the potential for screening of other toxicological endpoints. Escherichia coli has been studied for years; in-depth knowledge of its biochemistry and genetics makes it the most proficient prokaryote for the development of new toxicological assays. Several assays have been designed with E. coli as a bioreporter, and the recent trend to develop novel, better advanced reporters makes bioreporter development one of the most dynamic in ecotoxicology. Based on in-depth knowledge of E. coli, new assays are being developed or existing ones redesigned, thanks to the availability of new reporter genes and new or improved substrates. The technological evolution towards easier and more sensitive detection of different gene products is another important aspect. Often, this requires the redesign of the bacterium to make it compatible with the novel measuring tests. Recent advances in surface chemistry and nanoelectronics open the perspective for advanced reporter based on novel measuring platforms and with an online potential. In this article, we will discuss the use of E. coli-based bioreporters in ecotoxicological applications as well as some innovative sensors awaited for the future.     

A Virulence and Antimicrobial Resistance DNA Microarray Detects a High Frequency of Virulence Genes in Escherichia coli Isolates from Great Lakes Recreational Waters

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.     

Microarray Analysis of Escherichia coli Strains from Interstitial Beach Waters of Lake Huron (Canada)

DNA microarray analyses revealed that clusters of repetitive extragenic palindromic PCR-related Escherichia coli isolates were isogenic only within interstitial Lake Huron beach water samples and not in surrounding waters. This suggested that adaptation and growth occurred within the interstitial water sites tested. All isolates were nonpathogenic, and three lake isolates possessed tetracycline resistance genes.     

Novel Method for Rapid Assessment of Antibiotic Resistance in Escherichia coli Isolates from Environmental Waters by Use of a Modified Chromogenic Agar

We validated a novel method for screening Escherichia coli resistance to antibiotics in environmental samples using modified Difco MI agar (Becton Dickinson) impregnated with selected antibiotics (tetracycline, ampicillin, cephalexin, and sulfamethoxazole), termed MI-R. This method combines an existing rapid assessment technique for E. coli enumeration with clinical reference data for breakpoint analysis of antibiotic resistance and was developed to address issues encountered when clinical methods are used with environmental samples. Initial trials conducted using strains of E. coli with resistance to the selected antibiotics showed that this method was reproducible and accurate with respect to antibiotic resistance. Trials using wastewater effluent demonstrated the precision of the method, and the levels of resistance found in effluent were directly comparable to the levels of antibiotic resistance determined using the more traditional CLSI (formerly NCCLS) disk susceptibility test. All wastewater isolates growing on MI-R plates were confirmed to be resistant using the CLSI disk susceptibility test. Bacterial resistance to ampicillin (38% ± 4% overall), sulfamethoxazole, tetracycline (21% ± 3% overall), and ciprofloxacin (6% ± 1%) were found in wastewater effluent. A successful trial was also conducted with water collected from the Brisbane River, Australia. The levels of antibiotic resistance in E. coli ranged from 0 to 47% for ampicillin, from 0 to 24% for tetracycline, from 0 to 63% for sulfamethoxazole, and from 0 to 1% for ciprofloxacin, with the highest incidence of resistance associated with wastewater treatment plant discharges. This method has great potential for rapid and representative assessment of antibiotic resistance in E. coli and could allow increased sample analysis, resulting in greater confidence in spatial analysis in environmental studies.     

Comparison of Rapid Quantitative PCR-Based and Conventional Culture-Based Methods for Enumeration of Enterococcus spp. and Escherichia coli in Recreational Waters

Recreational water quality is currently monitored using culture-based methods that require 18 to 96 h for results. Quantitative PCR (QPCR) methods that can be completed in less than 2 h have been developed, but they could yield different results than the conventional methods. We present two studies in which samples were processed simultaneously for Enterococcus spp. and Escherichia coli using two culture-based methods (EPA method 1600 and Enterolert/Colilert-18) and QPCR. The proprietary QPCR assays targeted the 23S rRNA (Enterococcus spp.) and uidA (E. coli) genes and were conducted using lyophilized beads containing all reagents. In the first study, the QPCR method developers processed 54 blind samples that were inoculated with sewage or pure cultures or were ambient beach samples. The second study involved 163 samples processed by water quality personnel. The correlation between results of QPCR and EPA 1600 during the first study (r²) was 0.69 for Enterococcus spp., which was less than that observed between the culture-based methods (r², 0.87). During the second study, the correlations were similar. No false positives occurred in either study when QPCR-based assays were used with blank samples. Levels of reproducibility measured through coefficients of variation were similar for results by Enterococcus QPCR and culture-based methods during both studies but were higher for E. coli QPCR results in the first study. Regarding the concentration at which beach management decisions are issued in the State of California, the agreement between results of Enterococcus QPCR and EPA method 1600 was 88%, compared to 94% agreement between EPA method 1600 and Enterolert. The beach management decision agreement between E. coli QPCR and Colilert-18 was 94%. The samples showing disagreement suggested an underestimation bias for QPCR.Fecal indicator bacteria (FIB) are presently measured to assess recreational water quality using one of three U.S. Environmental Protection Agency (EPA)-approved method classes: membrane filtration, multiple-tube fermentation, or defined-substrate technology (DST). The membrane filtration approach is based on passing water through a filter that is placed on a medium selective for the bacterial group of interest. Multiple-tube fermentation relies on quantification via most-probable number (MPN) using serial dilutions within replicate tubes incubated with selective media. The DST methods are also typically used in an MPN approach, where water samples are incubated with specific media in a tray with replicate wells. These methods are detailed by the American Public Health Association (1, 2) and in the U.S. Federal Register (29). These culture-based methods are widely accepted because of their relative ease of use, low cost, and demonstrated relationship to health risk (6, 7, 9, 11). However, the time required for sample processing ranges from 18 to 96 h, with confirmation and verification steps taking even longer.Advances in technology provide new opportunities to measure bacterial water quality more rapidly (4, 16, 19). While currently used methods rely on bacterial growth and metabolic activity, these new methods directly measure DNA, RNA, or surface immunological properties. This is important because FIB concentrations have been shown to change substantially on a time scale of hours (3). Thus, contaminated beaches remain open during the laboratory processing period, but the contamination event has often passed by the time warnings are posted (20). By eliminating the need for a lengthy incubation step, results from rapid methods are available in several hours, enabling managers to take action to protect public health (i.e., post warnings or close beaches) on the same day that water samples are collected. Rapid quantitative PCR (QPCR) methods, such as the Enterococcus sp. assay developed by Haugland et al. (16), have also exhibited significant relationships with the risk of gastrointestinal illness in beachgoers (31, 32).While QPCR-based methods are promising, their results may differ from those of the conventional culture-based methods that they are intended to replace. Since QPCR measures genetic material rather than the viable cells quantified by culture-based methods, it may overestimate FIB concentrations because of the inclusion of target DNA from dead or dying cells in the measurement. Differences may also be related to chemical inhibition of the amplification, assay design, or challenges in technology transfer to personnel with little or no molecular biology-based experience. Acceptance of new methods by water quality professionals with a long history of using culture-based methods will depend on understanding the frequency and the underlying causes of these differences. Whereas a number of studies have assessed the relative performance of the three most commonly used culture-based methods (13, 25, 30), there have been few comparisons of QPCR- and culture-based method performance, especially with marine beach samples. Here, quantification of FIB by Enterococcus species QPCR (here referred to as simply Enterococcus QPCR) and Escherichia coli QPCR is compared to that by their respective culture-based assays. We also quantify the effect of two different QPCR sample processing approaches and assess the ability of personnel from a state-certified water quality laboratory to implement the rapid QPCR-based methods.     

Escherichia coli with a linear genome

Chromosomes in eukaryotes are linear, whereas those of most, but not all, prokaryotes are circular. To explore the effects of possessing a linear genome on prokaryotic cells, we linearized the Escherichia coli genome using the lysogenic lambda-like phage N15. Linear genome E. coli were viable and their genome structure was stable. There were no appreciable differences between cells with linear or circular genomes in growth rates, cell and nucleoid morphologies, genome-wide gene expression (with a few exceptions), and DNA gyrase- and topoisomerase IV-dependent growth. However, under dif-defective conditions, only cells with a circular genome developed an abnormal phenotype. Microscopy indicated that the ends of the linear genome, but not the circular genome, were separated and located at each end of a new-born cell. When tos - the cis-element required for linearization - was inserted into different chromosomal sites, those strains with the genome termini that were more remote from dif showed greater growth deficiencies.     

Stable-light-emitting Escherichia coli as a biosensor

We have studied possibilities for constructing Escherichia coli strains capable of producing stable light. Light production in E. coli is achieved by cloning the genes encoding bacterial luciferase from Vibrio harveyi. To gain the advantage of sensitive detection of light we transferred the genes under the control of a strong, regulatable promoter system. Stabilization of light produced by E. coli clones was accomplished by finding the optimal plasmid construction and growth conditions as well as suitable measuring buffers. The adjustment of the luciferase synthesis for bioluminescence measurements to a high but not harmful level gives healthy cells and stable luciferase. Cultivation at 30 °C in an uninduced state was found to be the most important factor in getting stable-light production. The overall cell metabolism being unstressed gives us the possibility of monitoring cell physiology and factors affecting it via bioluminescence reactions in vivo. To make the results easy to interpret the light emission has to be stable during a measurement period of one to several hours. In the case of the original light-producing bacteria, Vibrio and Photobacterium strains it has not thus far been possible to find conditions where light emission would be stable for several hours. Based on our findings an automated biosensor system can be developed to monitor the effects of biologically active compounds against stable-light-producing bacteria.